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https://openalex.org/W4244572299	https://esurf.copernicus.org/articles/9/1239/2021/esurf-9-1239-2021.pdf	English	null	Short communication: Analytical models for 2D landscape evolution	null	2,021	cc-by	9,745	Short communication: Analytical models for 2D landscape evolution Philippe Steer Univ. Rennes, CNRS, Géosciences Rennes, UMR 6118, 35000 Rennes, France Correspondence: Philippe Steer (philippe.steer@univ-rennes1.fr) Received: 14 April 2021 – Discussion started: 21 April 2021 Revised: 12 July 2021 – Accepted: 2 August 2021 – Published: 15 September 2021 Philippe Steer Univ. Rennes, CNRS, Géosciences Rennes, UMR 6118, 35000 Rennes, France Correspondence: Philippe Steer (philippe.steer@univ-rennes1.fr) Received: 14 April 2021 – Discussion started: 21 April 2021 Revised: 12 July 2021 – Accepted: 2 August 2021 – Published: 15 September 2021 Abstract. Numerical modelling offers a unique approach to understand how tectonics, climate and surface pro- cesses govern landscape dynamics. However, the efﬁciency and accuracy of current landscape evolution models remain a certain limitation. Here, I develop a new modelling strategy that relies on the use of 1D analytical solutions to the linear stream power equation to compute the dynamics of landscapes in 2D. This strategy uses the 1D ordering, by a directed acyclic graph, of model nodes based on their location along the water ﬂow path to propagate topographic changes in 2D. This analytical model can be used to compute in a single time step, with an iterative procedure, the steady-state topography of landscapes subjected to river, colluvial and hillslope erosion. This model can also be adapted to compute the dynamic evolution of landscapes under either heterogeneous or time-variable uplift rate. This new model leads to slope–area relationships exactly consistent with predictions and to the exact preservation of knickpoint shape throughout their migration. Moreover, the absence of numerical diffusion or of an upper bound for the time step offers signiﬁcant advantages compared to numerical models. The main drawback of this novel approach is that it does not guarantee the time continuity of the topography through successive time steps, despite practically having little impact on model behaviour. Published by Copernicus Publications on behalf of the European Geosciences Union. 1 Introduction to a non-linear kinematic wave equation, which offers sim- ple ﬁnite-difference or ﬁnite-volume solutions in 1 and 2D (e.g. Pelletier, 2008; Braun and Willett, 2013; Campforts and Govers, 2015; Campforts et al., 2017). Despite these beneﬁts, these numerical solutions have several drawbacks: (1) their stability or consistency requires the use of a small time step that must respect the Courant condition, i.e. that an erosional wave cannot travel over a distance greater than one or a few node spacings during one time step, and (2) they are prone to numerical diffusion and therefore only offer approximate solutions. Numerical schemes in 2D have recently been de- veloped to reduce the time-step dependency on grid spac- ing (Braun and Willett, 2013) or numerical diffusion (Camp- forts and Govers, 2015). In 1D, evolution of river proﬁles can be derived using analytical solutions determined by the method of the characteristics (Luke, 1972, 1974, 1976; Weis- sel and Seidl, 1998; Whipple and Tucker, 1999; Lavé, 2005; Pritchard et al., 2009; Royden and Taylor Perron, 2013). These solutions have been successfully used in formal inver- While the elevated but incised landscapes of mountain belts testify to the cumulated actions of tectonics, erosion and cli- mate, unravelling how these processes act and interact to shape the Earth’s surface remains one of the most challeng- ing issues in Earth sciences (e.g. Molnar and England, 1990; Willett, 1999; Whipple, 2009; Steer et al., 2014; Croissant et al., 2019). Numerical models have been pivotal to under- standing how topography and erosion respond to spatial and temporal changes in climate and tectonics (e.g. Howard et al., 1994; Whipple and Tucker, 1999; Tucker and Whipple, 2002; Carretier and Lucazeau, 2005; Thieulot et al., 2014; Crois- sant et al., 2017). At the mountain scale, numerical mod- els generally account for geomorphological processes using effective and reduced-complexity erosion laws such as the stream power incision model (SPIM) for rivers (e.g. Howard et al., 1994) and diffusion for hillslopes (e.g. Roering et al., 1999). In particular, the SPIM is popular in landscape evo- lution models (LEMs) as its physical expression resolves Published by Copernicus Publications on behalf of the European Geosciences Union. 1240 P. Steer: Short communication: Analytical models for 2D landscape evolution where U is the uplift rate, K′ the erodibility, Qw = rA the water discharge with A the drainage area, r the mean daily runoff, and m and n are two exponents. 2 From a 1D to a 2D analytical solution to the stream power law Under this condition and assuming a constant and homogeneous uplift rate U, the steady-state river proﬁle ele- vation is Using this response time and assuming a constant but poten- tially heterogeneous uplift rate U(l) or a uniform but poten- tially variable uplift rate U(t), river proﬁle elevation can be derived analytically assuming A is known (see derivation in Royden and Taylor Perron, 2013). As I intend to implement a solution in a LEM, the solution needs to remain practical. In particular, it is noticeable that the response time and celer- ity become independent of local river slope S(l) = ∂z(l,t)/∂l when n = 1, which is a classical choice in forward or inverse landscape evolution models (e.g. Goren et al., 2014a; Fox et al., 2014). Under this condition and assuming a constant and homogeneous uplift rate U, the steady-state river proﬁle ele- vation is z(l) =z(0) + Uτ(l) = z(0) + U l Z 0 1 KA(l′)m dl′, with z(0) = zbase, (4) with z(0) = zbase, (4) and with zbase the base-level elevation. Note that this solution is asynchronous as steady state is achieved for an increas- ing response time in the upstream direction. Importantly, as the ﬂow network is not known a priori, this integral solution still requires one to numerically compute a ﬂow network and drainage area over a discretized grid. In the following, I adapt this formalism to develop two modelling approaches which either computes the steady-state topography of a landscape or solves for its dynamic evolution (Fig. 1). and with zbase the base-level elevation. Note that this solution is asynchronous as steady state is achieved for an increas- ing response time in the upstream direction. Importantly, as the ﬂow network is not known a priori, this integral solution still requires one to numerically compute a ﬂow network and drainage area over a discretized grid. In the following, I adapt this formalism to develop two modelling approaches which either computes the steady-state topography of a landscape or solves for its dynamic evolution (Fig. 1). In 1D, a classical detachment-limited approach to describe the rate of change in river elevation change z with time t is the SPIM (Howard and Kerby, 1983; Howard, 1994; Whipple and Tucker, 1999; Lague, 2014): 3 A single time step iterative solution to topographic steady state in 2D ∂z(l,t) ∂t = U(l,t) −K′(l)Qw(l)m ∂z(l,t) ∂l n , (1) This solution (Eq. 1 Introduction This equation can be cast in a more commonly used form, as a function of drainage area, by deﬁning an effective erodibility K = K′rm: sion of river proﬁles (Goren et al., 2014a; Fox et al., 2014; Goren, 2016), but they have been largely ignored in forward landscape evolution models, despite their inherent exact ac- curacy. This likely results from the apparent absence of an analytical solution in 2D. In this study, I extend the applicability of these 1D ana- lytical solutions to 2D problems by developing a new type of landscape evolution model based on analytical solutions. I ﬁrst demonstrate how this model, that I refer to as Salève, can be used to compute – in a single time step – a steady- state topography in 2D. I then develop a dynamic version of Salève to solve for transient landscape changes under hetero- geneous or time-variable uplift. Last, I demonstrate the abil- ity of Salève to accurately model the propagation of knick- points in LEMs and to account for river, colluvial and hills- lope erosion. ∂z(l,t) ∂t = U(l,t) −K(l)A(l)m ∂z(l,t) ∂l n . (2) (2) This equation corresponds to a non-linear kinematic wave equation with a celerity C(l) = K(l)A(l)m(∂z(l,t)/∂l)n−1 representing the speed at which information propagates along the river (e.g. Rosenbloom and Anderson, 1994; Weis- sel and Seidl, 1998; Whipple and Tucker, 1999; Royden and Taylor Perron, 2013). Following Royden and Taylor Per- ron (2013), this migrating information can be referred to as slope patches. Integrating the inverse of this celerity along the river path, from the river outlet at l = 0 to a point of co- ordinate l along the river, deﬁnes the river response time: 2 From a 1D to a 2D analytical solution to the stream power law τ(l) = l Z 0 1 C(l′)dl′ = l Z 0 1 K(l′)A(l′)m(∂z(l′,t)/∂l′)n−1 dl′. (3) (3) Most LEMs require the computation of river water discharge as the main driver of river erosion and sediment transport. While ﬂow algorithms based on physical considerations of- fer more accurate solutions (e.g. Davy et al., 2017), wa- ter routing in 2D LEMs is generally achieved using sim- ple ﬂow algorithms, like the steepest slope (O’Callaghan and Mark, 1984) or the multi-ﬂow direction (Quinn et al., 1991; Freeman, 1991). The Fastscape algorithm, and other graph-based approaches, offers a very efﬁcient means to or- der nodes along the steepest water ﬂow path and to compute river discharge and drainage area (Braun and Willett, 2013; Schwanghart and Scherler, 2014). A single receiver and po- tentially several donors are attributed to each node of the to- pographic grid to recursively build a node stack (or graph) from the outlet node to the crest nodes of each catchment. Each node is therefore associated with its outlet node through a single ﬂow path. These ﬂow paths represent 2D trajectories in the (x, y) space that can be converted to pseudo-1D trajec- tories (i.e. to directed acyclic graphs) using the node order- ing of the stack. For instance, local river slope along the wa- ter ﬂow can be computed by simply differentiating elevation over the distance along the river length l between successive nodes. The 2D LEMs solving for river erosion using a single ﬂow algorithm and local river slope or water discharge are therefore fundamentally solving a 1D problem, based on a 2D description of the ﬂow. To be more accurate, they actu- ally solve for a series of 1D problems, with one 1D problem for each catchment connected to an outlet. Using this response time and assuming a constant but poten- tially heterogeneous uplift rate U(l) or a uniform but poten- tially variable uplift rate U(t), river proﬁle elevation can be derived analytically assuming A is known (see derivation in Royden and Taylor Perron, 2013). As I intend to implement a solution in a LEM, the solution needs to remain practical. In particular, it is noticeable that the response time and celer- ity become independent of local river slope S(l) = ∂z(l,t)/∂l when n = 1, which is a classical choice in forward or inverse landscape evolution models (e.g. Goren et al., 2014a; Fox et al., 2014). P. Steer: Short communication: Analytical models for 2D landscape evolution 1241 Figure 1. Overview of the algorithms used for the (a) steady-state and (b) dynamic simulations. convergence of this iterative procedure, I deﬁne the degree of crest disequilibrium 1zcrest as being the average of the abso- lute difference of elevation between crest nodes of juxtapos- ing catchments. I ﬁnd that 1zcrest follows a rapid decay with Niter until reaching a slower decay phase when Niter ≥40. 1zcrest never reaches 0 m, even after 100 iterations, as dif- ferences of elevation can remain along the two sides of the crests, as in other LEMs, due to the non-continuity of the spa- tial discretization for grid-based models (Fig. 2c). However, the model reaches a stable solution at Niter = 127. Note that running the same model but with a different initial topogra- phy leads to a variability of this required number of iterations due to the initial conﬁguration of the ﬂow network. Changing U, K and L while keeping npt constant does not lead to a signiﬁcant change in the number of iterations required to reach steady state (Fig. 3). This shows that the convergence of this algorithm is independent of the model parametrization. However, increasing the number of points npt leads to an increase in the number of iterations required to reach steady state, which scales with n0.5 pt , or in other words with the number of nodes in one of the horizontal dimen- sions (Fig. 3). This scaling emerges due to the more numer- ous numbers of local (i.e. among direct neighbours) permu- tations of the crest location required to reach a stable ﬂuvial organization when increasing npt. Figure 1. Overview of the algorithms used for the (a) steady-state and (b) dynamic simulations. ceiver node τR(l) and its elevation zR(l): z(l) =zR(l) + U(l)(τ(l) −τR(l)) for l > 0, and z(0) = zbase. (5) (5) Obviously, this operation needs to be performed iteratively and in the correct node order, from the outlet node towards the upstream direction using the node stack or graph (Braun and Willett, 2013; Schwanghart and Scherler, 2014). Ignor- ing hillslope processes, I use this solution to attempt com- puting the steady-state topography with Salève in a single iteration (Fig. 2). The initial topography consists of a ﬂat sur- face with a random noise discretized by a regular grid. P. Steer: Short communication: Analytical models for 2D landscape evolution by com- puting the steepest slope, node order, and drainage area or discharge) after each iteration leads to a steady-state topog- raphy after a few tens of iterations Niter (Fig. 2). To assess the The obtained solution looks very roughly like a clas- sical steady-state topography, and yet it is not strictly at steady state (Fig. 2). Indeed, during this ﬁrst iteration, the scheme used (Fig. 1a) imposes the constraint that rivers de- velop over the ﬂow network deﬁned by the initial topogra- phy and, in turn, does not ensure that the nodes located on the same crest of two juxtaposing catchments share the same response time or the same elevation. This leads to an exces- sive elevation as some rivers have planar length greater than predicted. This is the main limit of this 1D algorithm that cannot ensure the optimality of the 2D organization of the river network at steady state after only one iteration (Fig. 2). P. Steer: Short communication: Analytical models for 2D landscape evolution I use m = 0.5, corresponding to the classical unit stream power, U = 10 mm yr−1, K′ = 1×10−6 yr−1, r = 5/365 m d−1 and a square model domain of extent L = 10 km with a reso- lution of 50 m, corresponding to npt = 40.401 points. Flow over the topography is computed using the single-ﬂow algo- rithm provided by TopoToolbox (Schwanghart and Scherler, 2014), which efﬁciently exploits the directed acyclic graph structure of the ﬂow network (Phillips et al., 2015). The new algorithm developed in Salève presents sig- niﬁcant advantages compared to ﬁnite-difference schemes, which are fundamentally limited by the time step 1t that must respect the Courant conditions 1t < k1x/max(C(l)), with k equal to ∼0.1 or to 100 for explicit or implicit schemes, respectively (e.g. Braun and Willett, 2013). There- fore, these ﬁnite-difference solutions are doomed to use shorter time steps and a larger number of iterations when considering ﬁner resolutions. On the contrary, this analyt- ical LEM converges towards steady state with roughly the same number of iterations, independently of the celerity C(l), which is set by K, A (i.e. L2) and m. The number of required iterations, however, increases with n0.5 pt , which is equivalent to an increase with decreasing 1x = L/n0.5 pt when L is con- stant, as in classical ﬁnite-difference schemes. Moreover, this steady-state modelling approach is compatible with spatially variable U, K and r. ( p ) The obtained solution looks very roughly like a clas- sical steady-state topography, and yet it is not strictly at steady state (Fig. 2). Indeed, during this ﬁrst iteration, the scheme used (Fig. 1a) imposes the constraint that rivers de- velop over the ﬂow network deﬁned by the initial topogra- phy and, in turn, does not ensure that the nodes located on the same crest of two juxtaposing catchments share the same response time or the same elevation. This leads to an exces- sive elevation as some rivers have planar length greater than predicted. This is the main limit of this 1D algorithm that cannot ensure the optimality of the 2D organization of the river network at steady state after only one iteration (Fig. 2). However, repeating this operation by computing the to- pography and then updating the ﬂow network (i.e. 2 From a 1D to a 2D analytical solution to the stream power law 4) can be extended to spatially variable uplift rate U(l) by simply using the response time of the re- (1) ∂t Earth Surf. Dynam., 9, 1239–1250, 2021 Earth Surf. Dynam., 9, 1239–1250, 2021 https://doi.org/10.5194/esurf-9-1239-2021 P. Steer: Short communication: Analytical models for 2D landscape evolution https://doi.org/10.5194/esurf-9-1239-2021 4 A 2D dynamical model with analytical accuracy I now explore the use of this analytical model in dynamic simulations with Salève (Fig. 1b). I ﬁrst consider the case of potentially heterogeneous but constant uplift rate U(l). A transient solution for river elevation z(l,t) at a speciﬁc time t can be computed using Eqs. (4) or (5) by simply thresholding the response time so that for every node τ(l,t) = min(τ(l),t). It results in the following: However, repeating this operation by computing the to- pography and then updating the ﬂow network (i.e. by com- puting the steepest slope, node order, and drainage area or discharge) after each iteration leads to a steady-state topog- raphy after a few tens of iterations Niter (Fig. 2). To assess the z(l,t) =zR(l,t) + U(l)(τ(l,t) −τR(l,t)) for l > 0, and z(0,t) = zbase. (6) (6) Earth Surf. Dynam., 9, 1239–1250, 2021 Earth Surf. Dynam., 9, 1239–1250, 2021 https://doi.org/10.5194/esurf-9-1239-2021 1242 P. Steer: Short communication: Analytical models for 2D landscape evolution Figure 2. Modelled steady-state topographies obtained after (a) 1 (left panel), 10 (middle panel) and 50 (right panel) iterations. (b) The steady-state topography is obtained after 127 iterations. (c) Convergence of the iterative algorithm inferred from the degree of crest dis- equilibrium 1zcrest, computed as the average of the absolute difference of elevation between crest nodes of juxtaposing catchments. Red dot indicates model shown in panel (b). Note that in panel (a), the colour map is bounded by the maximum elevation of the steady-state topography shown in panel (b). Figure 2. Modelled steady-state topographies obtained after (a) 1 (left panel), 10 (middle panel) and 50 (right panel) iterations. (b) The steady-state topography is obtained after 127 iterations. (c) Convergence of the iterative algorithm inferred from the degree of crest dis- equilibrium 1zcrest, computed as the average of the absolute difference of elevation between crest nodes of juxtaposing catchments. Red dot indicates model shown in panel (b). Note that in panel (a), the colour map is bounded by the maximum elevation of the steady-state topography shown in panel (b). This solution therefore enables computation of the time evo- lution of a landscape under potentially heterogeneous erodi- bility, uplift and runoff (or precipitation) rates. Thresholding the response time enforces that the uplift rate is considered null before the beginning of the simulation. The limitation of non-optimality of the planar organization of ﬂow network remains as in the steady-state solution. https://doi.org/10.5194/esurf-9-1239-2021 Earth Surf. Dynam., 9, 1239–1250, 2021 4 A 2D dynamical model with analytical accuracy However, this limita- tion can be solved by simply updating the river network, the node order, the steepest slope and water discharge after each time step, as in any other LEMs. As the time step is not con- strained by numerical stability issues, such as the Courant condition, it can be chosen only based on the rate of ﬂow network reorganization linked to river capture and piracy. Note, however, that in the dynamic Salève models, ﬂow net- work reorganization will lead to an immediate topographic reorganization, to respect Eq. (6). Indeed, time evolution of the elevation in Salève should not be seen as a continuous time evolution of a same topography, which would evolve by erosion under different time and space distributions of water discharge (e.g. as in other LEMs), but as a succession of to- pographic realizations which respect that the distribution of elevation is set by the ﬂow network. In other words, Salève does not fully guarantee the time continuity of the topogra- phy through successive time steps, despite practically having a limited impact on model behaviour as I will demonstrate later. I here run a simulation, using the same parameters as in the steady-state simulation, over a duration of 500 kyr (Fig. 4). The time steps 1t = 2 and 0.2 kyr correspond to about 45 and 4.5 times the Courant condition, respectively. Because implicit ﬁnite-difference solutions to the SPIM also remain numerically stable for time steps longer than the one imposed by the Courant condition, I also run simulations us- ing an implicit solution with the same parameters and time steps and compare them to the results of the Salève simula- tions. The implicit solution is computed following Eq. (22) in Braun and Willett (2013). I also compare Salève with results obtained with an implicit solution using a time step 1t = 0.002 kyr, corresponding to a Courant condition of 0.45. The ﬁnal topographies, i.e. at steady state, obtained with Salève or with the implicit solution share roughly the same statistical properties in terms of vertical and horizontal orga- Earth Surf. Dynam., 9, 1239–1250, 2021 https://doi.org/10.5194/esurf-9-1239-2021 P. Steer: Short communication: Analytical models for 2D landscape evolution P. Steer: Short communication: Analytical models for 2D landscape evolution 1243 Figure 3. Inﬂuence of model parameters and geometry on the con- vergence towards a steady-state landscape. (a) Uplift rate U was varied between 10−4 and 1 m yr−1. 4 A 2D dynamical model with analytical accuracy (b) Erodibility K was varied between 1 × 10−7 and 1 × 10−3 yr−1. (c) Model length L was var- ied between 0.1 and 100 km. (d) The number of model points npt was varied between 1.2 × 103 and 0.4 × 106. (e) The relationship between the number of iterations required to reach steady state and npt follows a power law with an exponent 0.5 (blue line). models continues to evolve after steady state, in particular the maximum elevation max(z), with larger variations for mod- els with longer time steps, which occurs due to catchment reorganization and numerical noise. g Moreover, erosion rates E ﬁrst increase more slowly and then more rapidly in Salève than with the implicit solu- tions before reaching steady state (Fig. 3d). In particular, the second phase is due to longer upstream distances and erosional response times in the topographies simulated with the implicit solution than with Salève (Fig. 3f). This is at least partly due to the dependency of the transient phase du- ration on 1t for ﬁnite-difference models (Braun and Wil- lett, 2013). Geometrically, longer transient phases are as- sociated with ﬂuvial networks with longer upstream dis- tances, i.e. distances to the outlet, in the implicit models with longer time steps compared to implicit models with shorter time steps or to Salève models (Fig. 3e). These re- sults also show that the response time of the landscapes is shorter than with other 2D LEMs as it is equal to the 1D re- sponse time, based on the ﬂow network length at steady state, when the time step used is sufﬁciently short to allow progres- sive reorganization of the ﬂuvial network (e.g. model with 1t = 0.2 kyr). Erosion rates in Salève, calculated by differencing eleva- tion between successive time steps and subtracting the con- tribution of uplift, are signiﬁcantly more variable, in partic- ular for the model with the shorter time step 1t = 0.2 kyr than with the implicit solutions. This variability highlights phases of ﬂuvial network reorganization which lead to im- mediate topographic reorganization, due to time discontinu- ity, and therefore to an immediate increase in erosion rates. Figure 3. Inﬂuence of model parameters and geometry on the con- vergence towards a steady-state landscape. (a) Uplift rate U was varied between 10−4 and 1 m yr−1. (b) Erodibility K was varied between 1 × 10−7 and 1 × 10−3 yr−1. 4 A 2D dynamical model with analytical accuracy (c) Model length L was var- ied between 0.1 and 100 km. (d) The number of model points npt was varied between 1.2 × 103 and 0.4 × 106. (e) The relationship between the number of iterations required to reach steady state and npt follows a power law with an exponent 0.5 (blue line). In terms of horizontal organization, all the Salève and im- plicit models lead to the same Hack’s law (Hack, 1957), which relates, through a power-law relationship, the down- stream maximum river length Lr with catchment area: Lr ∝ Ah. A least-square ﬁtting gives an exponent h of 0.65 ± 0.01 for Salève and the implicit models at steady state, with no dependency over 1t. In terms of vertical organiza- tion, the slope–discharge relationship obtained with Salève at steady state ﬁts perfectly with the predicted one, S = (U/K)1/nQ−m/n, while the implicit solution shows a signif- icant spread, in particular at low drainage area or discharge, which increases with 1t (Fig. 3d). Using 1t = 0.02 instead of 2 kyr leads to a slightly better consistency between the im- plicit and Salève solution, including the slope–discharge re- lationship and the temporal evolution of elevation. nization. The time evolution of the mean elevation (mean(z)) and maximum elevation (max(z)) is similar in all the mod- els, even if the steady-state value is higher by ∼50 for mean(z) and ∼500 m for max(z), with the implicit solution (Fig. 4c). Moreover, the ﬂuvial network and hence the to- pography modelled with Salève reach a stable conﬁguration once at steady state, with no subsequent vertical or horizon- tal changes. Topographic stability occurs when the model time t becomes greater or equal to the response time τ(l) of all the model nodes, and in particular the ones located on crests (Fig. 4b). This is particularly true if 1t is small enough to allow the horizontal organization of the ﬂuvial network to evolve concomitantly with its vertical component. On the contrary, the topography simulated by the ﬁnite-difference 5 Application: time-variable uplift and knickpoint propagation I now investigate the case of time-variable but homogeneous uplift rate U(t). Following Royden and Taylor Perron (2013), this case leads to additional complexity as the uplift rate, when the slope patches were initiated, must be tracked during https://doi.org/10.5194/esurf-9-1239-2021 Earth Surf. Dynam., 9, 1239–1250, 2021 1244 P. Steer: Short communication: Analytical models for 2D landscape evolutio Figure 4. Dynamic behaviour of the Salève model. (a) Time evolution of the modelled topography after 50 (left panel), 150 (middle pane and 500 kyr (right panel). Time evolution of the (b) max elevation, (c) mean elevation and (d) mean erosion rate for Salève, using a time ste of 1t = 2 kyr (black line) and 0.2 kyr (dashed black line) and for the implicit solution with 1t = 2 kyr (blue line), 0.2 kyr (green line) an 0.02 kyr (red line). The uplift rate U is shown in panel (d) with a cyan line. (e) Slope–discharge distributions at steady state (at 500 kyr) f the three models compared to the predicted relationship (cyan line). No binning is done, and all the model nodes are represented in panel (d (f) Histograms of upstream distances d (left panel) and response times τ (right panel) for the different models at steady state. upstream migration. In a LEM, this is performed by com- i h iﬁ i h I f h lif computed in the upstream direction, starting with the riv l P. Steer: Short communication: Analytical models for 2D landscape evolution 1244 Figure 4. Dynamic behaviour of the Salève model. (a) Time evolution of the modelled topography after 50 (left panel), 150 (middle panel) and 500 kyr (right panel). Time evolution of the (b) max elevation, (c) mean elevation and (d) mean erosion rate for Salève, using a time step of 1t = 2 kyr (black line) and 0.2 kyr (dashed black line) and for the implicit solution with 1t = 2 kyr (blue line), 0.2 kyr (green line) and 0.02 kyr (red line). The uplift rate U is shown in panel (d) with a cyan line. (e) Slope–discharge distributions at steady state (at 500 kyr) for the three models compared to the predicted relationship (cyan line). No binning is done, and all the model nodes are represented in panel (d). (f) Histograms of upstream distances d (left panel) and response times τ (right panel) for the different models at steady state. 6 Solving for river and hillslope dynamics p Discrete temporal changes in uplift rates or in base-level elevation can lead to sharp ruptures in the slope of river pro- ﬁles, generally referred to as knickpoints (e.g. Rosenbloom and Anderson, 1994; Whipple and Tucker, 1999; Steer et al., 2019). Finite-difference solutions to the stream power equa- tion inherently lead to a progressive numerical diffusion of knickpoints during their migration, even with n = 1, while the algorithm developed here preserves the shape of knick- points. To illustrate this advantage, I run a simulation with the same parameters as in the steady-state case, except that U is raised from 10 to 20 mm yr−1 at 250 kyr for a total model duration of 500 kyr (Fig. 5). Compared to previous models, the model is here restricted to an extent of 10 km over 2 km, with only one boundary (left) that is considered as possible outlets for water. This setting limits ﬂuvial network reorgani- zation (or time discontinuity) and in turn allows for tracking geomorphological features during the evolution of the land- scape. I use a time step of 25 kyr, which is about 500 times greater than the time step imposed by the Courant condition, clearly above the range of time steps compatible with nu- merical solutions. Despite this, the knickpoints formed at the outlets of the model at 250 kyr, at the onset of the increase in uplift rate, are accurately modelled, i.e. with analytical ac- curacy, throughout their propagation (Fig. 5c). The shape of In previous sections, I have considered the steady-state and dynamic solutions of landscapes subjected only to river ero- sion following the SPIM. However, these analytical solutions can be extended to simulate the dynamics and morphology of colluvial valleys and hillslopes. Indeed, a power-law scaling for the slope–area relationship is observed in colluvial val- leys, which suggest they could obey a similar erosion law as Eq. (1), but with different m and n exponents (Lague and Davy, 2003). A solution with m = 0.24 and n = 1, but considering a non-negligible incision threshold, was found to best explain the geometry of colluvial valleys in the Si- walik Hills of Nepal for drainage area between 7 × 10−3 and 1 km2, representing the thresholds in drainage area be- tween colluvial valleys and hillslopes or rivers, respectively (Lague and Davy, 2003). 5 Application: time-variable uplift and knickpoint propagation computed in the upstream direction, starting with the river outlets, as upstream migration. In a LEM, this is performed by com- puting, at the speciﬁc time t, what I refer to as the uplift memory map Umem(l,t) = U(t −τ(l,t)). It is not equivalent to a classical uplift map and corresponds to the uplift rate when the slope patches were formed. I remind the reader here that the response time is bounded by actual model time τ(l,t) = min(τ(l),t). The elevation at a time t is then simply z(l,t) =zR(l,t) + Umem(l,t)(τ(l,t) −τR(l,t)) for l > 0, and z(0,t) = zbase. (7) (7) As in the heterogeneous uplift case, this solution is easily implemented in a LEM by updating the river network and its As in the heterogeneous uplift case, this solution is easily implemented in a LEM by updating the river network and its Earth Surf. Dynam., 9, 1239–1250, 2021 https://doi.org/10.5194/esurf-9-1239-2021 P. Steer: Short communication: Analytical models for 2D landscape evolution 1245 Figure 5. Dynamic evolution of the topography and knickpoint migration over 500 kyr. The initial uplift rate U = 10 mm yr−1 is doubled after 250 kyr. (a) Topography before the increase in uplift at 50 (top panel), 125 (middle panel) and 225 kyr (bottom panel). (b) Topography after the increase in uplift at 300 (top panel), 400 (middle panel) and 500 kyr (bottom panel). (c) Temporal evolution of the longest river proﬁle shown at every time step (except the ﬁrst one), with the “winter” and “autumn” colour map showing river proﬁles before and after the increase in uplift. (d) Temporal evolution of the uplift (blue squares) and erosion (red circles) rates. Figure 5. Dynamic evolution of the topography and knickpoint migration over 500 kyr. The initial uplift rate U = 10 mm yr−1 is doubled after 250 kyr. (a) Topography before the increase in uplift at 50 (top panel), 125 (middle panel) and 225 kyr (bottom panel). (b) Topography after the increase in uplift at 300 (top panel), 400 (middle panel) and 500 kyr (bottom panel). (c) Temporal evolution of the longest river proﬁle shown at every time step (except the ﬁrst one), with the “winter” and “autumn” colour map showing river proﬁles before and after the increase in uplift. (d) Temporal evolution of the uplift (blue squares) and erosion (red circles) rates. the knickpoint is also kept throughout its migration. 5 Application: time-variable uplift and knickpoint propagation I also highlight here that, due to the model setting with only one outlet boundary, which limits river reorganization (and time discontinuity), erosion rates are smoother than in Fig. 4. properties after each time step. I also emphasize here that the previous model example (Fig. 4) is already a speciﬁc case of a time-variable uplift rate, with a change in uplift rate which occurs at the beginning of the simulation, leading in turn to a simpler formalism (Eq. 6). In the following, I focus on demonstrating the ability of the model to simulate and track knickpoints. 7 Discussion and conclusion hillslope erosion (e.g. Tucker and Bras, 1998; Densmore et al., 1998; Roering et al., 1999; Lague and Davy, 2013; Jean- det et al., 2019) but that this framework can approximate the observed geometrical relationships between slope and area. Based on previous analytical developments (e.g. Royden and Taylor Perron, 2013), I have designed a new method to solve for the steady-state topography or the dynamic evolution of a landscape in 2D, following the SPIM, with analytical preci- sion. The model can solve in a single time step, using an iter- ative scheme, the steady-state topography of a landscape un- der homogeneous or heterogenous conditions (i.e. uplift rate, erodibility and runoff). Iterations are required to optimize the planar organization of the river network and crest positions, starting from a random network. The number of iterations required for the convergence of the scheme only depends on the number of nodes discretizing the surface topography and only scales with n0.5 pt , independently of other model parame- ters. Moreover, the model can also solve for the dynamic evo- lution of a landscape under either heterogeneous but constant or time-variable but homogeneous conditions. The dynamic and steady-state Salève models can solve for river, colluvial and hillslope erosion, if the associated erosion laws lead to slope–area (or discharge) relationships that can be modelled using a linear SPIM. The two main beneﬁts of this new model are (1) its analytical accuracy that enables suppression of nu- merical diffusion and, for instance, the maintenance of the shape of knickpoints and (2) the absence of an upper bound for the time step that is not limited by the Courant condi- tion. Contrary to any other state-of-the-art LEMs using the SPIM (e.g. Braun and Willett, 2013; Carretier et al., 2016; Campforts et al., 2017; Hobley et al., 2017; Salles, 2018), the time-stepping strategy in Salève can be chosen only based on physical considerations, such as the rate of river network reorganization, and not on numerical ones. All these advan- tages make Salève unique in its ability to efﬁciently model landscape evolution. In addition to its use in landscape evolu- tion modelling, Salève could offer new opportunities to gen- erate terrains for applications in computer graphics (e.g. Cor- donnier et al., 2016); to infer the time and space evolution of uplift by inverting landscapes in 2D (e.g. 6 Solving for river and hillslope dynamics Below the area transition between colluvial valleys and hillslopes, the power-law scaling for the slope–area relationship becomes ﬂat, due to landsliding and mass wasting processes, or reverts where hilltops are convex (Ijjasz-Vasquez and Bras, 1995; Tarolli and Dalla Fontana, 2009). Once again, this hillslope domain could be geometri- cally modelled using the SPIM with different m and n, e.g. with m = 0 and n = 1, to model hillslopes following a critical angle of repose Sc. I do not argue here that these laws nec- essarily encapsulate the processes controlling colluvial and https://doi.org/10.5194/esurf-9-1239-2021 Earth Surf. Dynam., 9, 1239–1250, 2021 P. Steer: Short communication: Analytical models for 2D landscape evolution P. Steer: Short communication: Analytical models for 2D landscape evolution P. Steer: Short communication: Analytical models for 2D landscape evolution 1246 https://doi.org/10.5194/esurf-9-1239-2021 Earth Surf. Dynam., 9, 1239–1250, 2021 7 Discussion and conclusion Other analytical solutions can be considered to account for hillslope processes such as the one developed in the DAC model (Goren et al 2014b) τ(l) = l Z 0 1 K1(l)A(l)m1 dl′ for l < l1, τ(l) = l1 Z 0 1 K1(l)A(l)m1 dl′ + l Z l1 1 K2(l)A(l)m2 dl′ for l1 < l ≤l2, τ(l) = l1 Z 0 1 K1(l)A(l)m1 dl′ + l2 Z l1 1 K2(l)A(l)m2 dl′ + l Z l2 1 K3(l)A(l)m3 dl′ for l > l2, (8) τ(l) = l Z 0 1 K1(l)A(l)m1 dl′ for l < l1, τ(l) = l1 Z 0 1 K1(l)A(l)m1 dl′ + l Z l1 1 K2(l)A(l)m2 dl′ for l1 < l ≤l2, τ(l) = l1 Z 0 1 K1(l)A(l)m1 dl′ + l2 Z l1 1 K2(l)A(l)m2 dl′ + l Z l2 1 K3(l)A(l)m3 dl′ for l > l2, (8) (8) where A(l1) and A(l2) are model parameters that deﬁne the threshold areas for river to colluvial valley and for colluvial valley to hillslope transitions, and (K1, m1), (K2, m2), and (K3, m3) are the K values and m exponents for rivers, col- luvial valleys and hillslopes, respectively. I emphasize here that the colluvial law used here is only inspired from the col- luvial law described in Lague and Davy (2003), as it neglects the incision threshold which lead to a non-linear behaviour. Figure 6 shows the steady-state topographies obtained when considering river, colluvial and hillslope erosion. Consider- ing these additional erosion laws leads, as expected, to dif- ferent scaling in the slope–discharge relationships, separated by thresholds in discharge or drainage area. These thresholds should be chosen to ensure (1) the continuity of the slope– discharge relationship and (2) the slope is equal to Sc when A ≤A(l2). I emphasize, once again, that the models devel- oped here lead to slope–discharge relationships with exact accuracy, at steady state, due to the use of analytical solu- tions. Other analytical solutions can be considered to account for hillslope processes such as the one developed in the DAC model (Goren et al., 2014b). where A(l1) and A(l2) are model parameters that deﬁne the threshold areas for river to colluvial valley and for colluvial valley to hillslope transitions, and (K1, m1), (K2, m2), and (K3, m3) are the K values and m exponents for rivers, col- luvial valleys and hillslopes, respectively. 7 Discussion and conclusion Pritchard et al., 2009; Roberts and White, 2010; Goren et al., 2014a; Fox et al., 2014; Croissant and Braun, 2014) including river, collu- vial valleys, and hillslopes; to predict thermochronological ages from landscape evolution (e.g. Braun et al., 2014); or to validate the accuracy of numerical schemes used in other LEMs. The model is fast as it makes use of the optimized ﬂow routing algorithm provided by TopoToolbox (Schwang- hart and Scherler, 2014). The developed scheme that uses 1D analytical solutions Practically, considering three different erosion laws, for river, colluvial valleys and hillslopes, simply requires chang- ing the value of K, m and n in the deﬁnition of celerity in the response time equation (Eq. 3) for each of the different do- mains, separated by thresholds in discharge or drainage area. Keeping n = 1 for simplicity leads to the following set of re- sponse time equations: τ(l) = l Z 0 1 K1(l)A(l)m1 dl′ for l < l1, τ(l) = l1 Z 0 1 K1(l)A(l)m1 dl′ + l Z l1 1 K2(l)A(l)m2 dl′ for l1 < l ≤l2, τ(l) = l1 Z 0 1 K1(l)A(l)m1 dl′ + l2 Z l1 1 K2(l)A(l)m2 dl′ + l Z l2 1 K3(l)A(l)m3 dl′ for l > l2, (8) where A(l1) and A(l2) are model parameters that deﬁne the threshold areas for river to colluvial valley and for colluvial valley to hillslope transitions, and (K1, m1), (K2, m2), and (K3, m3) are the K values and m exponents for rivers, col- luvial valleys and hillslopes, respectively. I emphasize here that the colluvial law used here is only inspired from the col- luvial law described in Lague and Davy (2003), as it neglects the incision threshold which lead to a non-linear behaviour. Figure 6 shows the steady-state topographies obtained when considering river, colluvial and hillslope erosion. Consider- ing these additional erosion laws leads, as expected, to dif- ferent scaling in the slope–discharge relationships, separated by thresholds in discharge or drainage area. These thresholds should be chosen to ensure (1) the continuity of the slope– discharge relationship and (2) the slope is equal to Sc when A ≤A(l2). I emphasize, once again, that the models devel- oped here lead to slope–discharge relationships with exact accuracy, at steady state, due to the use of analytical solu- tions. 7 Discussion and conclusion I emphasize here that the colluvial law used here is only inspired from the col- luvial law described in Lague and Davy (2003), as it neglects the incision threshold which lead to a non-linear behaviour. Figure 6 shows the steady-state topographies obtained when considering river, colluvial and hillslope erosion. Consider- ing these additional erosion laws leads, as expected, to dif- ferent scaling in the slope–discharge relationships, separated by thresholds in discharge or drainage area. These thresholds should be chosen to ensure (1) the continuity of the slope– discharge relationship and (2) the slope is equal to Sc when A ≤A(l2). I emphasize, once again, that the models devel- oped here lead to slope–discharge relationships with exact accuracy, at steady state, due to the use of analytical solu- tions. Other analytical solutions can be considered to account for hillslope processes such as the one developed in the DAC model (Goren et al., 2014b). The developed scheme, that uses 1D analytical solutions, is limited to ﬂow networks that can be topologically classi- ﬁed as 1D node stacks or graphs (Braun and Willett, 2013), as resulting from a steepest slope ﬂow routing algorithm. This excludes, for instance, recent models accounting for ﬂow algorithms based on physical considerations (Davy et al., 2017). The main limitation of this new approach is that re- organizations of the river network, such as catchment piracy, will not lead to transient phases of erosion, as the river el- Earth Surf. Dynam., 9, 1239–1250, 2021 https://doi.org/10.5194/esurf-9-1239-2021 1247 P. Steer: Short communication: Analytical models for 2D landscape evolution Figure 6. Steady-state topographies obtained with Salève considering only (a) stream power incision (m = 0.5) in rivers (like in Fig. 1), (b) stream power incision (m = 0.5) in rivers and colluvial erosion (m = 0.24), and (c) stream power incision in rivers (m = 0.5), colluvial erosion (m = 0.24), and hillslopes following a critical slope (m = 0) of Sc = 30◦. To better highlight relief, elevation is represented by transparency over the raster of hillshade. (d) Slope–discharge distributions for these three models. Figure 6. Steady-state topographies obtained with Salève considering only (a) stream power incision (m = 0.5) in rivers (like in Fig. https://doi.org/10.5194/esurf-9-1239-2021 References Barnes, R., Lehman, C., and Mulla, D.: Priority-ﬂood: An optimal depression-ﬁlling and watershed-labeling algorithm for digital elevation models, Comput. Geosci., 62, 117–127, 2014. Braun, J. and Sambridge, M.: Modelling landscape evolution on ge- ological time scales: a new method based on irregular spatial dis- cretization, Basin Res., 9, 27–52, 1997. Braun, J. and Willett, S. D.: A very efﬁcient O(n), implicit and parallel method to solve the stream power equation governing ﬂuvial incision and landscape evolution, Geomorphology, 180, 170–179, 2013. Braun, J., Simon-Labric, T., Murray, K. E., and Reiners, P. W.: To- pographic relief driven by variations in surface rock density, Nat. Geosci., 7, 534–540, 2014. Campforts, B. and Govers, G.: Keeping the edge: A numerical method that avoids knickpoint smearing when solving the stream power law, J. Geophys. Res.-Earth, 120, 1189–1205, 2015. Campforts, B., Schwanghart, W., and Govers, G.: Accurate simu- lation of transient landscape evolution by eliminating numerical diffusion: the TTLEM 1.0 model, Earth Surf. Dynam., 5, 47–66, https://doi.org/10.5194/esurf-5-47-2017, 2017. Carretier, S. and Lucazeau, F.: How does alluvial sedimentation at range fronts modify the erosional dynamics of mountain catch- ments?, Basin Res., 17, 361–381, 2005. Code availability. A MATLAB version of the model can be accessed through a Zenodo repository: https://doi.org/10.5281/zenodo.4686733 (Steer, 2021). It is delivered with a routine to solve for the stream power law using an implicit ﬁnite-difference solution. Code availability. A MATLAB version of the model can be accessed through a Zenodo repository: https://doi.org/10.5281/zenodo.4686733 (Steer, 2021). It is delivered with a routine to solve for the stream power law using an implicit ﬁnite-difference solution. Carretier, S., Martinod, P., Reich, M., and Goddéris, Y.: Modelling sediment clasts transport during landscape evolution, Earth Surf. Dynam., 4, 237–251, 2016. Cordonnier, G., Braun, J., Cani, M. P., Benes, B., Galin, E., Pey- tavie, A., and Guérin, E.: Large scale terrain generation from tectonic uplift and ﬂuvial erosion, Comput. Graph. Forum, 35, 165–175, 2016. Competing interests. The author declares that there is no con- ﬂict of interest. Croissant, T. and Braun, J.: Constraining the stream power law: a novel approach combining a landscape evolution model and an inversion method, Earth Surf. Dynam., 2, 155–166, https://doi.org/10.5194/esurf-2-155-2014, 2014. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Croissant, T., Lague, D., Steer, P., and Davy, P.: Rapid post- seismic landslide evacuation boosted by dynamic river width, Nat. Geosci., 10, 680–684, 2017. 7 Discussion and conclusion lapping or stretching river proﬁles for n > 1 or n < 1, re- spectively (Royden and Taylor Perron, 2013). Using Salève to simulate the impact of both a heterogeneous and time- variable uplift rate has not been attempted and might also result in convergence issues. Moreover, using an even more efﬁcient algorithm to route water also represents a promising avenue (e.g. Barnes et al., 2014). This is critical for Salève that can use a time step much greater than the Courant con- dition and for which the main computational limit is the ﬂow routing algorithm. Therefore, no computational time bench- mark was done for this new model, as the computation of elevation changes, even on large grids, is negligible com- pared to ﬂow routing. In turn, solving for individual time steps in this model takes a similar amount of computational time as in other similar LEMs using the same ﬂow routing algorithm (e.g. Braun and Willett, 2013; Schwanghart and Scherler, 2014). Yet, the advantage of this new model is its ability to use longer time steps while preserving analytical accuracy and consistency. Lastly, Salève represents the ﬁrst attempt to use analytical solutions to model the dynamics of landscapes in 2D using the SPIM. Because little modiﬁca- tions are required to implement this solution in other LEMs, I believe the strategy developed in this paper could be adapted and further developed to make LEMs more efﬁcient and ac- curate. Review statement. This paper was edited by Greg Hancock and reviewed by Sebastien Carretier and Liran Goren. 7 Discussion and conclusion 1), (b) stream power incision (m = 0.5) in rivers and colluvial erosion (m = 0.24), and (c) stream power incision in rivers (m = 0.5), colluvial erosion (m = 0.24), and hillslopes following a critical slope (m = 0) of Sc = 30◦. To better highlight relief, elevation is represented by transparency over the raster of hillshade. (d) Slope–discharge distributions for these three models. evation is directly updated to its optimal elevation for each node where t ≤τ(l,t). The response time of the modelled landscapes is therefore shorter than with other 2D LEMs as it is equal to the 1D response time based on the ﬂow network length at steady state. Moreover, the ﬂow network topology is updated at every iteration or time step, in the steady-state or dynamic modes, respectively, while other strategies, based on physical criterion, could be adopted (Goren et al., 2014b). If many dynamic LEMs use the same approach (e.g. Braun and Willett, 2013), this is a critical aspect of the convergence speed and computational time in the steady-state mode, and future work should focus on accelerating it. tive to the location of the base-level condition. Moreover, Salève is a purely detachment-limited model which does not consider the role of sediment transport and deposition in landscape dynamics. Only the linear SPIM with n = 1 has been considered in this study, while some observations sup- port non-linear models with greater values for n (e.g. Lague, 2014). These limitations also emphasize that analytical so- lutions to landscape dynamics, such as Salève, represent a complementary approach to other “numerical” LEMs, which are in essence more versatile and allow for tackling coupled or complex scientiﬁc problems which characterize geomor- phological systems. The Salève model is also not designed for horizontal tec- tonic displacement (e.g. Braun and Sambridge, 1997; Steer et al., 2011; Miller et al., 2007) that displaces nodes rela- Extending the Salève algorithm to non-linear SPIMs rep- resents a challenging and non-trivial perspective that requires accounting for more complex analytical solutions with over- https://doi.org/10.5194/esurf-9-1239-2021 Earth Surf. Dynam., 9, 1239–1250, 2021 P. Steer: Short communication: Analytical models for 2D landscape evolution P. Steer: Short communication: Analytical models for 2D landscape evolution P. Steer: Short communication: Analytical models for 2D landscape evolution 1248 Review statement. 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https://openalex.org/W2564807617	https://oatext.com/pdf/CRT-2-158.pdf	English	null	Randomized observational multicenter study to assess the efficacy and safety of the association of Fortigel (10 Gr) and Fucoidan (100 Mg) in patients with Gonarthrosis	Clinical research and trials	2,016	cc-by	4,608	Abstract Purpose: In order to further elucidate the efficacy and safety of some nutritional supplements on gonarthrosis, we have conducted a preliminary randomized multicenter (n=9) observational study comparing the effects of an association of Fortigel® (10 gr) and Fucoidan (100 mg) (ACTEN®) versus another commonly therapeutically used formulation based on Glucosamine (500 mg), Chondroitin Sulfate (400mg) hyaluronic Acid (50 mg) and Vitamin C (100 mg) (COMBIART). Patients and methods: The protocol was administered over a 12-weeks period in a population (n=126) aged 40-65 years, with diagnosed mild-to-moderate osteoarthritis (OA) of the knee (grade 2-3 of Kellgren Lawrence grading scale). Safety was measured by closely monitoring adverse events. Efficacy was measured by grading evaluations, at basal, 1 month and 3 months controls, of the Visual Analog Scale (VAS) and the Lequesne algofunctional index for severity of osteoarthritis (LAI) for articular functionality. Results: Both groups showed an important reduction (P < 0.0001) in the mean visual analog scale values at T1 (28.5% ACTEN®, 21.3% COMBIART at 1 month) and T3 (49.4% ACTEN®, 40.1% COMBIART at 3 months), as well as a marked reduction in the Lequesne algofunctional index means (P < 0.0001) (ACTEN® 28.9% T1 44.9% T3, COMBIART T1 21.3% T3 37%). The effect seems to be time dependent, as the mean values decrease further for both parameters from T1 to T2 (P < 0.0001, for VAS for both groups; P 0.0011 for ACTEN® group, P 0.0064 Control group for LAI). No statistically significant difference was found between the ACTEN® group and the COMBIART group at time T1 or T3. These interesting preliminary data will be further investigated on a larger scale. Conclusions: Fortigel® (10gr) and Fucoidan (100 mg) (ACTEN®) taken as oral nutritional supplements have a significant impact as therapeutic intervention for knee osteoarthritis as indicated by the marked decrease in VAS and LAI values over the course of the treatment. A similar effect, as expected, has been confirmed in the COMBIART group, and no statistically significant difference has been detected between the two groups. Acetaminophen and nonsteroidal anti-inflammatory drugs (NSAIDs) are common treatments for patients with osteoarthritis to cope with pain and stiffness, in severe conditions on a daily basis. For this reason, in recent years many studies have focused on alternative molecules and nutritional supplements as therapeutic options to achieve cartilage tissue repair, while bypassing the negative effects of cyclooxygenase 2 (COX-2) inhibitors and other NSAIDs. Research Article Research Article ISSN: 2059-0377 Randomized observational multicenter study to assess the efficacy and safety of the association of Fortigel (10 Gr) and Fucoidan (100 Mg) in patients with Gonarthrosis Martin-Martin LS1*, Pierluigi B2, La Medica C3, Melis G4, Nuvoli G5, Piccinni V6, 1Department of Internal Medicine, Regina Apostolorum Hospital, Albano Laziale, Rome-Italy 2Department of Orthopedics, San Giovanni Calibita-Fatebenefratelli Hospital, Rome-Italy 3Department of Orthopedics, Madre Giuseppina Vannini Hospital, Rome-Italy 4Department of Orthopedics, Marino Hospital, Alghero, Sassari-Italy 5Department of Rheumatology, ASL 1, Sassari, Italy 6Department of Orthopedics, Rome-Italy 7Department of Orthopedics, ASL Rome G, Rome-Italy 8Department of Orthopedics, ASL Rome H, Rome-Italy 9Department of Rheumatology, IRCSS Fondazione Santa Lucia, Rome-Italy 9Department of Rheumatology, IRCSS Fondazione Santa Lucia, Rome-Italy 9Department of Rheumatology, IRCSS Fondazione Santa Lucia, Rome-Italy Abstract Glucosamine has been extensively studied [2-18] showing positive results in the Received: November 02, 2016; Accepted: November 11, 2016; Published: November 14, 2016 Clinical Research and Trials Clinical Research and Trials Intervention clinical management of pain and structural improvements [19]. Chondroitin sulfate coupled to Glucosamine, or by itself, has been the center of significant studies [20], but many other compounds synthetic or natural have been investigated, including Vitamin C [21- 25]. Particular interest has been paid to Collagen hydrolysates (CHs) and Fucoidans [26-44]. CH is obtained by the enzymatic hydrolysis of collagenous tissues from mammals. The main characteristic of CH is its amino acid composition, which is identical to type II collagen, thus providing high levels of glycine and proline, essential amino acids for the stability and regeneration of cartilage [28]. This product is recognized as a safe food ingredient by regulatory agencies. CH is well digested and is preferentially accumulated in cartilage [29]. Clinical use of CH has not been associated with adverse effects, aside some gastro- intestinal side effects, such as fullness or unpleasant taste. Participants underwent a 4-week washout period during which they stopped taking all dietary supplements for bones, joints, and inflammation as well as non-prescription drugs. Prescription drugs that were not related to joint health were allowed, as was OTC rescue medication that was taken only on an as- needed basis. Participating individuals were also asked not to change any aspects of their lives during the 4-week trial. Lifestyle variables were unaltered, including but not limited to diet, fitness regimens, work and family-related tasks. Participants who passed the initial screening at the baseline appointment were provided with one of the two randomly assigned nutritional supplements (ACTEN® or COMBIART), paper copies of a visual analog scale (VAS) questionnaire and the Lequesne algo- functional index (LAI). Each participant was asked to fill out the baseline VAS and LAI questionnaire and return it to the investigator before beginning to take the supplements. The supplements were consumed daily for both groups for the first month. The remaining eight weeks ACTEN® group used the supplement every other day, while COMBIART continued on a daily basis. Fucoidans, on the other side, are a class of sulfated, fucose- rich polymers found in several types of brown macroalgae [41,42] used in a variety of different medical conditions [38]. Several recent studies indicate a role of fucoidan in addressing the symptoms of osteoarthritis. Animal models of collagen induced arthritis showed that orally administered fucoidans successfully inhibited pain [39]. Introduction Osteoarthritis (OA) is a very common, slowly progressive joint condition, that causes significant discomfort and disability in the adult population. The prevalence of the condition increases with age, with well-known risk factors such as genetics, obesity, local trauma, and occupation. Important radiographic changes of knee OA are found in 1% of adults of 25–35 years of age and increase almost to 50% in elders aged 75 years and older. OA of the hips and knees are the most common complaint in the adult population. Osteoarthritis has an inflammatory component, and a tissue breakdown component, which result in pain and stiffness [1] in these crucial weight-bearing joints. This often imposes an important disability, leading to an increased difficulty in undertaking normal activities of daily life, up to requiring joint replacement surgery in the most severe of cases. Correspondence to: Martin-Martin LS, Department of Internal Medicine, Regina Apostolorum Hospital, Albano Laziale, Rome-Italy, Tel: 06 93298240; E-mail: severino.martin@gmail.com Received: November 02, 2016; Accepted: November 11, 2016; Published: November 14, 2016 Clin Res Trials, 2016 doi: 10.15761/CRT.1000158 Volume 2(6): 253-257 Martin-Martin LS (2016) Randomized observational multicenter study to assess the efficacy and safety of the association of Fortigel (10 Gr) and Fucoidan (100 Mg) in patients with Gonarthrosis Martin-Martin LS (2016) Randomized observational multicenter study to assess the efficacy and safety of the association of Fortigel (10 Gr) and Fucoidan (100 Mg) in patients with Gonarthrosis Intervention In a small human clinical study osteoarthritis symptoms were inhibited by 12 weeks oral administration of fucoidan rich seaweed extracts by 52% [40]. There was no reduction in TNF alpha as inflammation marker, but an accompanying study in healthy volunteers showed a decrease in Interleukin 6, a marker for chronic inflammation [43]. Fucoidan’s effect on pain has been linked to its selective blockade on neutrophils accumulation [44,45]. Data were collected again at 1 month and 3 months (12 weeks). This approach was chosen to reflect the higher absorption of gel (ACTEN®) compared to capsules (COMBIART) [46]. Outcome measures A VAS scale of 0 to 10 for pain, and the LAI scale of 0 to 24 for pain and functionality were employed to evaluate the 2 measures before and after 4 and 12 weeks of supplementation. VAS scores were reported by asking the participants to mark responses on a 10-cm line. The line contained both end anchor points and 3 additional descriptors that were evenly spaced along the VAS scale at 2.5-cm intervals to help orient the participant. Definitions for the descriptors for each scale are listed in Table 2. In order to test the clinical efficacy of the combined use of these compounds, CH (Fortigel® 10gr.) and Fucoidan (100mg) (ACTEN®), we have designed a preliminary multicenter randomized clinical trial. Testing its clinical efficacy against another formulation used in clinical practice, based on well-known substances: Glucosamine (500mg), Chondroitin Sulfate (400mg) hyaluronic Acid (50 mg) and Vitamin C (100mg) (COMBIART). Material and methods At baseline evaluation, participants were told to notify the research team immediately if any mild, moderate, or severe adverse events occurred during the period of supplementation. During the 1 month follow-up, and again at 3 months participants were once again asked whether they had experienced any adverse events or required any form of rescue treatment (pain or anti-inflammatory drugs) during the supplementation period. No complaints were reported. This trial was designed as a randomized preliminary study. It was conducted over 12 weeks (84 days) by a single research group in nine centers (Lazio and Sardinia regions, in Italy) between Oct 2015 and May 2016 and involved 126 participants aged 40-65 years, with mild- to-moderate osteoarthritis (OA) of the knee (grade 2-3 of Kellgren Lawrence grading scale). Individuals were excluded from the study if they 1) were aged 39 years or younger at the start of the study; 2) had rheumatoid or other forms of arthritis; 3) had joint pain as a result of nerve or muscle damage, accidents, falls, trauma, etc. (4) had comorbidities often associated with osteoarthritis, such as diabetes, cardiovascular disease, elevated cholesterol requiring medical intervention, renal insufficiencies, asthma, or hypertension requiring medical intervention; 5) had cancer within the prior 5 years; 6) were taking any other herbal product or other supplement for pain or inflammation of joints health, such as glucosamine/chondroitin/methylsulfonylmethane, S-adenosylmethionine, or omega-3; 7) were smokers; 8) were heavy alcohol consumers; 9) were pregnant or nursing women; 10) had shellfish allergies; and 11) had a BMI lower than 18.5 (underweight) or greater than 40.0 (morbidly obese) or a body weight exceeding 225 lbs (102 kg). The demographics of the patient population are listed in Table 1. Population Age Mean 57.10 SD 8.04 Men 48 Women 78 VAS cm Mean 6.595 SD 1.318 LAI (Lequesne index) Mean 10.595 SD 3.519 Table 1. The demographics of the patient population. Population Age Mean 57.10 SD 8.04 Men 48 Women 78 VAS cm Mean 6.595 SD 1.318 LAI (Lequesne index) Mean 10.595 SD 3.519 Table 1. The demographics of the patient population. VAS Value (cm) Pain Level 0 None 2.5 Mild 5 Moderate 7.5 Severe 10 Worst possible Table 2. Internal descriptor for the VAS Scale, descriptors for LAI are as previously published [45]. Table 1. The demographics of the patient population. Table 2. Internal descriptor for the VAS Scale, descriptors for LAI are as previously published [45]. Results Of the 126 participants enrolled in the study, none withdrew or were removed due to medical complications or lack of compliance with the study’s protocol. In comparison with baseline, the results for all participants at the end of the 4/12 weeks for all measures were statistically significant. The ACTEN® group showed a 28.5% decrease in joint pain at 1 month (T1), from a mean VAS score of 6.68 (1.38SD) at baseline to 4.77 (1.98SD) (P = 0.0001; 95% CI, 1.57, 2.25) and a -49.4% at T3, mean VAS 3.38 (1.78SD) (P = 0.0001; 95% CI, 2.95, 3.65). LAI was also reduced by -28.9% at T1, from a mean baseline of 11.28 (3.69SD) to 8.01 (P = 0.0001; 95% CI, 2.72, 3.82) and by -44.9% at T3 mean LAI 6.21 (3.01SD) (P = 0.0001; 95% CI, 4.42, 5.71). Baseline T1 VAS Leq Ind Figure 3. ACTEN Group. T1 T3 VAS Leq Ind Figure 3. ACTEN Group. Leq Ind Figure 3. ACTEN Group. The COMBIART group showed a 21.3% decrease in joint pain, from a mean VAS score of 6.5 (1.25SD) at baseline to 5.11 (1.53SD) at T1 (P = 0.0001; 95% CI, 1.09, 1.69) and a – 40.1% at T3 mean VAS 3.89 (1.81SD) (P = 0.0001; 95% CI, 2.97, 4.33). LAI is again reduced by -21.3% at T1 from a mean baseline of 9.84 (3.18 SD) to 7.74 (3.08 SD) (P = 0.0001; 95% CI, 1.63, 2.57) and by -37% at T3, mean LAI 6.19 (3.04 SD) (P = 0.0001; 95% CI, 2.97, 4.33) The ACTEN® group seems to work more efficently than the COMBIART group (VAS T3 means -49.4%/-40.1%; LAI T3 means -44.9%/-37%), but no statistically relevant significance has been detected comparing the two groups [Figure 1-4]. T3 Summary of adverse effects No adverse effects were reported during the study. Material and methods VAS Value (cm) Pain Level 0 None 2.5 Mild 5 Moderate 7.5 Severe 10 Worst possible Clin Res Trials, 2016 doi: 10.15761/CRT.1000158 Volume 2(6): 253-257 Martin-Martin LS (2016) Randomized observational multicenter study to assess the efficacy and safety of the association of Fortigel (10 Gr) and Fucoidan (100 Mg) in patients with Gonarthrosis Martin-Martin LS (2016) Randomized observational multicenter study to assess the efficacy and safety of the association of Fortigel (10 Gr) and Fucoidan (100 Mg) in patients with Gonarthrosis Discussion Osteoarthritis is the most common form of arthritis in Italy and in the USA [47]. Glucosamine and chondroitin sulfate and hyaluronic acid are natural substances present in and around cartilage cells and, as such are the most commonly used natural supplements for OA and 0 2 4 6 8 10 12 Baseline T1 T3 Acten Control group Figure 2. Lequesme Index Acten vs COMBIART. 0 2 4 6 8 10 12 Baseline T1 T3 VAS Leq Ind Figure 3. ACTEN Group. 0 5 10 15 Baseline T1 T3 VAS Leq Ind Figure 4. COMBIART Group. 0 2 4 6 8 10 12 Baseline T1 T3 Acten Control group Figure 2. Lequesme Index Acten vs COMBIART. The criteria for mild, moderate, and severe adverse events were as follows: (1) mild: an adverse event that does not interfere with usual day-to-day activities and requires no special intervention or treatment; (2) moderate: an adverse event that can affect usual daily activities and that can be addressed with simple therapeutic treatments; (3) severe: an adverse event that requires therapeutic intervention. 0 2 4 6 8 Baseline T1 T3 Statistical analysis Data gathered from the VAS and LAI questionnaires underwent statistical analysis using a 2-tailed, paired t test to test baseline versus T1 (1 month) and T2 (3 months) for both treatments. Data were cross- analyzed using a 2-tailed non-paired t test to compare efficacies. The analyses were performed with the Graphpad PRISM statistical analysis software, version 5.0 (La Jolla, CA, USA). The alpha that was used for statistical significance was 0.05. Acten Control group Acten Control group Figure 2. Lequesme Index Acten vs COMBIART. 0 2 4 6 8 10 12 Baseline T1 T3 Acten Control group Figure 2. Lequesme Index Acten vs COMBIART. 0 2 4 6 8 10 12 Baseline T1 T3 VAS Leq Ind Figure 3. ACTEN Group. 0 5 10 15 Baseline T1 T3 VAS Leq Ind Figure 4. COMBIART Group. Figure 2. Lequesme Index Acten vs COMBIART. Discussion 0 0.51 1.52 2.53 3.54 4.55 5.56 6.57 7.5 Baseline T1 T3 Acten Control group Figure 1. VAS Acten vs Control Group. 0 0.51 1.52 2.53 3.54 4.55 5.56 6.57 7.5 Baseline T1 T3 Osteoarthritis is the most common form of arthritis in Italy and in the USA [47]. Glucosamine and chondroitin sulfate and hyaluronic acid are natural substances present in and around cartilage cells and, as such, are the most commonly used natural supplements for OA and joint pain. Previous studies have found them to be effective and safe for addressing the symptoms of OA [48,49]. In particular, the Glucosamine/ Chondroitin Arthritis Intervention Trial in 2006, sponsored by the National Institutes of Health (NIH), found that glucosamine combined with chondroitin sulfate provided statistically significant pain relief for a subset of participants in comparison with a placebo [20]. T3 In this study we have evaluated the clinical safety and efficacy of ACTEN® (CH 10gr and Fucose 100 mg) and compared it with a supplement (COMBIART) based on the well- studied effects: glucosamine, chondroitin sulfate, hyaluronic acid and the added value of Vitamin C [21]. As expected the COMBIART group showed a Acten Control group Figure 1. VAS Acten vs Control Group. Figure 1. VAS Acten vs Control Group. Clin Res Trials, 2016 doi: 10.15761/CRT.1000158 Volume 2(6): 253-257 Volume 2(6): 253-257 Martin-Martin LS (2016) Randomized observational multicenter study to assess the efficacy and safety of the association of Fortigel (10 Gr) and Fucoidan (100 Mg) in patients with Gonarthrosis Martin-Martin LS (2016) Randomized observational multicenter study to assess the efficacy and safety of the association of Fortigel (10 Gr) and Fucoidan (100 Mg) in patients with Gonarthrosis 13. Rovati L (1997) Clinical development of glucosamine sulfate as selective drug in osteoarthritis. Rheumatol Eur pp: 26:70. marked reduction in pain and functionality indexes. The decrease in pain and functionality is established in the first 4 weeks of treatment (-21.3% for both VAS and LAI) and is further increased in the following weeks, up to a -40.1% in VAS and -37% in LAI at T3 (12 weeks). ACTEN® showed similar results, with a reduction in VAS and LAI at T1 of 28.5% and 28.9% respectively. Again the highest efficacy is reached with prolonged exposure, reaching a -49.4% in VAS and -44.9% in LAI at T3. References 1. Litwic A, Edwards MH, Dennison EM, Cooper C (2013) Epidemiology and burden of osteoarthritis. Br Med Bull 105: 185-199.[Crossref] 23. Oben J, Enonchong E, Kothari S, Chambliss W, Garrison R, et al. (2009) Phellodendron and Citrus extracts benefit joint health in osteoarthritis patients: a pilot, double-blind, placebo-controlled study. Nutr J 8:38. 2. Reginster JY, Bruyere O, Fraikin G, Henrotin Y (2005) Current concepts in the therapeutic management of osteoarthritis with glucosamine. Bull Hosp Jt Dis 63: 31- 36.[Crossref] 24. Witt CM, Michalsen A, Roll S, Morandi A, Gupta S, et al. (2013) Comparative effectiveness of a complex Ayurvedic treatment and conventional standard care in osteoarthritis of the knee. Study protocol for a randomized controlled trial.Trials14:149. 3. Largo R, Alvarez-Soria MA, Diez-Ortego I, Calvo E, Sanchez-Pernaute O, et al. (2006) Glucosamine inhibits IL-1beta-induced NFkappaB activation in human osteoarthritic chondrocytes.Osteoarthr Cartil 11: 290-298. 25. Ezaki J, Hashimoto M, Hosokawa Y, Ishimi Y (2013) Assessment of safety and efficacy of methylsulfonylmethane on bone and knee joints in osteoarthritis animal model. J Bone Miner Metab 31: 16-25.[Crossref] 4. Alvarez-Soria MA, Largo R, Calvo E, Herrero-Beaumont G (2005) Differential anticatabolic profile of glucosamine sulfate versus other anti-osteoarthritic drugs on human osteoarthritic chondrocytes and synovial fibroblasts in culture. Osteoarthr Cartil13:S153. 26. McAlindon TE, Nuite M, Krishnan N, Ruthazer R, Price LL, et al. (2011) Change in knee osteoarthritis cartilage detected by delayed gadolinium enhanced magnetic resonance imaging following treatment with collagen hydrolysate: a pilot randomized controlled trial. Osteoarthritis Cartilage 19: 399-405.[Crossref] 5. Uitterlinden EJ, Jahr H, Koevoet JL, Jenniskens YM, Bierma-Zeinstra SM, et al. (2006) Glucosamine decreases expression of anabolic and catabolic genes in human osteoarthritic cartilage explants. Osteoarthr Cartil 14: 250-257. 27. Bannuru RR, Osani M, Vaysbrot EE, McAlindon TE (2016) Comparative safety profile of hyaluronic acid products for knee osteoarthritis: a systematic review and network meta-analysis. Osteoarthritis Cartilage S1063-4584. 6. Taniguchi S, Ryu J, Seki M, Sumino T, Tokuhashi Y, et al. (2012) Long-term oral administration of glucosamine or chondroitin sulfate reduces destruction of cartilage and up-regulation of MMP-3 mRNA in a model of spontaneous osteoarthritis in Hartley guinea pigs. J Orthop Res 30: 673-678.[Crossref] 28. Walrand S, Chiotelli E, Noirt F, Mwewa S, Lassel T (2008) Consumption of a functional fermented milk containing collagen hydrolysate improves the concentration of collagen-specific amino acids in plasma. J Agric Food Chem 56: 7790-7795. 7. Conclusions 20. Clegg DO, Reda DJ, Harris CL, Klein MA, O’Dell JR, et al. (2006) Glucosamine, chondroitin sulfate, and the two in combination for painful knee osteoarthritis. N Engl J Med 354: 795-808.[Crossref] Both nutritional supplements (ACTEN® and COMBIART) are safe, well tolerated, and able to exert an important pain reduction and improved functionality in the OA of the knee. Further studies are required to establish pathways for the shown efficacy in pain reduction and radiological studies to check on the structural changes behind the improved functionality of the articulation. 21. Peregoy J, Wilder FV (2011) The effects of vitamin C supplementation on incident and progressive knee osteoarthritis: a longitudinal study. Public Health Nutr 14: 709-715. [Crossref] 22. Malek Mahdavi A, Mahdavi R, Kolahi S, Zemestani M, Vatankhah AM. L-Carnitine supplementation improved clinical status without changing oxidative stress and lipid profile in women with knee osteoarthritis. Nutr Res 35:707-715. Discussion Notably the ACTEN® group in the last 8 weeks only assumed the nutritional supplement once every other day, without any loss of efficacy. No statistically relevant difference has been found between the two groups, but larger numbers or prolonged exposures to the supplements might uncover subtler differences. 14. Houpt JB, McMillan R, Wein C, Paget-Dellio SD (1999) Effect of glucosamine hydrochloride in the treatment of pain of osteoarthritis of the knee. J Rheumatol 26: 2423-2430.[Crossref] 15. Rovati L (1993) Clinical efficacy of glucosamine sulfate in osteoarthritis of the spine. Rev Esp Reumatol 20(S1): 325. 16. Müller-Fassbender H, Bach GL, Haase W, Rovati LC, Setnikar I (1994) Glucosamine sulfate compared to ibuprofen in osteoarthritis of the knee. Osteoarthritis Cartilage 2: 61-69.[Crossref] 17. Qiu GX, Gao SN, Giacovelli G, Rovati L, Setnikar I (1998) Efficacy and safety of glucosamine sulfate versus ibuprofen in patients with knee osteoarthritis. Arzneimittelforschung 48: 469-474.[Crossref] It has to be taken in consideration that, a daily regimen of ACTEN®, instead of every other day regimen as used in this trial, it is likely to produce further benefits, and it should be investigated. 18. Rovati L (1997) The clinical profile of glucosamine sulfate as a selective symptom modifying drug in osteoarhtritis: current data and perspective. Osteoarthr Cartil 5(SA):72. 19. Reginster JY, Neuprez A, Lecart MP, Sarlet N, Bruyere O (2012) Role of glucosamine in the treatment for osteoarthritis. Rheumatol Int 32: 2959-2967.[Crossref] References Imagawa K, Andres MC, Hashimoto K, Pitt D, Itoi E, et al. (2011) The epigenetic effect of glucosamine and a nuclear factor-kappa B (NF-kB) inhibitor on primary human chondrocytes-implications for osteoarthritis. Biochem Biophys Res Commun 405: 362-367. 29. Bello AE, Oesser S (2006) Collagen hydrolysate for the treatment of osteoarthritis and other joint disorders: a review of the literature. Curr Med Res Opin 22: 2221-2232. [Crossref] 30. Oesser S, Seifert J (2003) Stimulation of type II collagen biosynthesis and secretion in bovine chondrocytes cultured with degraded collagen. Cell Tissue Res 311: 393-399. [Crossref] 8. Altman RD, Abramson S, Bruyere O, Clegg D, Herrero-Beaumont G, et al. (2006) Commentary: osteoarthritis of the knee and glucosamine. Osteoarthr Cartil 14: 963– 966. 31. Schunck M, Schulze CH, Oesser S (2006) Disparate efficacy of collagen hydrolysate and glucosamine on the extracellular matrix metabolism of articular chondrocytes. OA and Cartilage 14: S114. 9. Reginster JY, Bruyere O, Neuprez A (2007) Current role of glucosamine in the treatment of osteoarthritis. Rheumatology (Oxford) 46: 731-735.[Crossref] 10. Reichelt A, Förster KK, Fischer M, Rovati LC, Setnikar I (1994) Efficacy and safety of intramuscular glucosamine sulfate in osteoarthritis of the knee. A randomised, placebo- controlled, double-blind study. Arzneimittelforschung 44: 75-80.[Crossref] 32. Oesser S, Proksch E, Schunck M (2008) prophylactic treatment with a special collagen hydrosylate decreases cartilage tissue degeneration in the knee joints. OA and Cartilage 16: S45. 11. Setnikar I, Palumbo R, Canali S, Zanolo G (1993) Pharmacokinetics of glucosamine in man. Arzneimittelforschung 43: 1109-1113.[Crossref] 33. Nakatani S, Mano H, Sampei C, Shimizu J, Wada M (2009) Chondroprotective effect of the bioactive peptide prolyl-hydroxyproline in mouse articular cartilage in vitro and in vivo. Osteoarthritis Cartilage 17: 1620-1627. 12. Leffler CT, Philippi AF, Leffler SG, Mosure JC, Kim PD (1999) Glucosamine, chondroitin, and manganese ascorbate for degenerative joint disease of the knee or low back: a randomized, double-blind, placebo-controlled pilot study. Mil Med 164: 85-91.[Crossref] 34. Clark KL, Sebastianelli W, Flechsenhar KR, Aukermann DF, Meza F, et al. (2008) 24- Week study on the use of collagen hydrolysate as a dietary supplement in athletes with activity-related joint pain. Curr Med Res Opin 24: 1485-1496.[Crossref] Clin Res Trials, 2016 doi: 10.15761/CRT.1000158 Volume 2(6): 253-257 Martin-Martin LS (2016) Randomized observational multicenter study to assess the efficacy and safety of the association of Fortigel (10 Gr) and Fucoidan (100 Mg) in patients with Gonarthrosis 42. References Berteau O, Mulloy B (2003) Sulfated fucans fresh perspectives: Structures, functions, and biological properties of sulfated fucans and an overview of enzymes active toward this class of polysaccharide. Glycobiolog13:29–40. 35. Zuckley L, Angelopoulou K, Carpenter MSS, Meredith BA, et al. (2004)Collagen hydrosylate improves joint function in adults with mild symptoms of osteoarthritis of the knee. Medicine& Science in Sports & Exercise 36: S153–S154. 36. Moskowitz RW (2000) Role of collagen hydrolysate in bone and joint disease. Semin Arthritis Rheum 30: 87-99.[Crossref] 43. Myers SP, O’Connor J, Fitton JH, Brooks L, Rolfe M, et al. (2011) A combined Phase I and II open-label study on the immunomodulatory effects of seaweed extract nutrient complex. Biologics 5:45–60. 37. Benito-Ruiz P, Camacho-Zambrano MM, Carrillo-Arcentales JN, Mestanza-Peralta MA, Vallejo-Flores CA, et al. (2009) A randomized controlled trial on the efficacy and safety of a food ingredient, collagen hydrolysate, for improving joint comfort. Int J Food Sci Nutr 60: 99-113 44. Cunha TM, Verri WA Jr, Schivo IR, Napimoga MH, Parada CA, et al. (2008) Crucial role of neutrophils in the development of mechanical inflammatory hypernociception. J Leukoc Biol 83: 824-832.[Crossref] 45. Lequesne MG (1997) The algofunctional indices for hip and knee osteoarthritis. J Rheumatol 24: 779-781.[Crossref] 38. Fitton JH (2011) Therapies from fucoidan; multifunctional marine polymers. Mar Drugs 9: 1731-1760.[Crossref] 39. 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In: Beck SV (Ed) The Cambridge World History of Food. Cambridge University Press, Cambridge, UK 1: 231–249. 49. Clin Res Trials, 2016 doi: 10.15761/CRT.1000158 Copyright: ©2016 Martin-Martin LS. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. References Uebelhart D, Thonar EJ, Delmas PD, Chantraine A, Vignon E (1998) Effects of oral chondroitin sulfate on the progression of knee osteoarthritis: a pilot study. Osteoarthritis Cartilage 6 (suppl A): 39-46. Copyright: ©2016 Martin-Martin LS. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Clin Res Trials, 2016 doi: 10.15761/CRT.1000158 Clin Res Trials, 2016 doi: 10.15761/CRT.1000158 Volume 2(6): 253-257
https://openalex.org/W4213430415	https://tspace.library.utoronto.ca/bitstream/1807/110292/1/ijerph-19-02607.pdf	English	null	Acute Physiological Response to Different Sprint Training Protocols in Normobaric Hypoxia	International journal of environmental research and public health/International journal of environmental research and public health	2,022	cc-by	8,428	Citation: Maldonado-Rodriguez, N.; Bentley, D.J.; Logan-Sprenger, H.M. Acute Physiological Response to Different Sprint Training Protocols in Normobaric Hypoxia. Int. J. Environ. Res. Public Health 2022, 19, 2607. https://doi.org/10.3390/ ijerph19052607 Keywords: hypoxia; sprint training; physiological response Academic Editors: Chansol Hurr and Jordan Patik Academic Editors: Chansol Hurr and Jordan Patik International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health Article Naomi Maldonado-Rodriguez 1, David J. Bentley 1,2 and Heather M. Logan-Sprenger 1,2,3,* 1 Faculty of Kinesiology & Physical Education, University of Toronto, Toronto, ON M5S 1A4, Canada; nmrod@mail.ubc.ca (N.M.-R.); bentley.dj@gmail.com (D.J.B.) ( ) y j g ( J ) 2 Canadian Sport Institute of Ontario, Toronto, ON M1C 0C7, Canada y j g 2 Canadian Sport Institute of Ontario, Toronto, ON M1C 0C7, Canada 3 Faculty of Health Sciences, Ontario Tech University, Oshawa, ON L1H 7K4, Canada * Correspondence: heather.sprenger@ontariotechu.ca 3 Faculty of Health Sciences, Ontario Tech University, Oshawa, ON L1H 7K4, Canada * Correspondence: heather.sprenger@ontariotechu.ca Abstract: Background: the purpose of this study was to examine acute physiological responses to and the performance effects of two sprint training protocols in normobaric hypoxic conditions. Methods: Healthy competitive female (n = 2) and male (n = 5) kayakers (19 ± 2.1 years) performed four sprint training sessions on a kayak ergometer over a period of two weeks. Participants performed ﬁve sets of 12 × 5 s sprints or 3 × 20 s sprints in both normobaric normoxic (NOR, FiO2 = 20.9%) or normobaric hypoxic (HYP, FiO2 = 13.6%) conditions. The peak power output (PPO), rate of perceived exertion (RPE), and heart rate (HR) of each participant were monitored continuously. Their blood lactate concentrations ([BLa+]), in addition to their blood gas (mixed-venous partial pressure (p) of carbon dioxide (pCO2), O2 (pO2), and oxygen saturations (sO2)) were collected before and after exercise. Results: A signiﬁcantly greater RPE, HR, and [BLa+] response and a signiﬁcant decrease in pCO2, pO2, and sO2 were observed in HYP conditions versus NOR ones, independent of the type of training session. The PPO of participants did not differ between sessions. Their RPE in HYP12 × 5 was greater compared to all other sessions. Conclusions: The HYP conditions elicited signiﬁcantly greater physiological strain compared to NOR conditions and this was similar in both training sessions. Our results suggest that either sprint training protocol in HYP conditions may induce more positive training adaptations compared to sprint training in NOR conditions. Int. J. Environ. Res. Public Health 2022, 19, 2607. https://doi.org/10.3390/ijerph19052607 1. Introduction Traditional “altitude training”, where athletes live and train at real or simulated al- titudes of 1800–2400 m, has focused on enhancing sea level endurance performance by improving red cell mass [1]. Live high train low (LHTL) models are commonly used in preseason training to enhance aerobic and potentially anaerobic performance [2,3]. LHTL training approaches require athletes to adhere to prolonged and consistent expo- sure (≥3 weeks) to hypoxia, while training at sea level, in order to stimulate an increase in red blood cell (RBC) mass whilst reducing the potentially detrimental effects of con- tinuous altitude exposure on detraining [4]. However, such training is neither time- nor cost-effective [5]. While the positive effects on aerobic capacity are well-documented, the reported effects on anaerobic performance have been equivocal. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Traditional LHTL protocols, designed to induce improvements in aerobic capacity, typically do not result in meaningful anaerobic improvements, most likely as a result of training design that does not sufﬁciently stress the anaerobic system [6,7]. Recently, inter- mittent hypoxia training (IHT), “a method where athletes live at or near sea level but train under hypoxic conditions”, has been explored as a potential alternative to traditional train- ing at altitude to stimulate adaptations that may affect anaerobic performance [7]. Studies have shown that sport-speciﬁc IHT may lead to speed/power performance improvements Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Int. J. Environ. Res. Public Health 2022, 19, 2607. https://doi.org/10.3390/ijerph19052607 https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2022, 19, 2607 2 of 11 2 of 11 or repeated sprint ability via increased resting pH, enhanced buffering capacity, and im- proved blood perfusion [7–15]. However, despite many potential physiological adaptations, IHT has failed to show consistent performance improvements, such as in relation to power output or performance in 10 s or 30 s all out tests [13,16,17]. One important consideration is that training must be performed at a maximal intensity in order to augment anaerobic per- formance [5,7,15]. One way of adapting IHT for anaerobic performance-speciﬁc adaptations may be sprint training in hypoxia [7]. 1. Introduction The parameters of training design (e.g., modality, work-to-rest ratio, repetition duration) should be such that they result in incomplete re- covery, so that they induce glycolytic adaptations. One such parameter, sprint duration, largely inﬂuences the relative contribution of aerobic and anaerobic energy systems. A duration of 30 s is conventionally used in sprint training, with the ﬁrst 5–10 s representing the period during which peak power is achieved, with the latter 20–25 s constituting an effort to maintain this output [18]. Hazell and colleagues (2010), when comparing 10 s and 30 s sprint protocols, suggested that the initial generation of peak power output (PPO) was likely responsible for sprint interval training adaptations [19]. Sprint training is primarily dependent on the ability to match ATP resynthesis rate to utilization rate and altered intracellular and extracellular ion concentrations [12]. The added stimulus of hypoxia is likely to increase metabolic stress and, thus, hypothetically induce greater physiological adaptations and performance improvements. The sport of ﬂat water canoeing and kayaking encompasses events ranging between a ‘sprint’ (200 m) of <30 s to events that are 1–2 min in duration (1000 m). It has been shown that there is a large aerobic component to these events, especially 1000 m events [20]. In contrast, in the sprint event, there is a heavy anaerobic component, with athletes requiring considerable upper body anaerobic capacity. Competitive canoe and kayak athletes under- take sprint training and other forms of anaerobic conditioning in their preparation [20]. The acute response to different durations of anaerobic work intervals, with respect to work volume and work-to-rest ratio, performed in hypoxia, has not been previously investigated, and this is especially true in the context of kayak athletes, for whom anaerobic training is an important sport-speciﬁc consideration. The purpose of this study was to examine acute physiological responses to two sprint training protocols (sets of 5 s versus 30 s) in normobaric hypoxic conditions. We hypothesized that, (1) the hypoxic condition would induce greater physiological strain compared to normoxic conditions, and (2) longer duration sprints would result in a greater physiological response in terms of blood parameters. 2. Materials and Methods Seven healthy well-trained male or female kayakers (female: n = 2; male: n = 6) aged 15–24 (mean ± SD: 19 ± 2.1 years) were recruited from local canoe–kayak clubs (training 18 ± 2 h per week). All kayakers competed at a national and international level as part of their respective provincial or national federations in ﬂatwater kayaking. Participants were sea level natives with no exposure to hypoxia training. All participants were actively train- ing athletes in their off-season. Prior to the initial training sessions, a risk assessment was completed to identify any potential confounding respiratory, neurological, musculoskeletal, or circulatory conditions that may have been deemed a risk to sprint training or training in hypoxia. Parents (assent was required) and participants were informed both verbally and in writing of the experimental protocol and potential risks before giving their verbal and written assent and consent, respectively, to participate. The Research Ethics Board at the Canadian Sport Institute Ontario approved the study (REB#18-01). 2.3. Blood Sampling Blood samples (5 uL) were collected from participants’ ﬁngertips and analyzed for blood lactate concentration [BLa+] using a portable hand-held blood lactate analyzer (Lactate PRO, USA) pre-exercise (5 min prior to start), after set 3, and post-exercise (5 min post). Blood bicarbonate (HCO3−), pH, mixed-venous partial pressure of carbon dioxide (pCO2), oxygen (pO2), and oxygen saturation (sO2) were measured via capillary analysis using a blood gas analyzer (ABL80, Radiometer, Mississauga, ON, Canada). Various metabolites, including sodium (Na+), potassium (K+), calcium (Ca2+), and chloride (Cl−), were also assessed using a 125 uL plastic capillary tube with 70iu balanced heparin pre- and post-exercise and measured using a blood gas analyzer (ABL80, Radiometer, Mississauga, ON, Canada). All resting blood samples were taken in normoxia while the post-exercise blood draw was collected in either HYP or NOR conditions depending on the exercise session. 2.2. Training Protocol Participants performed ﬁve sets of 12 × 5 s sprints (protocol 1) or 3 × 20 s sprints (protocol 2) in both normobaric normoxia (NOR, 0 m altitude, FiO2 = 20.9%) and normobaric hypoxia (HYP, 3500 m altitude, FiO2 = 13.6%) (see Supplementary Materials). The warm-up consisted of 5-min of light intensity paddling at a self-selected pace, followed by 4 × 10 s preparatory ‘submaximal efforts’ with 20 s of light intensity work between each bout and 2 min of passive rest before the start of the sprint session (11 min total). Protocol 1 consisted of ﬁve sets of 12 × 5 s sprints with 15 s of active recovery between each repetition and 2 min of passive rest between sets. Protocol 2 consisted of ﬁve sets of 3 × 20 s sprints with 60 s active recovery between repetition and 2 min of passive rest between sets. Both protocols were matched for total work volume and work-to-rest ratio (1:3) based on recommendations set forth by Brocherie and colleagues (2017) [8]. The focus of the training was to maximize hypoxia exposure in order to improve anaerobic capacity by way of short high-intensity exercise, replicating the demands of kayaking. Athletes were asked to perform maximally for each sprint. p Training was completed in a normobaric hypoxic chamber that was purpose-built for intermittent hypoxia training (K2 Room, Storex Ca Inc., Montreal, QC, Canada). The chamber is a 5 × 5 m room with an airlock and a glass wall for viewing access. The temperature and humidity were set at approximately 20 ◦C and 20%, respectively, for consistency. Participants were monitored continuously using heart rate (HR) and rate of perceived exertion (RPE) [21]. Their peak HR was captured after every sprint using a Polar Strap (Polar H10, Polar Electro, Nassau, NY, USA) and recorded using the FIT IV Pulse application on an iPad (iPad version 3, Apple, Cupertino, CA, USA). Their RPE was recorded after every set on a scale of 6 to 20. Capillary blood samples for [BLa+], blood gas, and metabolite analysis were collected throughout. [BLa+] was collected pre-exercise, after set 3, and post-exercise. Blood gas and metabolites were collected immediately pre- and post-exercise. 2.1. Experimental Procedure Athletes completed four sprint training sessions on a kayak ergometer (K1 Speed Stroke Ergometer, KayakPro, Miami Beach, FL, USA) over a period of 2 weeks, with a minimum of 48 h between each session. Each session occurred in an environmentally controlled chamber (K2 Room, Storex Ca Inc., Montreal, QC, Canada). In a single-blind Int. J. Environ. Res. Public Health 2022, 19, 2607 3 of 11 3 of 11 fashion, each session was performed in the altitude chamber to ensure participants were blind to the environmental condition. The order of the training sessions was randomized to control for potential physiological adaptations that may have occurred throughout the duration of the testing. Previous studies have demonstrated that participants are not able to distinguish between hypoxia and normoxia during intermittent sprint training [7]. The trials were conducted at the same of time of the day. Any supplementary normoxic training that occurred outside the study was recorded (modality, duration, and intensity). 3. Results The mean ± SD values are outlined in Table 1. The PPO of participants did not differ between training protocols (Table 1). All participants trained at a very high intensity, with mean session RPEs ranging from 15 to 17 (Table 1). A signiﬁcant interaction was found between the environmental condition and training protocol with respect to RPE (p = 0.011, η2 = 0.76). Post-hoc analyses revealed that participants reported a signiﬁcantly greater RPE after the HYP12 × 5 protocol, compared to the NOR12 × 5 protocol (p = 0.003). In addition, we found a signiﬁcant interaction in terms of peak HR between training protocols (12 × 5 vs. 3 × 20) and environmental conditions (normoxia vs. hypoxia) (Table 1, p = 0.014, η2 = 0.73). Peak HR in both HYP12 × 5 and HYP3 × 20 was greater than its normoxia counterpart, with p = 0.029 and p = 0.025 respectively. Peak HR in NOR3 × 20 was signiﬁcantly greater than in NOR12 × 5 (p = 0.003). There were no differences in terms of hypoxia between training protocols. g p Post-exercise [BLa+] values were elevated in all training protocols, compared to pre- exercise resting values (refer to Table 1). Mean [BLa+] values were above the lactate threshold, which is generally deﬁned as between 4 mmol/L [20]. Post-exercise [BLa+] levels were signiﬁcantly greater in hypoxia, compared to normoxia (p = 0.029, η2 = 0.65), regardless of the training session (12 × 5 or 3 × 20) (Table 1). Additionally, post-exercise [BLa+] levels were signiﬁcantly greater following the 3 × 20 protocol, compared to the 12 × 5 protocol, regardless of the environmental condition (p = 0.016, η2 = 0.72). p g p No signiﬁcant differences were observed in pH between conditions or sprint ses- sions (p > 0.05). However, we did note an interaction that trended towards signiﬁcance (p = 0.085). For both the 3 × 20 sprint sessions (HYP and NOR), pH fell below 7.35, sug- gesting that the 3 × 20 sprint session elicited metabolic acidosis in both HYP and NOR environmental conditions. Post-exercise pCO2 was signiﬁcantly greater than pre-exercise values in the hypoxia conditions only (p = 0.05, η2 = 0.57), independent of the training session (Figure 1A,B). On the other hand, post-exercise pO2 was signiﬁcantly reduced in the hypoxia session (p = 0.001, η2 = 0.91), irrespective of the training session (Figure 1C,D). 2.5. Statistical Analysis Data are mean ± standard deviation (SD), unless otherwise stated. Changes in the mean and standard deviation of the variables representing between- and within-subject variability were assessed using a two-way repeated-measures ANOVA in SPSS (Version 24, IBM Corp., Armonk, NY, USA). RPE and peak HR were averaged to a session mean for all four trials. The difference between pre- and post-exercise values for [BLa+], blood gas, and metabolites was calculated to obtain a mean change over time, thus removing the third factor of time. Only mean values in PPO were compared using a three-way ANOVA with repeated measures model. Post-hoc analyses were conducted where applicable and adjusted for multiple comparisons using Bonferroni correction. Both HR and sO2 were not normally distributed; as both did not respond to transformation, data analysis was run with and without outliers (one in each). As they did not change, results from analysis with outliers are reported. To supplement important ﬁndings, effect sizes (η2) were calculated as the ratio of the mean difference to the pooled SD of the difference. The magnitude of the effect size was classed as trivial (<0.2), small (0.2–0.6), moderate (0.6–1.2), large (1.2–2.0), and very large (>2.0) based on previous published guidelines [22]. Moreover, exact p values and Cohen d are presented to show the magnitude of effect. 2.4. Performance Measures The participant’s peak power output (PPO) for each sprint was recorded using the kayak ergometer’s integrated monitor. The PPO was then averaged per set and per session and normalized to body mass (W/kg). 4 of 11 Int. J. Environ. Res. Public Health 2022, 19, 2607 3. Results There was no interaction between training protocol and environmental condition in either blood gases. Additionally, sO2 was signiﬁcantly lower in hypoxia than in normoxia, in- dependent of the training protocol (p = 0.001, η2 = 0.9, Figure 1E,F). The training protocol (12 × 5 or 3 × 20) had no statistical effect on pCO2 or pO2 response. Lastly, we found no signiﬁcant changes in the blood metabolites measured (Na+, K+, Ca2+, Cl−, HCO3−). While it did not reach signiﬁcance, HCO3−showed a trending decrease (p = 0.067, η2 = 0.52) in the NOR3 × 20 and both hypoxia sessions. Int. J. Environ. Res. Public Health 2022, 19, 2607 5 of 11 Table 1. Mean performance and physiological measures pre- and post-training. 12 × 5 Protocol 3 × 20 Protocol NOR HYP NOR HYP PPO (W/kg) 3.87 ± 1.04 3.84 ± 0.89 3.56 ± 0.32 4.14 ± 1.4 RPE 15 ± 1.21 17 ± 0.89 * 16 ± 0.73 16 ± 1.35 Peak HR (bpm) 159 ± 11 ˆ 167 ± 10 * 164 ± 8 170 ± 11 * [BLa+] (mmol/L) Pre 1.18 ± 0.2 1.9 ± 0.57 1.64 ± 0.6 1.44 ± 0.48 Post 4.23 ± 1.74 8.52 ± 2.5 * 9.02 ± 3.8 ˆ 10.28 ± 3.0 * Blood Gas/Metabolites pH Pre 7.41 ± 0.042 7.41 ± 0.013 7.43 ± 0.031 7.42 ± 0.035 Post 7.39 ± 0.051 7.36 ± 0.037 7.31 ± 0.010 7.34 ± 0.072 pCO2 (mmHg) Pre 36.33 ± 2.66 37.00 ± 2.92 37.00 ± 3.10 38.33 ± 4.32 Post 33.67 ± 3.33 25.8 ± 4.44 * 29.00 ± 3.39 29.33 ± 2.34 * pO2 (mmHg) Pre 76.33 ± 14.26 75.4 ± 9.26 65.8 ± 5.17 69.17 ± 9.79 Post 80.33 ± 16.75 57.8 ± 5.45 * 90.80 ± 11.26 57.17 ± 14.74 * HCO3−(mEq/L) Pre 22.6 ± 2.90 23.20 ± 1.63 24.38 ± 1.95 24.25 ± 1.71 Post 19.98 ± 2.69 14.18 ± 2.30 14.6 ± 4.81 15.7 ± 3.14 sO2 (%) Pre 95.8 ± 3.6 94.25 ± 5.75 95.28 ± 1.9 95.28 ± 2.84 Post 96.07 ± 3.7 89.2 ± 2.52 * 97.43 ± 1.0 87.97 ± 5.6 * Data are mean ± SD. 3. Results NOR, normobaric normoxia (20.9% O2); HYP, normobaric hypoxia (13.6% O2); PPO, peak power output; W/kg, watts per kilogram body mass; HR, heart rate; bpm, beats per minute; [BLa+], blood lactate concentration; pCO2, partial pressure of carbon dioxide; pO2, partial pressure of oxygen; HCO3−, bicarbonate; sO2, saturation of oxygen. * Signiﬁcant difference between the normoxic and hypoxic response within the training protocol (p < 0.05); ˆ Signiﬁcant difference between training protocols (p < 0.05). 2, 19, × FOR PEER REVIEW 6 Table 1. Mean performance and physiological measures pre- and post-training. Data are mean ± SD. NOR, normobaric normoxia (20.9% O2); HYP, normobaric hypoxia (13.6% O2); PPO, peak power output; W/kg, watts per kilogram body mass; HR, heart rate; bpm, beats per minute; [BLa+], blood lactate concentration; pCO2, partial pressure of carbon dioxide; pO2, partial pressure of oxygen; HCO3−, bicarbonate; sO2, saturation of oxygen. * Signiﬁcant difference between the normoxic and hypoxic response within the training protocol (p < 0.05); ˆ Signiﬁcant difference between training protocols (p < 0.05). 19, × FOR PEER REVIEW Data are mean ± SD. NOR, normobaric normoxia (20.9% O2); HYP, normobaric hypoxia (13.6% O2); PPO, peak power output; W/kg, watts per kilogram body mass; HR, heart rate; bpm, beats per minute; [BLa+], blood lactate concentration; pCO2, partial pressure of carbon dioxide; pO2, partial pressure of oxygen; HCO3−, bicarbonate; sO2, saturation of oxygen. * Signiﬁcant difference between the normoxic and hypoxic response within the training protocol (p < 0.05); ˆ Signiﬁcant difference between training protocols (p < 0.05). 19, × FOR PEER REVIEW 6 Figure 1. Cont. Figure 1. Cont. Figure 1. Cont. 6 of 11 Int. J. Environ. Res. Public Health 2022, 19, 2607 Figure 1. Mean pre- and post-exercise blood gas measurements for the two sprint training pro (12 × 5 versus 3 × 20). (A) partial pressure of carbon dioxide (pCO2) in the 12 × 5 protocol; (B) pressure of carbon dioxide (pCO2) in the 3 × 20 protocol; (C) partial pressure of oxygen (pO2) 12 × 5 protocol; (D) partial pressure of oxygen (pO2) in the 3 × 20 protocol; (E) blood oxygen s tion (sO2) in the 12 × 5 protocol; (F) blood oxygen saturation (sO2) in the 3 × 20 protocol. Da mean ± SD. Significant difference versus normoxia, independent of training protocol (p < 0.0 Figure 1. 3. Results Mean pre- and post-exercise blood gas measurements for the two sprint training protocols (12 × 5 versus 3 × 20). (A) partial pressure of carbon dioxide (pCO2) in the 12 × 5 protocol; (B) partial pressure of carbon dioxide (pCO2) in the 3 × 20 protocol; (C) partial pressure of oxygen (pO2) in the 12 × 5 protocol; (D) partial pressure of oxygen (pO2) in the 3 × 20 protocol; (E) blood oxygen saturation (sO2) in the 12 × 5 protocol; (F) blood oxygen saturation (sO2) in the 3 × 20 protocol. Data are mean ± SD. Signiﬁcant difference versus normoxia, independent of training protocol (p < 0.05). igure 1. Mean pre- and post-exercise blood gas measurements for the two sprint training pro Figure 1. Mean pre- and post-exercise blood gas measurements for the two sprint training protocols Figure 1. Mean pre- and post-exercise blood gas measurements for the two sprint training pr (12 × 5 versus 3 × 20). (A) partial pressure of carbon dioxide (pCO2) in the 12 × 5 protocol; (B) pressure of carbon dioxide (pCO2) in the 3 × 20 protocol; (C) partial pressure of oxygen (pO2 12 × 5 protocol; (D) partial pressure of oxygen (pO2) in the 3 × 20 protocol; (E) blood oxygen tion (sO2) in the 12 × 5 protocol; (F) blood oxygen saturation (sO2) in the 3 × 20 protocol. D mean ± SD. Significant difference versus normoxia, independent of training protocol (p < 0.0 Figure 1. Mean pre- and post-exercise blood gas measurements for the two sprint training protocols (12 × 5 versus 3 × 20). (A) partial pressure of carbon dioxide (pCO2) in the 12 × 5 protocol; (B) partial pressure of carbon dioxide (pCO2) in the 3 × 20 protocol; (C) partial pressure of oxygen (pO2) in the 12 × 5 protocol; (D) partial pressure of oxygen (pO2) in the 3 × 20 protocol; (E) blood oxygen saturation (sO2) in the 12 × 5 protocol; (F) blood oxygen saturation (sO2) in the 3 × 20 protocol. Data are mean ± SD. Signiﬁcant difference versus normoxia, independent of training protocol (p < 0.05). 4. Discussion 4. Discussion The results of this study demonstrate that regardless of the sprint training stim (12 × 5 vs. 3 × 20), a controlled normobaric hypoxic environment of 13.6% O2 elicite nificantly greater acute responses in terms of RPE, HR, BLa concentration, and pCO2 significantly lower post-exercise pO2 and sO2, along with a trend of lower blood pH HCO3−, compared to a sprint training session in normobaric normoxic conditions. comparing the sprint training session stimulus (12 × 5 vs. 3 × 20), the results show th RPE of the participants was higher in the 12 × 5 sprint workout; however, their H BLa+ concentrations were significantly greater in the 3 × 20 sprint workout, with no d ence between the sprint training stimulus in terms of blood gases, pH, or blood m The results of this study demonstrate that regardless of the sprint training stimulus (12 × 5 vs. 3 × 20), a controlled normobaric hypoxic environment of 13.6% O2 elicited signiﬁcantly greater acute responses in terms of RPE, HR, BLa concentration, and pCO2, with signiﬁcantly lower post-exercise pO2 and sO2, along with a trend of lower blood pH and HCO3−, compared to a sprint training session in normobaric normoxic conditions. When comparing the sprint training session stimulus (12 × 5 vs. 3 × 20), the results show that the RPE of the participants was higher in the 12 × 5 sprint workout; however, their HR and BLa+ concentrations were signiﬁcantly greater in the 3 × 20 sprint workout, with no difference between the sprint training stimulus in terms of blood gases, pH, or blood metabolites. lites. 4.1. Peak Power Output during Sprint Training in Hypoxia and Normoxia Athletes were able to maintain their PPO, independent of the training session. This is congruent with the literature, which suggests that PPO is generated during the ﬁrst 5–10 s of exertion [19,23]. Thus, athletes would have had time to reach their maximal power output even during the short-duration sprint protocol. An athlete’s ability to generate power throughout a session is an important consideration when it comes to potential adaptations to the anaerobic system. It is well-documented that athletes must train at maximal intensities in order to induce improvement in anaerobic capacity, likely due to enhanced glycolytic activity in muscles via increased mRNA expression related to pH regulation, upregulation of anaerobic metabolism, increased buffering capacity, modiﬁed fast twitch (FT) ﬁber behavior, and increased end-product metabolite removal mechanisms [5,7,8,13–15,24,25]. In contrast, a reduction in PPO may have indicated that the training stimulus was too high. This has historically been an issue in this ﬁeld of research, where prolonged exposure to hypoxia leads to a reduction in training quality and intensity, which may explain the lack of performance improvements previously noted, despite physiological adaptations [26–28]. It is thus paramount that athletes work maximally. Our results demonstrate that the sprint prescription (12 × 5 or 3 × 20) did not impair repeated maximal power output over a single session. The question becomes whether the repeated maximal power output can be sustained over multiple sessions to elicit positive training adaptations. 7 of 11 7 of 11 Int. J. Environ. Res. Public Health 2022, 19, 2607 4.2. Physiological Responses to Sprint Training in Hypoxia and Normoxia 4.2. Physiological Responses to Sprint Training in Hypoxia and Normoxia Peak HR was greater in hypoxia than normoxia, independent of the training session. As expected, the reduction in the fraction of inspired oxygen (FiO2 = 13.6%) resulted in greater cardiovascular strain, which translated into an increase in HR. Moreover, peak HR in NOR3 × 20 was greater than in NOR12 × 5. Additionally, athletes rated the intensity of the exercise as very high. Only HYP12 × 5 resulted in a greater RPE compared to its normoxia counterpart, but this was not reﬂected in any PPO differences. Interestingly, no differences were noted between NOR3 × 20 and HYP3 × 20. Studies have reported that shorter sprint durations see a greater relative contribution from the anaerobic system [29]. lites. 4.1. Peak Power Output during Sprint Training in Hypoxia and Normoxia Thus, a 5 s sprint would be a greater stressor on the anaerobic system compared to a 20 s sprint. This trend is also seen in [BLa+], which was greater in HYP12 × 5 than in NOR12 × 5. Post-exercise [BLa+] values were greater than pre-exercise values, regardless of the training session. These results are not surprising, as the athletes were instructed to sprint maximally. A high lactate accumulation is consistent with the literature, which has shown that high intensity short duration exercise, such as sprint training, leads to [BLa+] accumulation above the lactate threshold (typically deﬁned as 4 mmol/L). Elevated lactate levels at the cessation of exercise is indicative of anaerobic metabolism [30]. Additionally, [BLa+] was higher in HYP12 × 5 compared to NOR12 × 5, but no differences were noted between NOR3 × 20 and HYP3 × 20. This appears to be the only physiological response that supports our RPE ﬁndings. Much of our data suggests that the acute physiological response to HYP12 × 5 and HYP3 × 20 was similar. It is possible that the increase in [BLa+] resulted in greater muscular discomfort and, consequently, an increase in RPE and [BLa+] [31,32]. 4.3. Blood Gas Responses to Sprint Training in Hypoxia and Normoxia Public Health 2022, 19, 2607 8 of 11 sprint training in hypoxia resulted in greater physiological strain compared to normoxia, with no differences seen between the training protocols. sprint training in hypoxia resulted in greater physiological strain compared to normoxia, with no differences seen between the training protocols. 4.4. Perceptual Response to Sprint Training in Hypoxia and Normoxia It is well-documented that many anaerobic performance improvements, such as speed or power development, are mediated via adaptations in the neuromuscular system by way of motor unit recruitment, activation, and ﬁring [37–40]. Studies show that maximal effort sprints require high levels of motor unit activation [40,41]. In this study, participants perceived the HYP12 × 5 session to be harder than the three other sessions, despite them resulting in a similar PPO. One potential reason for this may be that the greater repetition of sprints in this session produced a greater stressor on the neuromuscular system due to rapid acceleration and deceleration. There is evidence that neural fatigue may be caused by a decrease in reﬂex sensitivity, which has been associated with force production and propulsion [18,42]. Bowtell and colleagues (2014) noted signiﬁcantly reduced iEMG activity and running speed following sprint training in hypoxia [14]. These changes in neuromuscular activity may reﬂect fatigue development, possibly due to reduced central neural drive or impaired neuromuscular transmission [14,43]. Moreover, metabolic changes in the muscle, such as the [BLa+] accumulation and the decrease in pH seen in this study, may have accelerated the onset of muscle fatigue [40]. It is worth noting that in severely hypoxic conditions, neural fatigue may be a limiting factor and prevent athletes from training at the maximal intensities needed to obtain performance increments [3,44]. As such, the interaction between training protocol and altitude should be carefully considered when designing a program to achieve a stimulus great enough to induce adaptations but that does not impair performance. 4.5. Limitations This study included a relatively small sample size, which may partly explain why certain effects were observed and others were not. Additionally, the results may have been affected by the range of competition levels tested. Participants ranged from national level to internationally ranked athletes. However, research shows that elite and sub-elite athletes often have different strengths and anaerobic power proﬁles [45,46]. Therefore, testing a sample of athletes of mixed competition levels may have confounded the results, especially given the small sample size. Moreover, although an effort was made to standardize the encouragement given, it is possible that the encouragement provided varied between athletes and may have affected the PPO results [47]. We also did not control the athlete’s training outside of the four sessions. We asked athletes to record the modality, intensity, and duration of their training and maintain their training schedules consistently throughout the study. However, adherence to instructions is not always high and it is possible that supplementary training affected the athlete’s ability to perform maximally. 4.3. Blood Gas Responses to Sprint Training in Hypoxia and Normoxia A signiﬁcant decrease in pCO2 and pO2 was observed in the hypoxic conditions. However, this response did not differ between the training protocols. A decrease in pCO2 is in accordance with a left shift of the oxyhemoglobin curve, which is typically observed following acute exposure to hypoxia [33]. The increase in ventilation, and, resulting respi- ratory alkalosis, are the body’s attempt to increase O2 saturation in an environment with reduced oxygen availability. This reduction in pCO2 may also reﬂect increased buffering activity, whereby, in an attempt to maintain a stable pH, the body will employ various mechanisms to buffer and maintain its acid-base balance. However, given the fact that HCO3−did not decrease signiﬁcantly, ventilation may have played a greater role in the pCO2 response. Nonetheless, it is more likely that both contributed to this response. On the other hand, a decrease in mixed-venous pO2 is a typical response to high-intensity exercise, as oxygen consumption increases at the tissue level. The magnitude of this response is likely a reﬂection of hypoxic conditions. It is also important to note that in normoxia, the mean post-exercise O2 values increased (non-signiﬁcantly) compared to pre-exercise. Elevated pO2 levels following exercise are indicative of hyperventilation and are congruent with our pCO2 results [25]. The reason why pO2 decreased in hypoxia is unclear and may be related to the magnitude of the hypoxic stimulus delivered [34]. Similarly, sO2 signiﬁ- cantly decreased in hypoxia compared to normoxia, independent of the training session. This supports the literature, which has established that sO2 decreases acutely in hypoxia, reﬂecting the lower FiO2 and the increase in oxygen extraction during exercise [14,25]. g yg g While pH did not reach statistical signiﬁcance, the mean post-exercise values suggest that metabolic acidosis (pH < 7.35) was present or very close in three of the training protocols (HYP12 × 5, NOR3 × 20, and HYP3 × 20) but not in NOR12 × 5 (pH = 7.39) [35]. This is possibly a result of the body’s inability to eliminate or buffer waste metabolites or [BLa+], thereby leading to the accumulation of the measured metabolites and a decrease in pH [36]. Given the fact that one important adaptation induced from training in hypoxia is an increase in resting pH, it is possible that this acute response may indicate that this protocol provided an adequate stressor to stimulate changes. These results clearly demonstrate that Int. J. Environ. Res. 4.6. Future Directions and Practical Application Ultimately, our results suggest that sprint training in hypoxia elicits a greater physio- logical response compared to training in normoxic conditions, irrespective of the training protocol. However, athletes found HYP12 × 5 more difﬁcult, despite recording similar phys- iological responses to other sessions. Understanding the reasons why athletes’ perceived rate of exertion was higher in this training protocol, while eliciting a similar physiological response, has important implications for training prescription. Special care should be taken when selecting a protocol that is applicable to the sport in question (i.e., which considers the demands of the sport) and one that considers the contribution of metabolic systems. Lastly, given the cross-sectional design of this study, we are only able to comment on acute physiological responses. Future research should explore the physiological response and adaptation to different training protocols in hypoxia using a longitudinal study design. 9 of 11 Int. J. Environ. Res. Public Health 2022, 19, 2607 5. Conclusions Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Böning, D. Altitude and Hypoxia Training—A Short Review. Int. J. Sports Med. 1997, 18, 565–570. [CrossRef] [PubMed] 2. Wehrlin, J.P.; Zuest, P.; Hallén, J.; Marti, B. Live high-train low for 24 days increases hemoglobin mass and red cell volume in elite endurance athletes. J. Appl. Physiol. 2006, 100, 1938–1945. [CrossRef] [PubMed] 2. 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Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijerph19052607/s1, Figure S1: Study Timeline. Author Contributions: All authors contributed to this study. Conceptualization: N.M.-R., D.J.B. and H.M.L.-S.; methodology: N.M.-R. and D.J.B.; data collection: N.M.-R.; writing—original draft preparation: N.M.-R.; writing—reviewing and editing: N.M.-R., D.J.B. and H.M.L.-S.; supervision: D.J.B. and H.M.L.-S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: The Research Ethics Board at the Canadian Sport Institute Ontario approved the study (REB#18-01). Institutional Review Board Statement: The Research Ethics Board at the Canadian Sport Institute Ontario approved the study (REB#18-01). Institutional Review Board Statement: The Research Ethics Board at the Canadian Sport Institute Ontario approved the study (REB#18-01). Informed Consent Statement: Parents (assent was required) and participants were informed both verbally and in writing of the experimental protocol and potential risks before giving their verbal and written assent and consent, respectively, to participate. Written informed consent has been provided by all participants to publish this paper. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy restrictions. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy restrictions. Acknowledgments: We would like to thank the University of Toronto, Faculty of Kinesiology and Physical Education and the Canadian Sport Institute of Ontario (CSIO) for their support. Acknowledgments: We would like to thank the University of Toronto, Faculty of Kinesiology and Physical Education and the Canadian Sport Institute of Ontario (CSIO) for their support. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Geiser, J.; Vogt, M.; Billeter, R.; Zuleger, C.; Belforti, F.; Hoppeler, H. Training High—Living Low: Changes of Aerobic Performance and Muscle Structure with Training at Simulated Altitude. Int. J. Sports Med. 2001, 22, 579–585. [CrossRef] 27. Fulco, C.S.; Rock, P.B.; Cymerman, A. Maximal and submaximal exercise performance at altitude. Aviat. 69, 793–801. Available online: http://www.ncbi.nlm.nih.gov/pubmed/9715971 (accessed on 25 April 28. Ventura, N.; Hoppeler, H.; Seiler, R.; Binggeli, A.; Mullis, P.; Vogt, M. 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https://openalex.org/W4312736016	https://research.rug.nl/files/101556023/Beumer2019_Article_HowToIncludeSocio_economicCons.pdf	English	null	How to Include Socio-Economic Considerations in Decision-Making on Agricultural Biotechnology? Two Models from Kenya and South Africa	null	2,022	cc-by	15,278	University of Groningen How to include socio-economic considerations in decision-making on agricultural biotechnology? Beumer, Koen Published in: Agriculture & Human Values DOI: 10.1007/s10460-019-09934-1 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2019 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Beumer, K. (2019). How to include socio-economic considerations in decision-making on agricultural biotechnology? Two models from Kenya and South Africa. Agriculture & Human Values, 36(4), 669-684. https://doi.org/10.1007/s10460-019-09934-1 University of Groningen How to include socio-economic considerations in decision-making on agricultural biotechnology? Beumer, Koen Published in: Agriculture & Human Values DOI: 10.1007/s10460-019-09934-1 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2019 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Beumer, K. (2019). How to include socio-economic considerations in decision-making on agricultural biotechnology? Two models from Kenya and South Africa. Agriculture & Human Values, 36(4), 669-684. https://doi.org/10.1007/s10460-019-09934-1 University of Groningen How to include socio-economic considerations in decision-making on agricultural biotechnology? Beumer, Koen Published in: Agriculture & Human Values DOI: 10.1007/s10460-019-09934-1 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2019 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Beumer, K. (2019). How to include socio-economic considerations in decision-making on agricultural biotechnology? Two models from Kenya and South Africa. Agriculture & Human Values, 36(4), 669-684. https://doi.org/10.1007/s10460-019-09934-1 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Citation for published version (APA): Beumer, K. (2019). How to include socio-economic considerations in decision-making on agricultural biotechnology? Two models from Kenya and South Africa. Agriculture & Human Values, 36(4), 669-684. https://doi.org/10.1007/s10460-019-09934-1 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Abstract This article contributes to the debate about how regulatory science for agricultural technologies can be ‘opened up’ for a more diverse set of concerns and knowledges. The article focuses on the regulation of ‘socio-economic considerations’ for genetically modified organisms. While numerous countries have declared their intent to include these considerations in biotechnology decision-making, it is currently unclear both what counts as a socio-economic consideration and how such considerations should be assessed. This article provides greater clarity about how socio-economic considerations can be included in regulations by drawing upon the experience of two countries whose efforts in this field are particularly advanced: Kenya and South Africa. Based on extensive fieldwork, this article identifies the contours of an emerging regulatory regime by presenting two practice-based models for including socio-economic considerations in biotechnology decision-making. Whereas Kenya has taken a bottom-up process prior to assessing the first technologies and strongly emphasises scientific expertise, South Africa has instead established regulatory standards in an ad hoc fashion on a case-to-case basis, with a less prominent role for scientific evidence. The discussion of the distinct characteristics and tensions of both models provides insight into two potential pathways for including socio-economic considerations in the regulation of agricultural technologies. GMO · Regulation · Socio-economic considerations · Genetic modification · Kenya · South Africa Abbreviations ACB African Centre for Biodiversity ARC Agricultural Research Council Bt Bacillus thuringiensis CBD Convention on biological diversity COP-MOP Conference of Parties serving as the meeting of the Parties to the Protocol CPB Cartagena Protocol on Biodiversity DEAT Department of environmental affairs GMO Genetically modified organism KARI Kenyan Agricultural Research Institute NACBAA National Advisory Committee on Biotech- nology Advances and their Applications Abbreviations ACB African Centre for Biodiversity ARC Agricultural Research Council Bt Bacillus thuringiensis CBD Convention on biological diversity COP-MOP Conference of Parties serving as the meeting of the Parties to the Protocol CPB Cartagena Protocol on Biodiversity DEAT Department of environmental affairs GMO Genetically modified organism KARI K A i l l R h I i NBA National Biosafety Authority NGO Non-governmental organization SAGENE South African Committee for Genetic Experimentation SEC Socio-economic considerations SOP Standard operating procedure SPS Sanitary and phytosanitary measures USAID United States Agency for International Development WTO World Trade Organization How to include socio‑economic considerations in decision‑making on agricultural biotechnology? Two models from Kenya and South Africa Koen Beumer1,2 Accepted: 23 March 2019 / Published online: 3 April 2019 © The Author(s) 2019 Accepted: 23 March 2019 / Published online: 3 April 2019 © The Author(s) 2019 University of Groningen How to include socio-economic considerations in decision-making on agricultural biotechnology? Beumer, Koen Published in: Agriculture & Human Values DOI: 10.1007/s10460-019-09934-1 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2019 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Beumer, K. (2019). How to include socio-economic considerations in decision-making on agricultural biotechnology? Two models from Kenya and South Africa. Agriculture & Human Values, 36(4), 669-684. https://doi.org/10.1007/s10460-019-09934-1 Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Agriculture and Human Values (2019) 36:669–684 https://doi.org/10.1007/s10460-019-09934-1 Introduction One of the recurring criticisms of regulatory science is that it functions to erroneously ‘close down’ the appraisal of food and agricultural technologies (Stirling 2008). The starting point for these critiques is the recognition that discussions about new food and agricultural technologies are “not just about the pros and cons of a particular set of technologies, but about politics and values and the future of agrarian society” (Scoones 2008). From this perspective, regulatory :(012345 1 3 (0121 3456789) 3 670 K. Beumer science has been criticized for predominantly framing issues in terms of risk to human health and the environment. While these issues are certainly important, the predominant focus on risks to human health and the environment has been criti- cized for narrowing down the scope of societal appraisal, and for relegating issues of risk to the realm of specialists, thereby excluding other types of knowledge and expertise from the decision-making process (Levidow and Carr 1997; Chataway and Tait 1993; Wynne 2001, 2005; Jasanoff 2005). The issue is ‘scientized’, as one commentator described it (Kinchy 2010). for SEIA [socio-economic impact assessment].” Hence it remains unclear what impacts should be considered under Article 26, how such impacts should be assessed, and how decisions should eventually be reached. Actors across the board have cited this lack of clarity a cause of concern, amongst others because of fears that SECs will only act “as justifications for the imposition of trade barri- ers” (Kerr et al. 2014) and because the ensuing regulatory uncertainty may deter investments in potentially beneficial technologies. This article aims to provide greater clarity about how SECs can be regulated based on ethnographic observations in two countries whose efforts in this field are particu- larly advanced: Kenya and South Africa. In both coun- tries, there are significant societal controversies around GMOs, often revolving around socio-economic impacts of the technology, thus sparking the need for including these considerations in decision-making processes. The promi- nence of African countries in this endeavour fits within a larger trend in that was already visible during the negotia- tions for the Cartagena Protocol, when most developed countries argued that SECs should not be included because they could better be left to market conditions and hence were “of little relevance” to the Cartagena Protocol (Secre- tariat of the CBD 2003, p. 79), while African countries on the other hand presented the most comprehensive submis- sion of socio-economic factors. Introduction Although several devel- oped nations have meanwhile taken pro-active attempts to include SECs (for instance see COGEM (2009) for the Netherlands and Binimelis and Myhr (2016) for Norway), it is hence not surprising that Kenya and South Africa, frontrunners in African biotechnology, have been particu- larly advanced in shaping regulatory practices for SECs. y This article takes forward these criticisms and investi- gates attempts to expand regulatory science and include different types of concerns and knowledges. In particular, I focus on the regulation of ‘socio-economic considerations’ (SECs) for genetically modified organisms. The concept ‘socio-economic considerations’ emerged in biotechnology regulation in the context of the Cartagena Protocol on Bio- diversity (CPB), an international agreement that, amongst others, regulates the cross-border movement of genetically modified organisms (GMOs). Article 26 of the protocol provided countries the option to include “socio-economic considerations arising from the impact of living modified organisms” (Secretariat of the Convention on Biological Diversity 2000, p. 19). The ethical, social, and economic consequences of genetically modified organisms has been subject to intense societal controversy over the last decades, and Article 26 for the first time provides countries with the possibility to take into account these widely perceived soci- etal concerns into their regulations. Numerous countries have meanwhile declared their intent to include these con- siderations in biotechnology decision-making for regulating both the import and domestic development of genetically modified organisms, including Argentina, China, Mexico, the Philippines, the Netherlands, Norway, Kenya, and South Africa (Falck-Zepeda et al. 2013). y p g g y p By providing a detailed account the work done under Article 26 of the Cartagena protocol at the national level, this article identifies the contours of an emerging regula- tory regime. Based on extensive fieldwork in Kenya and South Africa, I will identify two practice-based models for including socio-economic considerations in biotech- nology decision-making. Kenya and South Africa have taken different approaches to settle what SECs should (and should not) be taken into account, how these considera- tions should be assessed, and how decisions are eventually reached. These experiences are set to become even more important as a new wave of technological developments in the field of genetic editing will raise new questions about the ethical, societal, and economic consequences of biotechnology. Socio‑economic considerations as a new object of regulation However, despite these efforts, and despite the fact that numerous countries have pledged to include SECs in their decision-making processes, these attempts are hampered because as of yet “there is little experience in dealing with these issues in actual decision- making processes” (Fransen et al. 2005, p. 7). Even in a country like Norway, which is often referred to as having a progressive system for including SECs, thus far did not experience cases where SECs played a decisive role in decision-making (Rosendal and Myhr 2009). Despite these efforts, overall little consensus has emerged over the defini- tion and scope of SECs, the way SECs should be assessed, and the distribution of roles between different stakeholders in the decision-making process. “may take into account, consistent with their inter- national obligations, socio-economic considerations arising from the impact of living modified organisms on the conservation and sustainable use of biologi- cal diversity, especially with regard to the value of biological diversity to indigenous and local commu- nities” (Secretariat of the CBD 2000, p. 19). Although the Cartagena Protocol focused on the trans- boundary movement of GMOs, discussions and regula- tions of SECs soon transcended this focus. As we will see later, also in Kenya and South Africa the regulation of SECs is not limited to transboundary movements. Never- theless, it was the adoption of the Cartagena Protocol that spurred debate about the definitions and scope of SECs— i.e. about what counts as a valid SECs. In the most narrow int erpretation, SECs only apply to the impact genetically modified organisms on the value of biological diversity to indigenous and local communities, as is described in Article 26 (Jaffe 2005). In its broadest interpretation, a wide variety of concerns can be shared under the heading of SECs. RAEIN-Africa (2012), COGEM (2009), Lud- low et al. (2014) and Fransen et al. (2005) for instance mention economic impacts such as the costs of inputs and labour, the impact on employment, food supply, and on recreation; social impacts such as freedom of consumer choice, equity issues along the lines of gender and age, and religious beliefs; and cultural impacts such as traditional seed exchange systems, cultural uses of biodiversity, and cropping practices associated with indigenous knowledge systems. All these considerations seem to share, is that their object of regulation is not risks to human health and the environment. Socio‑economic considerations as a new object of regulation Questions have furthermore been raised about how SECs should be assessed and how decisions should eventually be reached. Various scholars and organizations have for exam- ple discussed the benefits and drawbacks for ex ante or ex post assessments (Falck-Zepeda 2009) and the importance of carefully manage the expectations arising from regula- tory frameworks (COGEM 2014). Also different methodolo- gies have been proposed for performing these assessments, ranging from economics-oriented methods like cost–benefit analysis (Hall and Moran 2003), social-economic impact analysis (Stabinsky 2000), and effectiveness tests (Chatur- vedi et al. 2012), to broader frameworks that include dif- ferent types of public participation (Fransen et al. 2005), scenario planning methodologies (AHTEG-SEC 2014), and problem formulation tools (Tepfer et al. 2013). Socio-economic considerations emerged in a context where the public debate about agricultural biotechnology continuously ‘overflowed’ the confines of the regulatory focus on risks to human health and the environment. The concept of ‘socio-economic considerations’ can be under- stood as a response to this perceived gap between the nar- row focus on risks in regulation and the broader concerns that are expressed in public debates. The term entered bio- technology regulations as a compromise in negotiations in the context of the Cartagena Protocol on Biodiversity. As countries were unable to reach consensus over the inclusion of criteria other than those concerning risk and trade, the compromise was reached to provide countries the option to include SECs in decision-making (Secretariat of the CBD 2003). This was articulated in Article 26 of the Protocol, noting that signatories of the Protocol: There has hence been quite some work that enables coun- tries to shape their regulations for SECs. There have been a few attempts to sketch the broad outlines of existing regula- tory schemes that include SECs (COP-MOP 2008; Falck- Zepeda 2009; Falck-Zepeda and Zambrano 2011; Binimelis and Myhr 2016), including for example Argentina, where the assessment of SECs is limited to impacts on trade, which are assessed using principles from cost–benefit analysis. And the regulation of SECs has also repeatedly received a prominent place on the agenda of the Conference of Parties serving as the meeting of the Parties to the Protocol (COP- MOP), the European Committee, and the Food and Agricul- ture Organization, amongst others. Introduction These two models, along with a critical discussion of their benefits and drawbacks, can provide guidance to other countries by highlighting different strat- egies for dealing with the practical difficulties of including SECs into biotechnology decision-making. In practice, however, it is far from clear what it means to include SECs in decision-making. For example there are vast differences in the definition of socio-economic considerations. While narrow interpretations limit the definition of SECs to economic effects on farmers rela- tive to existing crops, wider definitions include anything from impacts on gender, income, indigenous communi- ties, and even cultural and religious concerns. Further- more, traditionally the regulation of GMOs has heavily focused on sanitary and phytosanitary measures and the possibility to include SECs in decision-making—rather than leaving these to the market—is a rather new devel- opment. As a consequence, not only is there considerable lack of clarity as to what counts as a valid socio-economic consideration, also, as RAEIN-Africa (2012, p. 1) notes, “whilst methodology for environmental and health risk assessment is well-developed the same cannot be said 1 3 3 3 671 How to include socio-economic considerations in decision-making on agricultural… Theoretical and methodological approach The practice-based approach taken in this article can best be explained by contrasting it to what will be called a ‘tradi- tional’ approach to regulation. In this traditional approach, a strong distinction is made between the ‘science-based’ regulation of health and environmental risks and the sup- posedly ‘non-scientific’ nature of regulations based on other concerns, like socio-economic considerations. The scientific basis of regulations of health and environmental risks is for instance articulated in the WTO agreement on the applica- tion of Sanitary and Phytosanitary Measures (SPS), which specifies that trade-curtailing measures may only be taken if “based on scientific principles” and “sufficient scientific evidence” (WTO 1995). From this perspective, including SECs in decision-making potentially opens the floodgates to various ‘non-scientific justifications’ that hamper trade. j p g Socio-economic considerations, in contrast, present regu- lators with a largely new concern for which it has not yet been crystallized what work is required to take them into account in decision-making. From this perspective, the dif- ference between objective/science-based/predictable regula- tion of risks and subjective/politics-based/unpredictable reg- ulation of SECs comes into view as the difference between a practice that is well-established and a practice that is only emerging. Although there is certainly no guarantee that the regulation of SECs will travel the same path as SPS, the practice-based perspective helps to highlight that in theory, SECs too can evolve over time from subjective to objective and from unpredictable to predictable. There are no inherent properties to SECs as an object of regulation that prevent a potential consensus to emerge over what counts as a valid SEC and how this can best be measured (Table 1). ii This view is founded upon the assumption that the objec- tive or subjective nature of regulatory practices are somehow inherent to the regulated concern. In this reading the very nature of SECs makes it impossible for them to be regulated in an objective and predictable manner because they amount to mere opinions, values, and political interests that cannot be dealt with through science. This article builds upon sev- eral authors who have earlier rejected this seemingly clear distinction. Kleinman and Kinchy (2007) for instance point out that this distinction itself rests upon views about the con- ditions under which circumvention of the market is deemed legitimate that are themselves informed by neoliberal ideol- ogy. Socio‑economic considerations as a new object of regulation In the absence of consensus over what counts as valid socio-economic considerations and how to assess them, countries are caught in between seemingly mutually exclu- sive desires. On the one hand, these countries wish to broaden the scope of valid regulatory concerns to include issues beyond risks to human health and the environment. Doing so, on the other hand, risks ruling out potentially ben- eficial technologies since SECs “may constitute an unwork- able hurdle if the assessment procedure is not clearly defined up front” (Falck-Zepeda 2009). Failing to define what SECs are taken into considerations and how these can best be assessed could bar potentially beneficial technologies to 1 3 672 K. Beumer as a consensus that has slowly evolved over course of the last centuries (Luhmann 1993; Boholm 2015), reflecting both the value that is attributed to human health and the environment, the objects that are considered to pose a risk to those values, and the particular types of knowledge that are considered credible (e.g. Porter 1996; Jasanoff 2005). enter countries and could hamper technological progress as it dis-incentivizes researchers to put time and effort in developing new agricultural biotechnologies that may later be rejected. This article suggests one way to overcome this conun- drum is to take a practice-based approach to regulation that draws on literature from the sociology of science and tech- nology. This will be set out in the next section. f This perspective furthermore draws attention to all the practices involved in producing the knowledge about the object of regulation that enables objective forms of decision- making (de Vries et al. 2011; Boholm 2015; Beumer 2017). For instance, in the case of risks to human health and the environment thousands of people work to isolate relevant properties, administer those in a particular dosage, carefully selected animal models, whose exposure to the risk object is in turn measured after a particular period, using care- fully calibrated machines, operated by staff with the right educational background, who look for damage done by the risk object in predetermined organs in the animal models. 3 Agricultural biotechnology regulation Researchers in Kenya have been working on genetic modi- fication since at least the early 1990s and the government has taken several steps to promote and regulate the tech- nology since. In 1991, the National Advisory Commit- tee on Biotechnology Advances and their Applications (NACBAA) was established to identify research priorities and 1 year later the Kenyan Agricultural Research Institute (KARI) formulated the first biosafety guidelines (Wafula 1995). More recently the government approved a National Biotechnology Development Policy in 2006, outlining how Kenya could move ahead with biotechnology, and in 2009 the Biosafety Act was approved by the Kenyan president. These events have been accompanied by significant resist- ance, culminating in the 2012 imposition of a moratorium on the import of GMOs. Interviews focused on actors that were closely involved in the regulation of SECs so as to gain a detailed understanding of the process through which particular considerations were included or excluded from the regulatory process and how decisions were taken to measure and assess these considera- tions in particular ways. These interviews were supplemented with several other interviews with key actors who have been active in discussions about biotechnology regulation but who were less closely involved in the regulation of SECs. This helped to gain an alternative perspective on the events described by those closely involved in the regulatory process. Interviewees were first identified through a preliminary docu- ment analysis and internet search and thereafter also by asking interviewees once in Kenya and South Africa to suggest names of other relevant actors. The 2009 Biosafety Act established the structures through which SECs were first explicitly addressed. The Act foresaw the establishment of a new competent author- ity that was charged with the responsibility to implement the Act. This institute was called the National Biosafety Authority (NBA) and was placed under the Ministry of Science and Technology. The decision-making power is situated with the board of the biosafety authority, which by law consists of the chairperson and chief executive officer, along with scientists, interest groups, and representatives of various ministries and government agencies. The Act set forth different requirements for the contained use, environmental release, import, export, transit, and plac- ing on the market of genetically modified organisms. SECs were solely included in the approval for the environmental release of genetically modified organisms. Theoretical and methodological approach Agreement over what counts as a risks and how to measure that risk is not a given that is implicated in the nature of the object of regulation: it instead comes into view 3 How to include socio-economic considerations in decision-making on agricultural… 673 How to include socio-economic considerations in decision-making on agricultural… 673 How to include socio-economic considerations in decision-making on agricultural… Table 1 Differences in framing the regulation of risks and SECs Sanitary and phytosanitary measures Socio-economic considerations Traditional frame Science-based Politics-based Objectivity follows from nature of object of regulation Subjectivity follows from nature of object of regulation Practice-based frame Product of both science and politics Product of both science and politics Well-established practice Newly emerging practice Objectivity product of agreement over what constitutes valid evidence Subjectivity product of agreement over what consti- tutes valid evidence Socio-economic considerations Politics-based Subjectivity follows from nature of object of regulation Product of both science and politics Newly emerging practice Subjectivity product of agreement over what consti- tutes valid evidence Politics-based Subjectivity follows from nature of object of regulation Product of both science and politics Newly emerging practice Subjectivity product of agreement over what consti- tutes valid evidence the regulations, as well as scientists and companies filing applications, peer reviewers, and local and international non- governmental organizations. The interviews were conducted in the home institute of the interviewee (in and around Nairobi and Eldoret in Kenya, in and around Pretoria, Onderstepoort, Midrand, Johannesburg, Cape Town, Somerset West and Stel- lenbosch in South Africa) and they were fully transcribed. The semi-structured nature of the interviews allowed for compari- sons between interviewees and countries while their open- ended nature was essential in capturing regulatory practices that are not formalized in written documents and allowed the analyst to go further into detail when merited. in the process of assessing concrete applications. The empiri- cal sections mirror these differences and hence are structured rather differently. Theoretical and methodological approach The attempt to entrench this ideologically informed dis- tinction by characterizing the regulation of risks as scientific and delegating all other forms of regulation as unscientific is a form of scientism that cannot be reasonably be defended (Kinchy et al. 2008). This has consequences for the purpose of identifying the contours of a new regulatory regime. If the establishment of what counts as a valid SECs and what counts as valid evidence is the outcome of hard work, then the contours of the new regulatory regime can best be identified by observ- ing the practices through which actors attempt to define and measure SECs. The question hence is what evolving prac- tices can be observed through which SECs as an object of regulation are made sufficiently objective and predictable? fi This article takes an ethnographic approach, which is par- ticularly suited to investigate emerging practices that have not yet been fully formalized. I mainly rely upon two types of sources to identify relevant regulatory practices: semi-struc- tured qualitative interviews and written documents. I spent a total of 3 weeks in Kenya in March 2016 and 3 weeks in South Africa in April 2016 to conduct semi-structured qualita- tive interviews with a total of 39 people. This included inter- views with government officials from different regulatory agencies and ministries, actors directly involved in drafting This article instead adopts a practice-based approach to regulation. This perspective helps to show that differences in objectivity or subjectivity (and predictability or unpredict- ability) of different objects of regulation are the outcome of particular practices through which actors either manage or fail to agree on what constitutes valid conditions for market intervention. Agricultural biotechnology regulation The Act notes that in reaching its decision, the Authority “shall take into The documents were gathered through an elaborate inter- net search for which both academic and non-academic search engines were used (including EBSCOHOST, Google Scholar, and Google). Additional documents were gathered during field work, amongst others by asking interviewees if they could identify further relevant documents. Both documents were gathered that play a role in the regulatory process itself (such as laws, regulations, or guidelines) and documents that com- ment upon the process (such as academic studies, newspaper articles, or organizational websites). Together the interviews and documents provide a good base for triangulating findings and tracing the emergence of a regulatory regime for SECs. It was found that the regulation of SECs in Kenya was established largely separate from concrete applications whereas in South Africa the regulatory practices only emerged 1 674 K. Beumer send the applications to for review to look for” (interview 31 March 2016a). Initially the NBA board considered drafting the guidelines themselves, ensuring that the SECs consid- ered “should not be varying from one applicant to another” (interview 17 March 2016a). As an example, a former board member pointed to the highland area of Kitale, where the bulk of maize in the country comes from, and suggested that “you can say for Kitale … we want that area to be free of any issues from GM” (interview 17 March 2016a). Instead the NBA opted for a more participative approach to develop the standard operating procedure by organizing a workshop that was meant to solicit views from various stakeholders about what SECs had to be included and how they should be assessed. account … socio-economic considerations arising from the impact of the genetically modified organism on the environment” (Government of Kenya 2011). As one civil servant highlighted, whereas the Cartagena protocol stated that parties may consider socio-economic factors, “when you look at our biosafety law, it says it is a mandatory thing” (interview 31 March 2016a). The newly established National Biosafety Authority was charged with the responsibility to shape the regulation of SECs. The application form In order to decide whether or not a genetically modified crop will be approved for environmental release, the National Biosafety Authority requires applicants to provide infor- mation about the crop. Applicants therefore have to fill in a form consisting of a long list of detailed questions. The application process thereby “passes the ball to those who have developed the product” (interview 18 March 2016). SECs are not included in the form itself but applicants are obliged to provide information on socio-economic impacts in an annex (interview 31 March 2016a). The Biosafety Act itself does not specify what SECs should be taken into consideration. As one industry actor told me, the passage about SECs in the Biosafety Act “is just a general statement that is put there but digging deeper into guidelines and everything else, there’s no other place you’ll find when looking into socioeconomic considerations” (interview 31 March 2016b). Different actors perceived this as problematic. For applicants this meant that there was lit- tle clarity as to whether or not the Act’s requirements had been met. And regulators did not trust that applicants would provide sufficient and credible information in the absence of more specific criteria for providing information on SECs. As one former board member of the NBA mentioned: “they will give you biased information. An applicant will never tell you that our product is going to affect the choice of varieties from farmers, or that it’s going to take away from a variety which has medicinal value” (interview 17 March 2016a). Eventually “it was up to the NBA to decide what to take and what not to take” (interview 18 March 2016). Issues that were occasionally mentioned in literature on SECs but that were not brought up during the stakeholder workshop, like the impact on local seed systems or indigenous knowledge, were not included. Facing the lack of consensus amongst workshop participants, the biosafety authority simply included all SECs that were raised during the stakeholder workshop. The scope of the standard operating procedure was subsequently very broad, ranging from issues concern- ing equal access of men and women to farmers income, and from enhanced nutritional composition (see Table 2). The biosafety authority therefore set out to create a stand- ard operating procedure (SOP) for SECs. Agricultural biotechnology regulation SECs were eventually included in the applica- tion procedure for environmental release in three distinct moments: first, through information provided by the appli- cant, which is reviewed by socio-economic experts; sec- ond, through public comments that are invited after every application; and third, in the final decision taken by the board of the biosafety authority about the environmental release. These three moments will now be discussed in greater detail. The workshop was facilitated by a well-known interna- tional expert on SECs and was attended by about 25 different stakeholders, from scientists to consumer organizations, and from industry to environmental NGOs (interview 18 March 2016). Both participants and the organizers of the workshop mentioned that the participants failed to reach consensus about how to regulate SECs. This was hardly surprising con- sidering the antagonistic biotechnology debate in Kenya. Even the format of the workshop was subject to contestation. Actors from civil society organizations for instance noted that the international expert facilitating the workshop “is known to be a pro-GM person, … and that in itself tells me NBA is not interested in being impartial (interview 17 March 2016b). In his introductory presentation the expert furthermore put a strong emphasis on economic issues, in particular on “comparing the economic benefit of growing Bt maize vis-à-vis growing normal hybrid maize” (interview 17 March 2016b), thereby foreclosing discussions on non- economic issues “like indigenous varieties, co-existence, … and the impact on non-target organism like butterflies and bees” (interview 17 March 2016b). Yet, despite the fact that “it was a wide group of people with various opinions” (interview 18 March 2016), as one biotechnology proponent tactfully noted, different participants that were interviewed noted that they were nevertheless able to openly put forward what SECs they thought should be included in the decision- making process (NBA 2013). 1 For instance a question like “are seeds and other farm inputs afford- able to ordinary farmers” may include information about the seed price, over which the applicant may have a certain amount of con- trol, but could also include information about other factors affecting the economic situation of farmers, since this may determines what is affordable for farmers. Similarly, a question like “will other technolo- gies be available to guarantee freedom of choice” may include infor- mation about other commodities currently offered by the applicant but could also include an assessment of government policies and the strategies of competitors that structure this availability. The application form This document, in the words of one NBA employee, was supposed to provide “a guideline on what issues as NBA we want applicants to report, [and on what issues we want] the experts that we 1 3 3 3 How to include socio-economic considerations in decision-making on agricultural… How to include socio-economic considerations in decision-making on agricultural… 675 Table 2 Socioeconomic consideration criteria in the standard operating procedure Concept Elements/criteria Food security and sustainability Will there be increase in yield/ha? Will there be surplus for individuals/country? Will the technology be con- tinuous? Will technology complement other income sources? Access to the technology Will other technologies be available to guarantee freedom of choice? Will SEC increase time and cost of regu- latory approvals hence delaying its adoption? Gender issues: women versus men Income to farmers Will farmers make more money using GM technology compared to status quo? Will cost–benefit analysis be beneficial to farmers? Cost of seeds and other inputs Are seeds and other farm inputs affordable to ordinary farmers? Does the technology reduce cost of produc- tion? Are there economies of scale? Co-existence How to ensure genetic integrity on organic and conventional food? How to deal with litigation? Trade implications How approval of GM technology will impact on exports and other trade factors? Will Kenyan products access EU market? Are we going to be competitive? How will the technology affect our branding? Will it affect surpluses too? Benefits and freedom of choice Any tangible benefits to consumer? - Reduced commodity prices, enhanced nutritional composition? Is the society healthier? Is there good labelling practices? Can consumers easily access conventional or organic products? Biosafety and stewardship What happens if adverse effects are detected later when the product is in the market and people have consumed the GMO? Will farmers be trained and follow laid down GAPs? Will there be increase in yield/ha? Will there be surplus for individuals/country? Will the technology be con- tinuous? Will technology complement other income sources? Any tangible benefits to consumer? - Reduced commodity prices, enhanced nutritional composition? Is the society healthier? Is there good labelling practices? Can consumers easily access conventional or organic products? What happens if adverse effects are detected later when the product is in the market and people have consumed the GMO? Will farmers be trained and follow laid down GAPs? The application form Significantly, the standard operating procedure takes the form of a series of questions, 25 in total. The questions are explicitly meant to provide guidance for applicants and regu- lators about the type of issues that will be assessed. How these questions should be answered thus remains open, since the document specifies neither the information nor the meth- ods required for answering these questions (with the possible exception of cost–benefit analysis).1 The standard operating procedure furthermore does not define clear benchmarks of what socio-economic impacts are considered desirable or unacceptable. The senior NBA official strongly emphasized this point: “Very importantly, if you remember the SOP, … the document is not saying ‘if it is so high, then it’s not acceptable’. There are questions that do not have limits as to how much” (interview 31 March 2016c).f should not influence my decision and … because if I recom- mend something negative then they can come for my neck” (interview 16 March 2016). The reviewers assess whether the information provided by the applicant is correct and whether all relevant information is provided. In reviewing the information on SECs, the experts contin- uously balance the ambitious assessment criteria with practi- cal limitations. On the one hand the requirements are very high. For instance in assessing the correctness of informa- tion provided, so one reviewer told me, evidence that has not been published in academic journals “may not be acceptable. … It’s just like I can sit down and write the paper” (inter- view 16 March 2016). And even if the application draws on evidence that has been published in a peer reviewed journal, the anonymous experts are still required to review the quality of the scientific evidence. Although one reviewer highlights that deciding whether the evidence is sufficiently convinc- ing is to some extent a subjective exercise, the reviewers generally follow basic scientific guidelines: “Was there a good sampling? Were the data collection methods good enough? Was the analysis good? Is the interpretation good? … It has to be good science” (interview 16 March 2016). Furthermore, in assessing the completeness of the informa- tion, reviewers are instructed by the NBA staff to “go beyond what the applicant has provided because the applicant some- times doesn’t have some information, or maybe they are shy- ing away from giving some information that they feel can limit the approval” (interview 31 March 2016a). Public consultation Next to the information that applicants have to provide, the second way that SECs enter the Kenyan decision-making process is through a public consultation. The Biosafety Act requires the NBA to inform the public about any applica- tion for environmental release in order to provide the public with the opportunity to voice their opinion and to “address appropriately any relevant concerns raised by such a person” (Government of Kenya 2011). Formally the Act requires the NBA to publish a summary of the application in the Kenya Gazette and at least two nationally circulated news- papers, after which concerns can be send to the NBA within 30 days (Government of Kenya 2011; interview 31 March 2016a). There are no restrictions on who can voice concerns, whether they are individual members of the public or repre- sentatives of public or private organizations. Nor are there restrictions as to what concerns can be raised during this The application form … We want consumers and producers to benefit” (inter- view 16 March 2016). The biosafety authority furthermore introduced an obliga- tory form for written responses only days before the dead- line for submitting comments, when most actors had already prepared their responses on their own terms. A civil society representative observed: “I think a lot still needs to be done in defining and actually organizing a meaningful public par- ticipation process for biotechnology” (interview 29 March 2016). In practice the reviewers strike a balance between the strict criteria of completeness and scientific validity on the one hand and practical limitations in providing that informa- tion on the other—a practice that raised no objections by any of the actors interviewed. The expert reviewers subsequently write a report, including a recommendation, and send this to the board of the biosafety authority. After receiving the public comments, staff from the biosafety authority analyzes them and makes a shift. Both NBA staff and outsiders mentioned that they proceed by first setting apart concerns and questions that can readily be answered. These public comments are addressed by the NBA itself. In all other cases the NBA asks the applicant to submit a written response to each of these issues. The public comments as well as the applicant’s reply are then sent to the NBA board, informing them that the “public listed the fol- lowing issues, then we took the issues to the applicant, and this is the response” (interview 31 March 2016a). The application form In reviewing the information on SECs, the experts contin- uously balance the ambitious assessment criteria with practi- cal limitations. On the one hand the requirements are very high. For instance in assessing the correctness of informa- tion provided, so one reviewer told me, evidence that has not been published in academic journals “may not be acceptable. … It’s just like I can sit down and write the paper” (inter- view 16 March 2016). And even if the application draws on evidence that has been published in a peer reviewed journal, the anonymous experts are still required to review the quality of the scientific evidence. Although one reviewer highlights that deciding whether the evidence is sufficiently convinc- ing is to some extent a subjective exercise, the reviewers generally follow basic scientific guidelines: “Was there a good sampling? Were the data collection methods good enough? Was the analysis good? Is the interpretation good? I h b d i ” (i i 16 M h 2016) After an application is submitted, the NBA staff screens whether the application is complete and sends the annex on SECs out for peer review. The review is done by social scientists specialized in socio-economic issues (hence not by biotechnology scientists). Their identities are kept confi- dential, as one reviewer said, “because I think other people … It has to be good science” (interview 16 March 2016). Furthermore, in assessing the completeness of the informa- tion, reviewers are instructed by the NBA staff to “go beyond what the applicant has provided because the applicant some- times doesn’t have some information, or maybe they are shy- ing away from giving some information that they feel can limit the approval” (interview 31 March 2016a). In practice, on the other hand, it is not always feasible to meet these stringent criteria. The expert reviewers take into account such practical limitations in forming their judgment. For instance a reviewer pointed out that they understand that studies on consumer perception could only provide rough 1 3 676 K. Beumer public consultation, including concerns about risks to human health and socio-economic considerations. indications since consumer behavior regularly changes after commodities enter the market. And several interviewees pointed out the difficulties in establishing the cross-national validity of socio-economic studies. The application form Also here reviewers con- sider the practical limitations of providing this information, noting that “it is preferable when we get continental data [from Africa], but if it is lacking then you can get other data from other countries” (interview 16 March 2016). Hence while preference is given to peer reviewed publications of the technology in question in Kenya, these are commonly not available, after which also evidence will be considered from similar technologies in the country, or otherwise of similar technologies in other countries. And despite the wide range of issues mentioned in the standard operating procedure, in practice the emphasis lies rather strongly on the economic impacts of the genetically modified crop. When asking a reviewer what information applicants have to provide, a reviewer noted: So far two applications for environmental release have gone through this procedure for a public hearing and in both cases the articulation of SECs by members of the public did not proceed without problems—an observation that was shared by the NBA itself. Several actors who wanted to sub- mit comments during the public hearing, especially from NGOs, for instance pointed out that it is not a simple task to go through the technical aspects of applications, especially “if you’re not a scientist” (interview 17 March 2016b), as an actor from an environmental NGO testified. And this task was made even harder because the application is not readily available: “The application is a big dossier, probably hundreds of pages. The National Biosafety Authority did not put this application document on their website. It was only available in their offices. … You can’t take it home, they couldn’t give you a copy, it was not available on their website” (interview 17 March 2016b). “They’re supposed to include reports to show that the biotechnology is going to generate more income for the farmers. … That’s the bottom line. … And then the other be on the consumer side they have to show that the consumers are willing to consume the product. … We want consumers and producers to benefit” (inter- view 16 March 2016). “They’re supposed to include reports to show that the biotechnology is going to generate more income for the farmers. … That’s the bottom line. … And then the other be on the consumer side they have to show that the consumers are willing to consume the product. The NBA board Finally, the board of the biosafety authority gathers all this information: the annex on SECs that is provided by the applicant, the report and recommendations of the expert reviewer, the comments from the public consultation, and the replies by the applicant. The NBA board is responsible for taking an overall decision about whether the application for environmental release is granted. The board first consid- ers the risks to human health and the environment. Once an application has met the criteria for sanitary and phytosani- tary impacts the board subsequently takes into account the 1 3 3 How to include socio-economic considerations in decision-making on agricultural… 677 different SECs that have been articulated by the applicant, the expert reviewers, and the public. of SECs in the standard operation procedure, this lack of transparency runs the risk of obscuring the potentially con- flicting social and economic interests that underlie the dif- ferent considerations. Crucially, thus far it has remained unclear how the board weighs SECs in the decision-making process. Both NBA staff members and current and former members of the board confirmed that such a procedure is currently missing. The preceding procedure for articulating and assessing SECs— the standard operating procedure, the application annex, the expert review and the public consultation—certainly shapes what considerations can and cannot be included by the board. But each of these steps is explicitly framed as pro- viding information to the board of the NBA, thereby situat- ing the final authority to include SECs into decision making with the board of the biosafety authority. Thus far two appli- cations have gone through the entire procedure—one for a Bt maize event and one for a Bt cotton event. While in both cases permission was granted for environmental release, it has remained curiously unclear what role socio-economic considerations played in reaching that decision, if any. In the absence of clarity about how the board reaches its final deci- sion, both applicants aiming to put GM crops on the market and actors concerned about the negative socio-economic impact of those GM crops are left in the dark as to what socio-economic considerations are decisive. The NBA board As for the evidence, although the expert reviewers for- mally require applicants to provide evidence published in peer reviewed scientific journals, it is unrealistic to expect that applicants can meet this criteria for all the questions listed in the standard operating procedure, nor does this cri- teria of scientific evidence clarify what kinds of evidence are deemed sufficient. The biosafety authority currently seems to take a pragmatic approach by using the criterion for sci- entific evidence more as an objective rather than a stringent requirement, accepting evidence from peer reviewed publi- cations on related technologies or on related countries, or even non-peer reviewed publications, in case peer reviewed publications on the technology in question in the Kenyan context is not available. It is not inconceivable that by tak- ing such a pragmatic approach, over time a consensus about the standards of evidence will emerge from the interaction between applicants, expert reviewers, public comments and the biosafety authority. Any such tacit agreement over the standards of evidence can only emerge when requests for additional information are balanced with the practical limita- tions and additional burden for applicants. Socio‑economic considerations in South African biotechnology The regulation of socio-economic considerations in Kenya was predominantly shaped by the National Biosafety Author- ity, who made a sustained effort to define what counts as a valid SEC through a bottom-up process, prior to assessing the first application. The biosafety authority formalized the selection of relevant SECs in a standard operating proce- dure and further shaped the process for assessing SECs so as to include input from socio-economic experts judging the completeness and robustness of the scientific information provided by the applicant, and to include public from other actors through a public consultation to which the applicants are invited to reply. Although the ways in which various actors are invited to participate is not without its problems, overall the process is designed with the intent to offer vari- ous moments for stakeholder inclusion. Agricultural biotechnology regulation Socio-economic considerations play a very different role in decision-making in South Africa. South Africa has been a pioneer in genetic modification on the South African continent. The country already had a regulatory body since 1979, the first field trials took place in 1987, and the first GMOs were allowed on the market in 1997, even if GMOs have been accompanied by significant soci- etal controversy up to this day. In 1999 the government enacted a GMO Act that established a new regulatory structure, that abolished the older regulatory body and established two new bodies: an Advisory Council consist- ing of biotechnology scientists that provides advice about the scientific validity of applications and an Executive Council with representatives from different government ministries. The Executive Council takes the final deci- sion and for applications to be granted, members of the Executive Council have to reach a consensus: if only one of the ministry representatives has doubts, applications will be refused. Despite these measures to ensure inclusion, expert review, and formalization, at this point it is not quite clear what evi- dence is required for the assessment and when SECs make a difference in the decision to approve or reject an application. It is thus far unclear what the thresholds are for SECs to make a difference in decision-making and how positive and negative socio-economic impacts are weighed against each other. This lack of transparency endangers the credibility of the regulatory procedure as there currently is no way of finding out whether SECs have indeed been taken into con- sideration. In combination with the seeming rationalization Both the SAGENE guidelines and the 1999 GMO Act were primarily concerned with risks to human health and 1 1 3 678 K. Beumer the environment and neither mentioned SECs. These first appeared in South African regulation in 2006, when the GMO Act was amended in order to align South African regulations to the Cartagena Protocol. The Act now notes that in reaching its decision, the Executive Council may consider “the potential socio-economic impact of such activities” (Republic of South Africa 2007, p. 11). And indeed SECs began to play an important role in decision- making around the same time. Even though South Africa is known as being rather permissive towards GMOs, they have nevertheless banned several genetically modified crops on socio-economic grounds, as we will see shortly. Agricultural biotechnology regulation taste of the wine and malolactic yeasts in particular are used by wine producers in the production of less acidic tasting wines like Chardonnay. Grapes naturally contain the acidic- tasting malic acid and the malolactic yeast converts malic acid into the softer-tasting lactic acid. In 2006, a group of Canadian researchers had genetically modified a yeast strain so as to perform both alcoholic and malolactic fermentation at the same time (Husnik et al. 2006), raising the interest of researchers at Stellenbosch University Institute for Wine Biotechnology. In 2006 they applied for field trials, claiming ‘proof of concept’ as the main purpose, and the Executive Council denied the application in September 2007 (EC min- utes of 18 September 2007).l crops on socio economic grounds, as we will see shortly. In the current regulatory procedure, like in Kenya, the applicant has to provide information on SECs when applying for environmental release, and like in Kenya, a public consultation forms part of this procedure. But other than that, the way SECs were included in South African decision-making strongly diverged from Kenya, and in this process, different conclusions were drawn about what counts as a valid concern and how this can best be measured. The information provided by the appli- cant is not send out for peer review, it is not assessed by socio-economic experts, there have been no stakeholder workshops to identify the most relevant considerations, and there is no Standard Operating Procedure to stipulate what information is required for what SECs, even though the 2006 amendment to the GMO Act noted that the Min- ister “may make regulations … prescribing the procedure to be followed by an applicant for the purpose of drawing up … socio-economic considerations” (Republic of South Africa 2007, p. 20). The possibility for contamination and gene flow to non- GM yeast varieties played an important role the rejection. Although the applicants argued that the trial site would be effectively contained by covering the grapevines with nets to prevent seed dispersal, both civil society organiza- tions and winemakers questioned the effectiveness of these measures (Jordan 2006). This gene flow was not considered important for maintaining biodiversity in yeast varieties: it was trade that was the central concern. The largest export markets for South African wineries were in Europe and it was perceived that the widespread resistance against geneti- cally modified crops in Europe could negatively affect South African exports. Agricultural biotechnology regulation Although the South African wine industry had long been supporting research into genetic modifica- tion, they were eventually not willing to take the risk. The head winemaker at Spier Estate, a large wine estate close to the envisioned test site, clearly formulated this when he said that “for us on the production side, GMOs are a no-go” (Jordan 2006). Instead the process for assessing SECs was established in an ad hoc fashion as the Executive Council looked at the considerations arising from new applications on a case-to-case basis. Hence the description of the South African regulation of SECs will take a different structure. There have been two instances in which SECs played a decisive role in deciding the faith of an application: the application for a GM yeast in 2007 and the application for a GM potato in 2009. It is through the assessment of these cases, that various standards were established for what concerns were and were not considered valid, and what evidence was deemed required to take an objective decision. In preparing its decision, the Executive Council sent the application to the Advisory Council, who recommended approval, and they received letters from both wine mak- ers and civil society organizations like Biowatch and the African Centre for Biodiversity (ACB). Although Biowatch also questioned the robustness of the risk assessment (ACB 2006), it was the socio-economic impact on trade that was the main concern of the Executive Council. Representa- tives from different ministries highlighted in the committee meeting that concerns existed surrounding the “impact on the marketability of the product, both within or outside the RSA” and the “demand for the product” (EC minutes of 15 May 2007). The Department of Trade and Industry sub- sequently sat down with the South African Wine Industry Council, the representative body of the wine industry, who clearly communicated: GM potatoes Whereas the Executive Council is usually rather scant in its comments, in this case it cited a wide range of reasons in its July 2009 decision. These reasons for rejecting the GM potato can be grouped into two main categories: the benefits of the GM potato were insufficiently proven and the potato could potentially have a negative effect on South African potato exports. The second case where SECs played a decisive role con- cerned an application for the general release of a genetically modified potato. This was by far the most high-profile case in the country, with active civil society protest and wide media coverage. The potato variety in question was modified in order to gain resistance to tuber moth, a moth whose lar- vae damage potato tubers in storage. By inserting a gene of the Bacillus thuringiensis (Bt), the protein would disrupt the digestion of the moths that eat the potato, thereby allowing farmers to grow potatoes using less pesticides. The project was funded by the United States Agency for International Development (USAID) and ran by the Agricultural Research Council (ARC), as part of a larger consortium that included the International Potato Centre, Syngenta, and Michigan State University, who had developed the original genetic modification (Douches et al. 2002). The concern about trade, to start with the latter, had been raised in the previous years by several different actors. In 2008, the African Centre for Biodiversity had published a book in which they pointed out that the great majority of South Africa’s potato exports are destined for various Southern African countries that had previously restricted the import of genetically modified products (Black 2008). And at least as important, in the same year various large retailers and processors in South Africa had publically stated their opposition to the GM potato. Woolworths and Pick & Pay, two of the largest supermarkets in South Africa, made clear they would not sell these potatoes and McDonalds wrote a letter to the Executive Council stating that they did not want to use genetically modified potatoes for their French fries (Hall 2008). Also McCain, a processing company from whom McDonalds purchased their potatoes, explicitly said that all their products are GM free. Their managing director was quoted saying: “we’re very much driven by consumer needs and they don’t want GM” (Gosling 2008). GM yeast The first time socio-economic considerations played a deci- sive role was in 2006, when the government received an application for a genetically modified strain of malolactic yeast that could be used in the production of wine, one of South Africa’s best-known export products. Besides their function in fermentation, yeasts can also have effects on the “that they are against a general release due to the mas- sive consumer resistance against GMOs, especially from the wine drinking consumers. The Council noted that the major export market for the wine industry is 1 3 How to include socio-economic considerations in decision-making on agricultural… 679 the United Kingdom who is against GMOs and the local wine industry indicated that the risk is just too high” (EC minutes of 18 September 2007). “improving storage conditions to prevent infection of tubers after harvesting” (Ashton et al. 2004, p. 4).i The ARC was nevertheless allowed to pursue field trials with Bt potato and between 2000 and 2007 the Executive Council granted another nine applications for field trials, contained use, and trial release. Yet as the ARC consor- tium moved closer towards environmental release, concerns about the socio-economic implications of the GM potato grew. When the Agricultural Research Council finally filed a request for the general release of the GM potato in July 2008, the Executive Council rejected the application by unanimous decision (EC minutes of 21 July 2009; inter- view 11 April 2016). The applicants subsequently filed an appeal in September 2009 and it took until March 2015 for the Minister of Agriculture of take a final decision on the application, eventually deciding to uphold the decision of the Executive Council not to allow the GM potato on the market (EC minutes of 24 March 2015). The representative of the DEAT, the only one who had voted in favour of approval in the May meeting, concluded that apparently “the international industry is not yet ready for a general release of malolactic wine yeast” (EC minutes of 18 September 2007). The letters and opinions of the wine industry and non- governmental organizations were deemed sufficient evidence for the proclaimed reluctance of European markets to buy the wines using the GM malolactic yeast and the Execu- tive Council subsequently rejected the application on trade grounds, noting that “we are major exporters of wine, we cannot jeopardize those markets” (interview 14 April 2016). GM potatoes When the Executive Council finally consulted with Potato South Africa, the body representing the South African potato industry, who had initially had supported the Bt potato pro- ject (interview 15 April 2016), they too said they would oppose GM potatoes. And so the government rejected the crop because the “the markets were not ready” (interview 14 April 2016). The Executive Council cited this concern in their rejection when noting that “entry of these GM potatoes i The ARC had been conducting field trials with the Bt potato since 2001 and, in contrast to the yeast case, SECs only played out when the ARC applied for environmental release. Outside of the regulatory process, however, these concerns were raised much earlier. Already in 2004 civil society organizations like ACB and Biowatch formally filed an objection to the Executive Council protesting against the request for continued field trials (Ashton et al. 2004). They mainly protested the design of the environmental risk assess- ment but also highlighted a wide range of SECs. They for instance argued that smallholder farmers could not benefit from a patented potato, which they would not be allowed to replant, and “which consumers in all likelihood will not buy” (Ashton et al. 2004, p. 6). The ACB furthermore questioned whether genetic modification was an effective solution to the tuber moth problem in the first place, pointing out that “tuber moths mainly affect tubers during storage” (Ashton et al. 2004, p. 4) and that it may be more effective to focus on 1 3 680 K. Beumer into the formal trade remains a particular concern” (EC min- utes of 21 July 2009). minutes of 21 July 2009). And smallholder farmers were cited to identify “more pressing challenges related to pro- duction such as lack of water, seed availability, fertilizers, etc.” (EC minutes of 21 July 2009). This was aggravated by the observation that tuber moth, the pest that was targeted through the genetic modification, “is not a major pest for stored potatoes but rather rodents” (EC minutes of 21 July 2009). y The ARC consortium had repeatedly emphasized that the potato was also meant to bring benefits to South African farmers, and to subsistence farmers in particular, who do not produce potatoes for export. GM potatoes To substantiate these claims, their application included two socio-economic studies that sought to identify the agricultural needs and constraints by performing surveys amongst commercial and smallholder farmers (Jordaan and Carstens 2007; Hart and Vorster 2006). But while the studies set out to identify “the socio-economic benefits of the Bt potato” (Black 2008, p. 57 [italics added]), the results were rather mixed, to say the least. The studies showed that commercial farmers were willing to grow GM potatoes if this improved their output and profitability yet that tuber moth was not a dire problem to them and that they were sceptical it about pesticide savings, domestic mar- ketability, and exportability (Jordaan and Carstens 2007). The survey amongst smallholder farmers, furthermore, con- cluded that “farmers indicated a range of problems, many of which might be simply and cost effectively reduced by means of adopting existing technology to local conditions and practices” (Hart and Vorster 2006, p. 3). Tuber moth was mentioned by smallholder farmers as a problem but other challenges were mentioned much more frequently, like access to water and transport and damage done by other pests like millipedes, moles and cutworm. In the interviews with key South African stakeholders, several other reasons for rejecting the potato were suggested. For instance one scientist highlighted that the genetic modi- fication was made in Spunta, a potato variety that was not widely grown by South African farmers (interview 15 April 2016). Potato South Africa had earlier noted that this variety “is not suitable for processing, so the big food chains would not use it anyway” (Gosling 2008). And several interviewees further raised the suspicion that it may have played a role that no genetically modified potatoes had been allowed at that time anywhere in the world (interview 7 April 2016; Pretoria, 11 April 2016; Pretoria, 15 April 2016). As one interviewee said: “We don’t want to be the first. Who dares to be the first?” (interview 15 April 2016). i However, these concerns and the evidence provided for them did not appear in any official documentation and did not seem to play a role in the discussions leading up to the decision, while the Executive Council did provide a list of other objections in their rejection. GM potatoes This claim effectively challenged the decision to ex ante assess the socio-economic impact of genetically modified organisms, claiming that this can only be done ex post (despite having performed ex-ante socio- economic evaluations themselves). One civil society organ- ization fiercely dismissed this proposal, noting that “it is shameful that the most vulnerable people in our society are being asked to take on the burden of experimenting with new and very expensive technology” (Stafford et al. 2008, p. 26). GM potatoes The main concerns were the potentially negative impact on trade and domestic mar- ketability, for which industry consultations provided suffi- cient evidence, and the lack of benefits for smallholder farm- ers, for which farmer’s surveys provided the most important evidence. Perhaps it is telling that although it only takes the objection of one member of the Executive Council for an application to be rejected, in the case of the GM potato the Executive Council rejected the application by an unanimous vote. p p The applicants concluded from this that a need for the GM potato had been proven. In addition, and perhaps in contradiction, they argued that proper “information on many of the socio-economic issues can only be collected if the application is approved” (Thomson et al. 2010), suggesting that these trials could be conducted with active participation by South African farmers. This claim effectively challenged the decision to ex ante assess the socio-economic impact of genetically modified organisms, claiming that this can only be done ex post (despite having performed ex-ante socio- economic evaluations themselves). One civil society organ- ization fiercely dismissed this proposal, noting that “it is shameful that the most vulnerable people in our society are being asked to take on the burden of experimenting with new and very expensive technology” (Stafford et al. 2008, p. 26). This more fundamental challenge of whether ex ante or ex post assessments were necessary in the regulatory process was never responded to, however. The Executive Council went ahead and included SECs into their decision. Draw- ing upon the very farmers surveys provided by the ARC, the Executive Council outright dismissed the applicants’ claims and decided that the benefits of the GM potato were insufficiently proven. The Executive Council concluded that commercial farmers did not expect the GM potato to be very beneficial “as the same spraying regime is required to manage other pests which this event does not target” (EC The applicants concluded from this that a need for the GM potato had been proven. In addition, and perhaps in contradiction, they argued that proper “information on many of the socio-economic issues can only be collected if the application is approved” (Thomson et al. 2010), suggesting that these trials could be conducted with active participation by South African farmers. 3 Discussion and conclusion This article has investigated both what SECs are (and are not) included, what evidence is required for these considerations to be deemed sufficiently credible, and how these considerations are eventually included in decision-making. By thus looking at the work that is put into crafting a regime of regulation in a context where standards for taking such decisions are lacking, I have identified two practice-based models for including SECs in biotechnology decision-making. These can be seen as two sets of practices by which subjective and politically contentious issues evolve over time towards more objective and predictable objects of regulation. The models, which can be labelled are sum- marized in Table 3. The regulatory regime for SECs are emerging in rather different ways in Kenya and South Africa. The Kenyan model is characterized by a sustained effort to define what counts as a valid socio-economic consideration through a bottom-up process prior to assessing the first application, with input from socio-economic experts judging the com- pleteness and robustness of the information provided, and with a public consultation to which applicants are invited to reply. This can be understood as a definitive approach to reg- ulate new object of concerns—as an attempt to include these considerations in specified and stable way, sealing the proce- dure before the first assessment. The South African model is characterized by a case-to-case assessment by the Executive Council, that takes into account views from other actors in an ad hoc manner, and that does not require scientific evi- dence of socio-economic impacts but instead largely draws upon farmer surveys and letters by representative organiza- tions and key stakeholders, thereby establishing regulatory standards in the process. This can be understood as a tenta- tive approach to regulate new objects of concerns—as an Even though these standards are not written down any- where, both regulators, civil society organizations, and industry organizations seem to know well what considera- tions are important, when there is enough evidence for those considerations, and hence what applications do not stand a chance. In the words of one industry representative: “the whole potato story, if I can say that, left a mark on the pub- lic companies” (interview 7 April 2016), a claim that rings equally true for the malolactic yeast story. Ever since these applications were rejected, no other applications have been filed that touched upon either of these issues. Discussion and conclusion This article started by observing that actors around the world are struggling to ‘open up’ regulatory science to different types of issues and knowledges (Stirling 2008). The inclu- sion of socio-economic considerations into biotechnology decision-making is a case in point. Omitting SECs is deemed undesirable because limiting decision-making to issues of risks to human health and the environment does not answer to widely held societal concerns about the impact of bio- technology on other aspects of life. Yet including SECs in the absence of formalized methods for identifying and assessing what counts as a valid SEC runs the risk of creat- ing a decision-making process that is both subjective and unpredictable. The decisions about SECs furthermore lacked transpar- ency. Like the board of the biosafety authority in Kenya, the Executive Council in South Africa provides little insight into how they arrived at their decision. They pub- lish minutes of their meeting but these generally provide only little information. The paradoxical outcome of this ad hoc process, how- ever, is that over the years very clear standards have been established for what SECs are deemed valid. Genetically modified organisms will not be allowed on the South Afri- can market if they adversely impact indigenous biodiver- sity, potentially endanger exports, are unlikely to realize proclaimed benefits for smallholder farmers, and if they are not supported by those that eventually have to sell them to consumers. And also the evidence required for these claims is relatively clear, with stakeholder views being considered as sufficient evidence for assessing ‘if the mar- kets are ready or not’. As one interviewee told me: “When someone says they don’t need something, they don’t neces- sarily need scientific evidence or anything” (interview 4 April 2016). Not all actors fully agreed on either the SECs included (the ACB and Biowatch argued also the relative efficacy vis-à-vis non-GMO solutions to improving potato yields should be considered)) nor on the type of evidence required (the ARC challenged the need for providing ex ante assessments). Yet after the yeast and potato decisions, it was relatively clear for all actors what role SECs play in decision-making. This article aims to contribute to solving this dilemma by investigating the way Kenya and South Africa, in prac- tice, have tried to move beyond the regulation of health and environmental risks by including SECs in biotechnology decision-making. South Africa: ad hoc and case‑to‑case The regulation of socio-economic considerations in South Africa took shape in an ad hoc fashion, on a case-to-case basis. At first sight this process was less inclusive than in Kenya and also scientific evidence played a much smaller role in South Africa. While the different considerations that were taken into account were certainly fed by con- cerns that were articulated by a variety of actors in South Africa, ranging from corporations like McKain to civil society organizations like the ACB, the decision about what considerations were and were not taken into account was exclusively taken by the Executive Council. These civil servants were accountable to their respective min- isters and they did not provide clarity to applicants about what information had to be provided with respect to SECs. f This more fundamental challenge of whether ex ante or ex post assessments were necessary in the regulatory process was never responded to, however. The Executive Council went ahead and included SECs into their decision. Draw- ing upon the very farmers surveys provided by the ARC, the Executive Council outright dismissed the applicants’ claims and decided that the benefits of the GM potato were insufficiently proven. The Executive Council concluded that commercial farmers did not expect the GM potato to be very beneficial “as the same spraying regime is required to manage other pests which this event does not target” (EC 3 3 How to include socio-economic considerations in decision-making on agricultural… 681 And in contrast to Kenya, the most decisive evidence con- sisted of non-academic papers (like the farmers’ surveys) and letters from industry, all of which varied from case to case, and none of which was subjected to external peer review to establish the credibility of the claims. Discussion and conclusion Table 3 Emerging regulatory regimes for socio-economic considerations Kenya Definitive approach South Africa Tentative approach Inclusion Establishment procedure Standard Operating Procedure drafted by NBA staff based on stakeholder workshop including all stakeholder concerns Ad hoc case by case assessment by executive committee that occasionally takes into account stakeholders views Socio-economic considerations Impact on food security and sustainability, access to the technology, income to farmers, cost of seeds and other inputs, co-existence, trade implications, benefits and freedom of choice, biosafety and stewardship Impact on trade, biodiversity, marketability, and smallholder farmers Assessment Responsible actor NBA board Executive Council Provision of evidence Application in application form, expert reviewer report, comments from public consultation Applicant in application form, Executive Council may request other actors fo additional information Type of evidence Peer reviewed scientific studies on Kenya or proxies thereof in case such studies are not available, report by export reviewer, comments from public consultation Public comments and statements by representative organizations and key stakeholders. Occasionally farmer surveys Assessment mechanism Unclear how evidence is weighed and what thresholds are Clear thresholds for negative impacts have been established through prec- edents, though unclear how evidence is weighed, though ) Perhaps more than anything else, the Kenyan and South African practices demonstrate the challenges pioneers face when translating concerns from subjective and politically contentious issues into more objective and predictable objects of regulation. This particularly becomes clear when observing the similarities between Kenya and South Africa compared to other countries, particularly Argentina (Falck- Zepeda and Zambrano 2011; Binimelis and Myhr 2016). Argentina, which has signed but not ratified the Cartagena Protocol, has limited the assessment of SECs to the potential impacts on trade, specifically the competitiveness of Argen- tinean exports (Falck-Zepeda and Zambrano 2011), and its regulations stipulate that the assessment of these consid- erations should be conducted by one specific government agency on the basis of cost–benefit principles (Binimelis and Myhr 2016). While the exclusive focus on trade and on a well-established assessment method like cost–benefit analysis makes it easier for regulatory procedures to become objective and predictable, this in turn makes it more difficult to be responsive to the wide range of different considerations that gave to the societal controversy around GMOs in the first place. Discussion and conclusion While the pro- cess through which these standards were established was marked by substantial uncertainty and unpredictability, the current situation comes remarkably close to Falck-Zepeda’s (2009, p. 91) requirements for a system including SECs where the rules of the game are “transparent, well defined, protective, and understood by all actors and stakeholders.” 1 3 682 K. Beumer Table 3 Emerging regulatory regimes for socio economic considerations Kenya Definitive approach South Africa Tentative approach nclusion Establishment procedure Standard Operating Procedure drafted by NBA staff based on stakeholder workshop including all stakeholder concerns Ad hoc case by case assessment by executive committee that occasionally takes into account stakeholders views Socio-economic considerations Impact on food security and sustainability, access to the technology, income to farmers, cost of seeds and other inputs, co-existence, trade implications, benefits and freedom of choice, biosafety and stewardship Impact on trade, biodiversity, marketability, and smallholder farmers Assessment Responsible actor NBA board Executive Council Provision of evidence Application in application form, expert reviewer report, comments from public consultation Applicant in application form, Executive Council may request other actors for additional information Type of evidence Peer reviewed scientific studies on Kenya or proxies thereof in case such studies are not available, report by export reviewer, comments from public consultation Public comments and statements by representative organizations and key stakeholders. Occasionally farmer surveys Assessment mechanism Unclear how evidence is weighed and what thresholds are Clear thresholds for negative impacts have been established through prec- edents, though unclear how evidence is weighed, though ongoing process that is prudent and preliminary (Kuhlmann et al. 2019). References Ad Hoc Technical Expert group on Socioeconomic Considerations (AHTEG-SEC). 2014. Report of the ad hoc technical expert group on socioeconomic considerations. UNEP/CBD/BS/AHTEG- SEC/1/3. Seoul: CBD. African Centre for Biosafety (ACB). 2006. South Africa’s wine industry threatened by GM grapevine trials. http://acbio.org.za/ south-africas-wine-industry-threatened-by-gm-grapevine-trials/. Accessed 19 Mar 2019.f i In many ways, creating regulations for socio-economic considerations is a learning process and these regulatory practices will therefore undoubtedly continue to evolve over time. This mirrors the trajectory taken by the regulation of risks to human health and the environment. These regula- tions too once emerged from a shared concern—in this case for human health and the environment—in a context where consensus over the scope of these concerns and the methods for assessing them was lacking. The practices of regulating these risks has only emerged over the course of the last dec- ades, as consensus slowly emerged over the scope of these considerations and the types of knowledge that are deemed credible in assessing them (Luhmann 1993; Boholm 2015). The regulation of SECs is arguably only at an early stage in this trajectory and subsequently is largely open to change. Ashton, G., G. Baker, M. Mayet, E. Pschorn-Strauss, and W. Stafford. 2004. Objections to application for a permit for additional tri- als with insect resistant Bt Cry V Genetically Modified Potatoes (Solanum Tuberosum L. Variety ‘Spunta’G2 and G3). http://acbio .org.za/wp-content/upload/2015/02/objection_bt_potato_g2_g3. pdf. Accessed 19 Mar 2019. Beumer, K. 2017. Travelling risks. How did nanotechnology become a risk in India and South Africa? Journal of Risk Research 21 (11): 1362–1383. Binimelis, R., and A.I. Myhr. 2016. Inclusion and implementation of socio-economic considerations in GMO regulations: Needs and recommendations. Sustainability 8 (1): 62. Black, V. 2008. Hot potato. GM potatoes in South Africa - a critical analysis. Johannesburg: ACB. y g Boholm, A. 2015. Anthropology and risk. London: Routledge.l Chataway, J., and J. Tait. 1993. Is risk regulation a strategic influence on decision making in the biotechnology industry? Agriculture and Human Values 10 (10): 60–67. Identifying these two models for including socio-eco- nomic considerations into biotechnology decision-making is therefore both relevant and timely. Many countries currently intend to take into account SECs but struggle to implement it as detailed information about practical experiences with SECs in biotechnology regulation are absent. References It should be clear that the two models for solving this problem presented in this article are shaped by the local contexts of Kenya and South Africa and therefore cannot unproblematically be copy-pasted to other countries. However, the in-depth description of the two models, as well as the identification of distinct tensions in each model, provides insight into two potential pathways for including SECs into biotechnology decision-making. This may help policy makers in devising robust regulations for SECs. Chaturvedi, S., K.R. Srinavas, R.K. Joseph, and P. Singh. 2012. Approval of GM crops. Socio-economic considerations in devel- oping countries. Economic and Political Weekly 47 (23): 53–61.i Chaturvedi, S., K.R. Srinavas, R.K. Joseph, and P. Singh. 2012. Approval of GM crops. Socio-economic considerations in devel- Approval of GM crops. Socio-economic considerations in devel- oping countries. Economic and Political Weekly 47 (23): 53–61.i Commissie Genetische Modificatie (COGEM). 2009. Sociaal-econo- mische aspecten van ggo’s. Bouwstenen voor een EU duurzaam- heidsbeoordeling van genetisch gemodificeerde gewassen. The Hague: COGEM.i Commissie Genetische Modificatie (COGEM). 2014. Report—Inter- national workshop on a socio-economic assessment framework for GMOs. https://www.cogem.net/showdownload.cfm?objec tId=0D11C206-1517-64D9-CC263B30A61B4F01&objectType =mark.hive.contentobjects.download.pdf. Accessed 19 Mar 2019. Conference of Parties serving as the meeting of the Parties to the Pro- tocol (COP-MOP). 2008. Socio-economic considerations (Article 26, Paragraph 2). UNEP/CBD/BS/COP-MOP/4/15 17 Mar 2008. De Vries, G., I. Verhoeven, and M. Boeckhout. 2011. Taming uncer- tainty: The WRR approach to risk governance. Journal of Risk Research 14 (4): 485–499. Douches, D.S., et al. 2002. Development of Bt-cry5 insect resistant potato lines Spunta-G2 and Spunta G3. Horticultural Science 37 (7): 1103–1107. Acknowledgements I would like to thank Dorington Ogoyi and Hennie Groenewald for their invaluable support during the fieldwork. Also I would like to thank Sjaak Swart for his valuable comments and support, as well as the participants of the various ERAfrica workshops and the 4S conference in Boston, and the S.NET conference in Bergen. The paper has been developed under the project titled ‘Addressing societal challenges of biotechnology in Africa. Towards balanced innovation’, funded by ERAfrica. Falck-Zepeda, J.B. 2009. Socio-economic considerations, Article 26.1 of the Cartagena Protocol on Biosafety: What are the issues and what is at stake? AgBioForum 12 (1): 90–107. Falck-Zepeda, J.B., and P. Zambrano. 2011. Socio-economic consid- erations in biosafety and biotechnology decision making: The Cartagena Protocol and national biosafety frameworks. Review of Policy Research 28 (2): 171–195. Funding Funding was provided by Nederlandse Organisatie voor Wetenschappelijk Onderzoek. Discussion and conclusion The regulatory practice in both Kenya and South Africa instead have dedicated places for societal actors to raise concerns and contribute evidence, and both countries subsequently consider a much broader spectrum of socio- economic impacts, thereby being more responsive to consid- erations raised by various societal actors. The two practice- based models reflect different strategies for dealing with this. lf The analysis shows that the two models produce distinct tensions. While the Kenyan ‘definitive model’ contains dif- ferent moments for stakeholder inclusion, the lack of trans- parency over how SECs are weighed into the decision-mak- ing results in a lack of clarity as to what voices matter. And while there are high standards to ensure the credibility of the information provided, which in the long term may be a com- mendable aim for creating objective regulations, in practice the emphasis on peer reviewed scientific publications cannot be realistically adhered to and does not specify the kinds of studies that are deemed best suited for the concerns in ques- tion. And finally, while formalizing the procedure prior to the first assessment may increase the predictability of the regulatory procedure, this also runs the risk of obscuring the way conflicts over conflicting concerns are contested. In the South African ‘tentative model’, on the other hand, clear standards and thresholds have emerged regarding negative socio-economic impacts but there is uncertainty about both what new issues may emerge in the future and about what thresholds are operated in cases where distinct negative and positive socio-economic impacts have to be weighed. And while the pragmatic reliance on statements by key stakehold- ers provides valuable information for the cases assessed, it Table 3 Emerging regulatory regimes for socio-economic considerations 1 3 How to include socio-economic considerations in decision-making on agricultural… 683 is unlikely that this type of evidence is sufficient in cases where the socio-economic impact of new crops is unclear or contested. 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https://openalex.org/W3210321740	https://ruc.udc.es/dspace/bitstream/2183/29351/3/Pereira-Ruisanchez_Dariel_2021_Deep_Learning-Based_Strategy.pdf	English	null	A Deep Learning-Based Strategy to Predict Self-Interference in SFN DTT	null	2,021	cc-by	1,968	Keywords: SFN; deep learning; broadcasting; self-interference Citation: Pereira-Ruisánchez, D.; Pérez-Adán, D.; Castedo, L. A Deep Learning-Based Strategy to Predict Self-Interference in SFN DTT. Eng. Proc. 2021, 7, 57. https://doi.org/ 10.3390/engproc2021007057 Proceeding Paper A Deep Learning-Based Strategy to Predict Self-Interference in SFN DTT † , Darian Pérez-Adán and Luis Castedo Dariel Pereira-Ruisánchez * Pereira-Ruisánchez * , Darian Pérez-Adán and Luis Castedo Department of Computer Engineering & CITIC Research Center, University of A Coruña, Campus de Elviña S/N, 15071 A Coruña, Spain; d.adan@udc.es (D.P.-A.); luis.castedo@udc.es (L.C.) * Correspondence: d.ruisanchez@udc.es † Presented at the 4th XoveTIC Conference, A Coruña, Spain, 7–8 October 2021. Department of Computer Engineering & CITIC Research Center, University of A Coruña, Campus de Elviña S/N, 15071 A Coruña, Spain; d.adan@udc.es (D.P.-A.); luis.castedo@udc.es (L.C.) * Correspondence: d.ruisanchez@udc.es † Presented at the 4th XoveTIC Conference, A Coruña, Spain, 7–8 October 2021. Department of Computer Engineering & CITIC Research Center, University of A Coruña, Campus de Elviña S/N, 15071 A Coruña, Spain; d.adan@udc.es (D.P.-A.); luis.castedo@udc.es (L.C.) * Correspondence: d.ruisanchez@udc.es † Presented at the 4th XoveTIC Conference, A Coruña, Spain, 7–8 October 2021. Department of Computer Engineering & CITIC Research Center, University of A Coruña, Campus de Elviña S/N, 15071 A Coruña, Spain; d.adan@udc.es (D.P.-A.); luis.castedo@udc.es (L.C.) * Correspondence: d.ruisanchez@udc.es † Presented at the 4th XoveTIC Conference, A Coruña, Spain, 7–8 October 2021. Abstract: A deep learning-based strategy for the analysis of the self-interference in single frequency networks (SFNs) for digital terrestrial television (DTT) broadcasting is considered. Several laboratory measurements were performed to create a dataset that relates the self-interference parameters and some quality metrics of the resulting received signal. The laboratory setup emulates an SFN scenario with two DTT transmitters. The strongest received signal and the relative values of attenuation and delay between the signals stand for the input parameters. The modulation error ratio (MER) of the strongest received signal, the MER of the resulting signal, and the SFN gain (SFNG) are the output parameters. This dataset is used to train four different multi-layer perceptron (MLP) models to predict accurate maps of interference and signal quality metrics. The considered models are suitable as complements for any multiple frequency network (MFN) coverage software with the capability to return the signal strength and the position data. This way, the SFN self-interference behavior can be predicted by considering only a proper description of the MFN coverage. 1. Introduction The remarkable growth of mobile services and wireless communication technologies has led to a revision of the way the available spectrum bands are allocated. In digital terrestrial television (DTT) broadcasting, the spectral efﬁciency achieved with multiple frequency networks (MFNs) is signiﬁcantly improved when moving to single frequency networks (SFNs). Furthermore, the deployment of SFNs leads to a more homogeneous distribution of the electric-ﬁeld strength in the coverage area and to savings in transmission power [1]. In previous works, self-interference in SFNs is only considered when the interfering signals arrive with a delay longer than the guard interval. However, this only represents a critical scenario where the interference is mostly destructive. Signals arriving within the guard interval also produce self-interference, and it can be either constructive or destructive. The effect of this kind of interference is called SFN gain (SFNG) and it must be properly controlled to obtain a good performance in SFN systems [2–4]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Several network planning strategies based on deep learning (DL) algorithms are being considered as a reasonable alternative for the conﬁguration of broadcasting systems. These strategies allow reducing the computational complexity of theoretical models and the planning cost of the ﬁeld-testing-based approaches [5–7]. The predictive capability of DL algorithms and the lack of works about using them for SFN interference analyses have motivated this research. The major contributions of this work can be summarized as follows: Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ https://www.mdpi.com/journal/engproc Eng. Proc. 2021, 7, 57. https://doi.org/10.3390/engproc2021007057 Eng. Proc. 2021, 7, 57 2 of 4 • The development of a laboratory test-based dataset that relates the parameters of the received signals to several metrics of interference and signal quality. • The development of a laboratory test-based dataset that relates the parameters of the received signals to several metrics of interference and signal quality. g g q y • The implementation of deep learning-based models to predict the interference and the resulting signal quality metrics. • The implementation of deep learning-based models to predict the interference and the resulting signal quality metrics. 2. Dataset and Proposed Deep Learning-Based Models The proposed laboratory setup emulates an SFN scenario with two interfering trans- mitters. The interfering signals were generated by using a Broadcast Test Center (BTC) from Rohde and Schwarz and the signal quality metrics were measured by using the S7000 TV Analyzer professional receiver. The electric-ﬁeld strength of the main signal (EMainSignal), and the values of Attenuation and Delay of the secondary signal, were conﬁgured to emulate self-interference scenarios. These parameters are the input features in the proposed dataset. The modulation error ratio (MER) was the metric employed to quantify the signal quality. The measured values of modulation error ratio (MER) of both the main signal (MERMFN) and the resulting received signal (MERSFN) are output features. The SFN gain (GSFN) is the third output feature, which is calculated as the difference between the MERSFN and the MERMFN parameters. p p p p The modulation error ratio (MER) was the metric employed to quantify the signal quality. The measured values of modulation error ratio (MER) of both the main signal (MERMFN) and the resulting received signal (MERSFN) are output features. The SFN gain (GSFN) is the third output feature, which is calculated as the difference between the MERSFN and the MERMFN parameters. p The resulting dataset was employed to train four multi-layer perceptron (MLP) models by using a supervised-learning strategy (Table 1). The ﬁrst models are regression models; thus, they were trained to predict the exact values of their respective output features. The last one is a binary classiﬁcation model and it was trained to predict whether the value of GSFN is positive or negative. Positive GSFN values stand for the cases where the received signal improves when moving to SFN while the negative values correspond to a signal degradation. Table 1. Proposed deep learning-based models. MLP Models Output Feature Type MLP_MfnMER MERMFN Regression MLP_SfnMER MERSFN Regression MLP_SfnG GSFN Regression MLP_SfnGclass GclassSFN Classiﬁcation Table 1. Proposed deep learning-based models. 3. Results Table 2 summarizes the accuracy values obtained by employing the proposed regres- sion models. The coefﬁcient of determination (R2), the mean absolute error (MAE), the mean square error (MSE) and the root mean square error (RMSE) are the metrics used to measure the performance. A lower accuracy is obtained with the MLP_SfnG model since the correspondence between GSFN and the input features cannot be easily determined. Table 2. Performance metrics of the regression models. MLP Models R2 MAE MSE RMSE MLP_MfnMER 0.998 0.159 0.036 0.191 MLP_SfnMER 0.997 0.134 0.041 0.203 MLP_SfnG 0.909 0.151 0.049 0.221 Table 2. Performance metrics of the regression models. In Figure 1, the predicted values are plotted versus the measured values. As expected, a higher dispersion can be observed in the GSFN predictions because the performance of this model is lower than the others. Some dispersion can also be observed in the edge values of the parameters due to the instrument measuring ranges. Figure 2 shows the confusion matrix for the MLP_SfnGclass classiﬁcation model. From the 317 samples considered for the validation process, the 91.5% were well predicted Eng. Proc. 2021, 7, 57 3 of 4 (184 true negatives and 106 true positives). The remaining 8.5% of the predictions were either false positives or false negatives. 18 20 22 24 26 28 30 18 20 22 24 26 28 30 MERMFN measurements (dB) MERMFN predictions (dB) (a) 18 20 22 24 26 28 30 18 20 22 24 26 28 30 MERSFN measurements (dB) MERSFN predictions (dB) (b) −2 −1 0 1 2 3 4 −2 −1 0 1 2 3 4 GSFN measurements (dB) GSFN predictions (dB) (c) Figure 1. Predicted values versus measured values of (a) MERMFN, (b) MERSFN and (c) GSFN. −2 −1 0 1 2 3 4 −2 −1 0 1 2 3 4 GSFN measurements (dB) GSFN predictions (dB) (c) 18 20 22 24 26 28 30 18 20 22 24 26 28 30 MERSFN measurements (dB) MERSFN predictions (dB) (b) 18 20 22 24 26 28 30 18 20 22 24 26 28 30 MERMFN measurements (dB) MERMFN predictions (dB) (a) (b) (c) Figure 1. Predicted values versus measured values of (a) MERMFN, (b) MERSFN and (c) GSFN. Figure 2. Confusion matrix of the MLP_SfnGclass model. Figure 2. Confusion matrix of the MLP_SfnGclass model. Figure 2. Confusion matrix of the MLP_SfnGclass model. 4. Conclusions This paper proposes a deep learning-based strategy to analyze the self-interference in SFNs for DTT broadcasting. Unlike most planning-oriented researches, interference in SFNs is analyzed over the entire overlapping area and not only in critical cases where delays are especially long. A dataset obtained from laboratory measurements is employed to train four MLP models for predicting signal quality parameters in an SFN DTT deployment. The prediction results exhibit the high degree of relation between the received signal’s parameters and the resulting signal quality. The proposed dataset and the MLP models are suitable for any SFN interference analysis since this approach is not limited to speciﬁc terrain or transmission variables. Author Contributions: Conceptualization, formal analysis, investigation, visualization and writing original draft, D.P.-R. and D.P.-A.; writing—review and editing original draft, D.P.-R., D.P.-A. and L.C.; project administration and funding acquisition, L.C. Funding: This work has been funded by the Xunta de Galicia (by grant ED431C 2020/15, and grant ED431G2019/01 to support the CITIC, Centre for Information and Communications Technology Research, from the University System of Galicia), the Agencia Estatal de Investigación of Spain (by grants RED2018-102668-T and PID2019-104958RB-C42) and ERDF funds of the EU (FEDER Galicia 2014–2020 & AEI/FEDER Programs, UE), and the predoctoral grant BES-2017-081955. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The dataset used to train the deep learning-based models is publicly available at https://github.com/DarielPereira/SFN_Dataset.git (accessed on 30 September 2021). Conﬂicts of Interest: The authors declare no conﬂict of interest. Eng. Proc. 2021, 7, 57 4 of 4 2. Plets, D. On the Methodology for Calculating SFN Gain in Digital Broadcast Systems. IEEE Trans. Broadcast. 2010, 56, 331–339. [CrossRef] 3. Plets, D.; Joseph, W.; Angueira, P.; Arenas, J.A.; Verloock, L.; Martens, L. SFN gain in broadcast networks. In Proceedings of the IEEE International Symposium on Broadband Multimedia Systems and Broadcasting (BMSB), Nuremberg, Germany, 8–10 June 2011. 1. Mattson, A. Single frequency networks in DTV. IEEE Trans. Broadcast. 2005, 51, 413–422. [CrossRef] References [ ] 3. Plets, D.; Joseph, W.; Angueira, P.; Arenas, J.A.; Verloock, L.; Martens, L. SFN gain in broadcast networks. In Proceedings of the IEEE International Symposium on Broadband Multimedia Systems and Broadcasting (BMSB), Nuremberg, Germany, 8–10 June 2011. J 4. Ruisánchez, D.P. Estudio de la interferencia mutua en SFN para DTMB. RIELAC 2020, 41, 73–88. J 4. Ruisánchez, D.P. Estudio de la interferencia mutua en SFN para DTMB. RIELAC 2020, 41, 73–88. , p , , 5. Moreta, C.E.G. Prediction of Digital Terrestrial Television Coverage Using Machine Learning Regression. IEEE Trans. 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https://openalex.org/W2969637735	https://europepmc.org/articles/pmc6769464?pdf=render	English	null	Creatine for the Treatment of Depression	Biomolecules	2,019	cc-by	17,203	Received: 31 July 2019; Accepted: 21 August 2019; Published: 23 August 2019 Abstract: Depressed mood, which can occur in the context of major depressive disorder, bipolar disorder, and other conditions, represents a serious threat to public health and wellness. Conventional treatments are not eﬀective for a signiﬁcant proportion of patients and interventions that are often beneﬁcial for treatment-refractory depression are not widely available. There is, therefore, an immense need to identify novel antidepressant strategies, particularly strategies that target physiological pathways that are distinct from those addressed by conventional treatments. There is growing evidence from human neuroimaging, genetics, epidemiology, and animal studies that disruptions in brain energy production, storage, and utilization are implicated in the development and maintenance of depression. Creatine, a widely available nutritional supplement, has the potential to improve these disruptions in some patients, and early clinical trials indicate that it may have eﬃcacy as an antidepressant agent. Keywords: major depressive disorder; bipolar disorder; creatine; phosphocreatine Brent M. Kious 1,, Douglas G. Kondo 1,2 and Perry F. Renshaw 1,2 2 George E. Wahlen Veterans Aﬀairs Medical Center, 500 Foothill Drive, Salt Lake City, UT 84148, USA Correspondence: brent.kious@hsc.utah.edu; Tel.: +1-801-585-1418 biomolecules Review Biomolecules 2019, 9, 406; doi:10.3390/biom9090406 www.mdpi.com/journal/biomolecules 1. Introduction Major depressive disorder (MDD) and associated syndromes, such as dysthymic disorder and bipolar depression, impact a substantial fraction of children and adults globally. Despite the widespread availability and utilization of conventional antidepressants such as the selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs), roughly 53% of persons with depression fail to respond to an initial trial of those medications [1], and as much as 35% of patients do not respond to multiple trials of diﬀerent antidepressants [2]. Therapies that may be beneﬁcial for treatment-refractory depression, such as electroconvulsive therapy or ketamine, are not widely available [3]. Most conventional antidepressants alter the release or reuptake of the monoamine neurotransmitters serotonin, norepinephrine, and dopamine. The limited eﬃcacy of conventional antidepressants and the limited availability of more novel treatments with diﬀerent mechanisms together demonstrate a crucial need to identify antidepressant interventions with diﬀerent mechanisms of action which also have the potential to be accessible to many patients. Although recently much work in the pharmacological treatment of depression has been devoted to studying the potential of medications that alter the activity of the glutamatergic system—especially, the anesthetic agent ketamine and its enantiomer esketamine, which are potent antagonists of the N-methyl-D-aspartic acid (NMDA) glutamatergic receptor [4,5]—other physiologic pathways may also contribute to the development of depression. In particular, as we review below, there is growing evidence that both unipolar and bipolar depression involve alterations in the regulation of brain energy stores, which could produce depression, or limit antidepressant response, by several routes. As a result of this research, a number of investigators have begun to examine the antidepressant potential of compounds that could improve brain bioenergetics—that is, the processes of brain energy storage, Biomolecules 2019, 9, 406; doi:10.3390/biom9090406 www.mdpi.com/journal/biomolecules www.mdpi.com/journal/biomolecules 2 of 25 Biomolecules 2019, 9, 406 transport, and utilization. In particular, there has been increasing interest in the possible antidepressant eﬃcacy of creatine (N-aminoiminomethyl-N-methylglycine). In what follows, we review the evidence that altered brain bioenergetics contribute to the pathogenesis of some cases of depression and to limitations on response to conventional antidepressants, examine the pharmacological properties of creatine relevant to its use as an antidepressant agent, examine pre-clinical evidence from animal studies and studies of healthy (non-depressed) humans that pertain to its potential antidepressant eﬃcacy, and consider the limited but growing cadre of clinical studies that have examined creatine for the treatment of depression. 2.1. Creatine Biochemistry as it Pertains to Depression Although the basic biochemistry of creatine is reviewed elsewhere in this issue (CITE), we will highlight a few facts that are pertinent to its possible role in the treatment of depression. Creatine is an organic acid that is synthesized from the amino acids arginine, glycine, and methionine. It is also derived from diet, particularly from foods containing meat and ﬁsh [8]. It is synthesized in brain to a limited degree, but brain levels are primarily maintained by active transport from the serum via the sodium- and chloride-dependent creatine transporter SLC6A8 [9,10]. The brain is an energetically demanding organ and accounts for approximately 20% of body energy consumption at rest, even though it accounts for roughly 2% of body mass [11,12]. The best-characterized role of creatine in energy metabolism is as an energy buﬀer: creatine is converted to phosphocreatine (PCr) by the creatine kinase reaction, thereby storing energy in a more stable form than is provided by adenosine triphosphate (ATP) [13]. The creatine kinase reaction occurs in muscles as well as in the brain [13]. Creatine also serves as an “energy shuttle,” as its more rapid rate of intracellular diﬀusion compared to ATP means that it is able to more eﬃciently transport energy from sites where it is synthesized (e.g., mitochondria) to sites where it is utilized (e.g., the neuronal membrane) along a concentration gradient [8,14]. Creatine kinase is most extensively expressed in brain regions that exhibit higher levels of activity, such as the hippocampus and cerebellum [15]. 1. Introduction This topic has previously been reviewed elsewhere [6,7]; the current review adds to these excellent papers in that it encompasses more information about neuroimaging ﬁndings in depression that indicate the presence of bioenergetics deﬁcits, considers additional background pertaining to the role of hypoxia in the production of depression, and examines the results of the relevant clinical studies in more detail. 2. Methods For this review, we identiﬁed empirical studies published in peer-reviewed journals in English using several search engines (PubMed, PsycINFO, and Google Scholar) encompassing publication dates up to June 30, 2019. The following initial search parameters were used: depression creatine OR major depressive disorder creatine OR bipolar creatine OR suicide creatine. The initial search revealed 733 records which were manually screened for relevance and duplication by the ﬁrst author, leaving 112 records. Additional studies of relevance were identiﬁed through review of the reference lists of the studies identiﬁed in the initial search. 2.2. Animal Studies of Depression-Like Behavior Ovariectomized rats who received estradiol exhibited signiﬁcantly fewer depressive symptoms than ovariectomized rats who received vehicle only. The investigators also found that creatine at both the 2% and 4% by weight doses reduced depressive behaviors in the ovariectomized rats who received estradiol and progesterone compared to no creatine; this eﬀect was not observed in the other groups [20]. These studies of the antidepressant eﬀects of creatine in animal models are supported by others that indicate that creatine and phosphocreatine levels are altered in animal models of depression. Using proton magnetic resonance spectroscopy (1H MRS), Kim et al. showed that mice exposed to the forced swim test exhibited reduced total creatine (creatine + phosphocreatine) levels in the left dorsolateral prefrontal cortex; this reduction was corrected by treatment with the tricyclic antidepressant desipramine [21]. Other studies of animal models of depression and chronic stress also tend to show reduced brain creatine concentrations. Rats exposed acutely to the forced swim test plus restraint stress and ether exhibit reduced creatine in the frontal cortex [22], including in the medial prefrontal cortex as measured by 1H MRS [23]. A social isolation model of depression, in which rats are reared in isolation from conspeciﬁcs for 8 weeks, showed that they exhibited reduced hippocampal (though not cortical) phosphocreatine, along with glutamate and glutamine, coupled with reductions in antioxidant enzymes and increases in brain levels of hydrogen peroxide [24]. Male tree shrews subjected to chronic social defeat stress (another model of depression) exhibited on average a 15% reduction in cerebral total creatine levels, which was associated with reductions in neurogenesis measured by immunohistochemistry for BrdUrd. These changes were prevented by treatment with tianeptine, a tricyclic antidepressant [25]. Other, similarly-designed studies using the social defeat stress model in tree shrews have also found reductions in cerebral creatine, which are attenuated by a variety of potential antidepressant compounds [26–29]. A large series of animal studies conducted by Rodriques and collaborators and designed to ascertain the mechanisms underpinning creatine’s antidepressant activity for rats in the tail suspension test (TST) also indicate that creatine has an antidepressant eﬀect. These studies suggest that creatine may, in addition to its role in energy storage, function as a neurotransmitter. Almeida et al. 2.2. Animal Studies of Depression-Like Behavior Animal studies clearly indicate the essential role of creatine, phosphocreatine, and the creatine kinase system in the regulation of behavior and in brain development [16,17], and have provided compelling evidence of the antidepressant eﬀect of creatine. Allen et al. [18] evaluated the eﬀect of creatine supplementation on depression-like behavior, measured via the forced swim test (FST), in rats. In their studies, the wire suspension test (WST) was used to control for motor ability. In one experiment, 30 female rats were given either no creatine, 2% creatine by weight, or 4% creatine by weight. In another experiment, 36 male rats were exposed to the same dietary protocols and behavioral tests. Female rats receiving 4% creatine exhibited signiﬁcantly longer latency to immobility on the FST than controls, suggesting reduced depression-like behavior, though there was no diﬀerence 3 of 25 Biomolecules 2019, 9, 406 between groups in the WST. Surprisingly, male rats maintained on 4% creatine showed reduced time to immobility and increased immobility in the FST, and again no diﬀerence in the WST. In a later study, the investigators used a similar protocol to assess the impact of creatine supplementation on response to the antidepressant ﬂuoxetine. They found that female rats maintained on 4% creatine by weight for 5 weeks exhibited reduction in depressive behavior on the FST, and that the addition of creatine to ﬂuoxetine enhanced the antidepressant eﬀect of ﬂuoxetine. Analysis of estrous cycle data for the animals indicated that ovarian hormones likely aﬀected the response to creatine, with the antidepressant eﬀects in females occurring in the proestrous and estrous phases [19]. To further explore the eﬀect of gonadal hormones on creatine’s antidepressant eﬃcacy, Allen and colleagues later conducted two related experiments. In the ﬁrst experiment, male rats underwent either gonadectomy or sham surgery. Gonadectomized rats were then implanted with a supplemental testosterone capsule or an empty capsule. Sham-treated males did not demonstrate any signiﬁcant change in performance on the FST with creatine supplementation, while gonadectomized males who received testosterone exhibited a non-signiﬁcant trend toward reduced depressive behavior on the FST with increasing doses of creatine, and gonadectomized males who did not receive testosterone exhibited a non-signiﬁcant trend toward worsening depressive behavior on the FST with increasing creatine doses. In the second experiment, female rats were either ovariectomized or sham-treated; a subset of those who were ovariectomized were treated with either estradiol, progesterone, the combination, or sesame oil vehicle only. 2.2. Animal Studies of Depression-Like Behavior [30] found that radiolabeled creatine is released from stimulated brain tissue in a fashion that appeared consistent with action-potential dependence—for instance, creatine was not released if the culture medium lacked calcium, which is necessary for the degranulation of synaptic vesicles. It has also been found that the antidepressant-like eﬀect of exogenous creatine in mice in the TST is blocked by compounds that inhibit PKA, PKC, CAMK-II, and MEK1/2, a group of protein kinases that have been implicated in 4 of 25 Biomolecules 2019, 9, 406 depression, suggesting that the antidepressant eﬀect of creatine is mediated by these pathways [31]. The group later showed that the antidepressant eﬀect of creatine in the TST was blocked by compounds that inhibit PI3K, such as wortmannin and rapamycin, indicating that its antidepressant eﬀect involves Akt, mTOR, and GSK3, among other intracellular signals [32]. In a related study, they found that the eﬀect of creatine in the TST in rats exposed to corticosterone was similar to that of ketamine (a novel antidepressant), and that these eﬀects were reduced for both compounds by substances that targeted the PI3K/Akt and mTOR pathways [33,34]. These intracellular signals have been implicated in the pathogenesis of depression [35,36], potentially because of their eﬀects on synaptic sprouting, mediated by BDNF [37]. Creatine may interact with other neurotransmitter systems, such as the monoamines and adenosine. The antidepressant eﬀect of creatine in the TST is blocked by compounds that inhibit serotonin synthesis [38], and enhanced by co-administration with SSRIs like ﬂuoxetine [38]. Likewise, haloperidol and other dopamine receptor antagonists reduce the anti-immobility eﬀect of creatine in the TST, while this eﬀect is enhanced by co-administration with dopaminergic compounds such as bupropion [39], implying that creatine may interact with the dopaminergic system. The antidepressant eﬀect of creatine in a rodent model is also attenuated by compounds that block adenosine receptors, and enhanced by compounds that agonize those receptors [40,41]; adenosine receptors, too, have been implicated in the etiology of depression [42]. Finally, creatine may contribute to an antidepressant response by reducing oxidative and nitrosative stress. It has been observed that creatine can reduce glutamate-induced neuronal excitotoxicity, as it reduced the production of reactive oxygen species and mono-nitrogen oxides; this ability appeared to be dependent on its antioxidant eﬀect, as it also reduced the eﬀects of exposure to hydrogen peroxide [43]. 2.3. Altered Brain Bioenergetics in Human Depression 2.3. Altered Brain Bioenergetics in Human Depression 2.3. Altered Brain Bioenergetics in Human Depression Hibbeln and colleagues [56] found, in a sample of 9668 male participants in the Avon Longitudinal Study of Parents and Children, that persons with vegetarian diets (n = 350) had greater depression scores (on the Edinburgh Postnatal Depression Scale or EPDRS) and a greater risk of clinically signiﬁcant depressive symptoms (EPDRS total score >= 10) than those with omnivorous diets, after adjusting for sociodemographic confounds. In a related study involving both men and women [57], however, there was no association between a vegetarian diet and developing clinically signiﬁcant depressive symptoms on the EPDRS, deﬁned in this case as scores > 12. Similarly, Jin et al. [58] noted that vegetarians in a sample of 892 participants in the Mediators of Atherosclerosis in South Asians Living in America (MASALA) study had 43% lower odds of exhibiting signiﬁcant depressive symptoms, while Beezhold et al. observed, in a study of 138 Seventh Day Adventists [59], that vegetarians reported less negative symptoms than omnivores on both the Proﬁle of Mood States (POMS) and the Depression Anxiety Stress Scale (DASS). In a related study, Beezhold and colleagues [60] conducted an online survey of persons who participated in diet-related social media sites and found that those who followed vegan and vegetarian diets had less anxiety and stress on the DASS than omnivores. Sanchez-Villegas and colleagues [61] reported that in the Seguimiento Universidad de Navarra (SUN) cohort study, adherence to a pro-vegetarian dietary pattern was associated with a reduced incidence of depression in 15,093 Spanish persons followed for an average of 8.5 years, while Velten et al. [62] found that having a non-vegetarian diet was associated with greater positive mental health in a sample of 15,396 German and Chinese students. Still, there is evidence that creatine supplementation can improve cognitive performance in vegetarians, as supplementation with creatine at 5 gm per day for 6 weeks improved performance on backward digit span and Raven’s Progressive matrices compared to placebo in vegetarian subjects [63]. Medical conditions associated with relative hypoxia, such as asthma and chronic obstructive pulmonary disease (COPD), are associated with increased rates of depression and suicide [64–68]. COPD is also linked to increased odds of suicidal ideation and suicide attempts compared to non-hypoxic chronic medical conditions [66,67], and the risk of depression in COPD is almost twice that in non-hypoxic illnesses [68]. 2.3. Altered Brain Bioenergetics in Human Depression Multiple sources of evidence, from epidemiology, genetics, biochemistry, and neuroimaging, indicate that bioenergetic abnormalities contribute to the development of depressive symptoms in both MDD and bipolar disorder (BD). Together, they suggest that compounds that might enhance brain energy storage, like creatine, could contribute to the treatment of depression. Many clinical conditions that are associated with impaired energy storage and bioenergetics synthesis are also associated with depression. Depression frequently aﬄicts persons suﬀering from chronic medical illness, including both type 1 and type 2 diabetes [44,45]. Depression is three times more prevalent in people with type 1 diabetes than in the general population, and twice as common in type 2 diabetes [46]. Similarly, type 1 diabetes is associated with changes in energy metabolism, and mitochondrial function plays a primary role in the treatment and prevention of long term consequences of the disorder [47]. A case-control study showed that brain energy metabolism is abnormal in type 1 diabetes as represented by the PCr/ATP ratio [48]. Furthermore, phosphorus-31 magnetic resonance spectroscopy (31P MRS) studies in the heart showed altered energy homeostasis and decreased PCr/ATP ratios in type 1 diabetes patients, identical to the pattern observed in the brain [49–51]. Dietary patterns that may reduce creatine intake are also associated with the risk of depression in some studies, although results are mixed. Li and colleagues [52] observed that elderly men who followed a vegetarian diet had a higher risk of depression, more severe symptoms of depression, and increased scores on the Geriatric Depression Scale, though a similar result was not found for women. Matta et al. [53] also found evidence that a vegetarian diet was associated with increased depressive symptoms on the CES-D in a large (n = 90380) cross-sectional study of French persons, but observed, in addition, that any dietary restrictions were associated with increased symptoms of depression, not merely restriction of the intake of meat, ﬁsh, eggs, or dairy. Larssen and colleagues [54] reported that among 2041 Swedish and Norwegian students, vegetarian diet was associated with increased self-reported frequency of depressive episodes in both males and females. Although the apparent associations between depression and vegetarian or vegan diets may be related to nutrient 5 of 25 Biomolecules 2019, 9, 406 deﬁciencies, including deﬁciencies of creatine intake, it has also been suggested that the onset of mental disorders may precede the adoption of a vegetarian diet in some cases [55]. 2.3. Altered Brain Bioenergetics in Human Depression Cigarette smoking, which causes relative hypoxia independent of associated lung diseases [69], is also linked to increased risk for suicide and depression [70]. In adolescents, smoking increases the odds of developing depression by 1.7 times compared to non-smokers [71]. Current smoking in adults is linked in a dose-dependent fashion to increases in suicide rates [72]; long-term abstinence reduces this risk, while relapse precedes a return to high risk [73]. Poorly-controlled asthma is also associated with an increased incidence of suicide rates compared to remitted asthma [64], and suicide rates for teens with asthma are more than double those of teens without asthma [65]. There is also extensive, and growing, evidence accumulated by our group and others that increased altitude of residence, which may be associated with chronic relative hypoxia, is a risk factor for depression, suicide, and related adverse psychiatric outcomes [74–88]. In conjunction with this, it has been show that simulated high altitude can produce depression in a rodent model, and that these symptoms are not responsive to most antidepressants [89–92]. 31P MRS studies have also indicated that increased altitude of residence is associated with alterations in cerebral bioenergetic signatures that are similar to those seen in depression (described below) [93,94]. There is increasing reason to believe that mood disorders themselves involve alterations in brain bioenergetics, in some cases related to underlying abnormalities in mitochondria and mitochondrial activity, including changes in the mitochondrial genome that aﬀect oxidative phosphorylation and mitochondrial proliferation, increased frequency of the common mtDNA deletion in depressed persons, and lower levels of mtDNA expression in depression [95–98]. Inherited mitochondrial disorders are associated with an increased risk of depression in both pediatric patients and adults [99], where MDD may be the initial symptom of a mitochondrial disorder; depression may aﬀect up to 54% of patients 6 of 25 Biomolecules 2019, 9, 406 with such disorders [100]. Children with mitochondrial disorders exhibit increased rates of depression compared to the general population [101], with higher rates of mood and anxiety disorders in matrilineal relatives (mitochondrial genes follow a matrilineal pattern of inheritance) [102]. There is also peripheral evidence of abnormal mitochondrial activity in depressed subjects. Gardner et al. [103] performed muscle biopsies on 28 patients with MDD and examined rates of mitochondrial ATP production and enzyme levels, as well as the frequency of mitochondrial genome deletions. 2.3. Altered Brain Bioenergetics in Human Depression They found that, compared to controls, ATP production rates and mitochondrial enzyme levels were lower, and the frequency of mitochondrial genome deletions higher, in the depressed patients. These diﬀerences may be due to alterations in mitochondrial genetics. g Biochemical studies of persons with depression indicate altered bioenergetic signatures. Agren and Niklasson demonstrated increased creatine levels in the CSF of persons with MDD, which were positively correlated with CSF levels of dopamine and serotonin metabolites [104]. The authors reported a similar ﬁnding in an earlier, smaller study [105]. It has also been shown that peripheral creatine kinase levels are signiﬁcantly higher in persons with non-psychotic major depression than in other groups of psychiatric patients with psychotic disorders [106]. Neuroimaging studies also indicate altered bioenergetics in depression. Cerebral glucose metabolism is frequently noted to be abnormal in persons with depression in positron emission tomography studies, particularly in the prefrontal cortex [107–112]. Proton (1H) and phosphorus (31P) magnetic resonance spectroscopy (MRS) studies that allow the measurement of cerebral metabolite concentrations indicate that both unipolar and bipolar depression are associated with alterations in metabolites related to cerebral energy utilization and storage (Figure 1). Although essentially all spectroscopic studies provide at least indirect information about brain energy homeostasis, as they report metabolite levels that indicate the rates of synthetic processes, synaptogenesis, or membrane turnover, we focus here on studies that report directly on the measurement of nucleotide triphosphate (NTP) (which include levels of adenosine triphosphate, the primary energetic compound in cells) and creatine or phosphocreatine (Table 1). Although several studies in persons with depression have reported no diﬀerence in total creatine concentrations ([tCr]), which include concentrations of both creatine ([Cr]) and phosphocreatine ([PCr]), in multiple brain regions [113–122], others indicate increased [tCr] in some regions, such as in the inferior prefrontal white matter (WM) [123] and left caudate [124]. Studies have shown reduced [tCr] in MDD in the left dorsolateral prefrontal cortex (PFC) [125], posterior cingulate cortex (PCC) [126], and left hippocampus (HC) [127]. A study in of geriatric depression found reduced [tCr] in the PFC in persons with remitted depression compared to healthy controls [128]. It was also found that after electroconvulsive therapy patients with MDD exhibited increases in [tCr] in the dorsal anterior cingulate cortex (ACC) and subgenual ACC. 31P MRS studies can measure [PCr] as well as total nucleotide triphosphates ([tNTP]), and beta nucleotide triphosphates ([β-NTP]) such as adenosine triphosphate ([ATP]). 2.3. Altered Brain Bioenergetics in Human Depression Abbreviations: PCr: phosphocreatine; α/β/γ-NTP: α/β/γ-nucleoside triphosphate; PME: phosphomonoester; PDE: phosphodiester; Pi: inorganic phosphate. Figure 1. Example phosphorus 31 magnetic resonance spectrum from the frontal lobes of a single subject. Abbreviations: PCr: phosphocreatine; α/β/γ-NTP: α/β/γ-nucleoside triphosphate; PME: phosphomonoester; PDE: phosphodiester; Pi: inorganic phosphate. Figure 1. Example phosphorus 31 magnetic resonance spectrum from the frontal lobes of a single subject. Abbreviations: PCr: phosphocreatine; α/β/γ-NTP: α/β/γ-nucleoside triphosphate; PME: phosphomonoester; PDE: phosphodiester; Pi: inorganic phosphate. Figure 1. Example phosphorus 31 magnetic resonance spectrum from the frontal lobes of a single subject. Abbreviations: PCr: phosphocreatine; α/β/γ-NTP: α/β/γ-nucleoside triphosphate; PME: phosphomonoester; PDE: phosphodiester; Pi: inorganic phosphate. 31P MRS studies can measure [PCr] as well as total nucleotide triphosphates ([tNTP]), and beta nucleotide triphosphates ([β-NTP]) such as adenosine triphosphate ([ATP]). Kato et al. (1992) found that [PCr] were significantly reduced in persons with depression compared to persons who were euthymic, with lower [PCr] in those with more severe depression. The study included persons with both BD and MDD [129]. Moore et al. first demonstrated that basal ganglia [β-NTP] were reduced in depressed subjects [130]. Later, it was shown that frontal cortical [β -NTP] were reduced in depressed subject [131]. Renshaw et al. [132] found that, although basal ganglia [β-NTP] and total purine levels did not differ between depressed subjects and healthy controls overall in their sample, in the subgroup of depressed subjects who responded to fluoxetine, [β-NTP] were 21% lower. In female adolescents with depression, baseline depression severity is negatively correlated with [β-NTP] [133]. Volz et al. (1998) found, in subjects with depression who were mostly taking antidepressants, that frontal cortical [ATP] were reduced in depression [131]. In some studies, [PCr] and [B-NTP] have been found to be unchanged in MDD [129,134,135]. In some studies, [PCr] and [B-NTP] have been found to be unchanged in MDD [129,134,135]. One reason for these negative ﬁndings, however, may be a failure to segment the brain regions studied into gray matter (GM) and white matter (WM). When segmentation is used, diﬀerences in brain energy storage that are intrinsic to the diﬀerent metabolic properties of GM and WM may be revealed. When patients with MDD were compared to healthy controls and whole brain metabolites were segmented into GM and WM, it was observed that total tissue (GM+WM) [β-NTP] and [tNTP] were lower in depressed subjects and that [tNTP] decreased after 12 weeks of treatment with sertraline [136]. 2.3. Altered Brain Bioenergetics in Human Depression Kato et al. (1992) found that [PCr] were signiﬁcantly reduced in persons with depression compared to persons who were euthymic, with lower [PCr] in those with more severe depression. The study included persons with both BD and MDD [129]. Moore et al. ﬁrst demonstrated that basal ganglia [β-NTP] were reduced in depressed subjects [130]. Later, it was shown that frontal cortical [β -NTP] were reduced in depressed subject [131]. Renshaw et al. [132] found that, although basal ganglia [β-NTP] and total purine levels did not diﬀer between depressed subjects and healthy controls overall in their sample, in the subgroup of depressed subjects who responded to ﬂuoxetine, [β-NTP] were 21% lower. In female adolescents with depression, baseline depression severity is negatively correlated with [β-NTP] [133]. Volz et al. (1998) found, in subjects with depression who were mostly taking antidepressants, that frontal cortical [ATP] were reduced in depression [131]. 31P MRS studies can measure [PCr] as well as total nucleotide triphosphates ([tNTP]), and beta nucleotide triphosphates ([β-NTP]) such as adenosine triphosphate ([ATP]). Kato et al. (1992) found that [PCr] were signiﬁcantly reduced in persons with depression compared to persons who were euthymic, with lower [PCr] in those with more severe depression. The study included persons with both BD and MDD [129]. Moore et al. ﬁrst demonstrated that basal ganglia [β-NTP] were reduced in depressed subjects [130]. Later, it was shown that frontal cortical [β -NTP] were reduced in depressed subject [131]. Renshaw et al. [132] found that, although basal ganglia [β-NTP] and total purine levels did not diﬀer between depressed subjects and healthy controls overall in their sample, in the subgroup of depressed subjects who responded to ﬂuoxetine, [β-NTP] were 21% lower. In female adolescents with depression, baseline depression severity is negatively correlated with [β-NTP] [133]. Volz et al. (1998) found, in subjects with depression who were mostly taking antidepressants, that frontal cortical [ATP] were reduced in depression [131]. 7 of 25 Biomolecules 2019, 9, 406 Figure 1. Example phosphorus 31 magnetic resonance spectrum from the frontal lobes of a single subject. Abbreviations: PCr: phosphocreatine; α/β/γ-NTP: α/β/γ-nucleoside triphosphate; PME: phosphomonoester; PDE: phosphodiester; Pi: inorganic phosphate. Figure 1. Example phosphorus 31 magnetic resonance spectrum from the frontal lobes of a single subject. Abbreviations: PCr: phosphocreatine; α/β/γ-NTP: α/β/γ-nucleoside triphosphate; PME: phosphomonoester; PDE: phosphodiester; Pi: inorganic phosphate. Figure 1. Example phosphorus 31 magnetic resonance spectrum from the frontal lobes of a single subject. 2.3. Altered Brain Bioenergetics in Human Depression It appears that [tCr] is reduced in several brain regions in BD, including in the frontal lobes [141], cerebellar vermis [142], hippocampi [143], caudate [144], medial PFC [145,146], dorsolateral PFC WM [146], and PCC [126], although some studies indicate that [tCr] is increased in BD in several brain regions [147–149]. Several studies have also failed to ﬁnd alterations in [tCr], including in the left dorsolateral PFC [122,150,151] and ACC [152,153]. One study found that there were no signiﬁcant diﬀerences in [tCr] between unmedicated BD subjects and controls in a variety of GM and WM regions including the medial frontal cortex, ACC, putamen, caudate, insula, thalamus, parietal cortex, and occipital cortex [147]. The study did, however, ﬁnd that the severity of bipolar depression was inversely correlated with [tCr]. Using 31P MRS, Kato et al. [129] found that [PCr] trended toward being reduced in euthymic subjects with a history of BD. They later demonstrated lower [PCr] in subjects with BD type II compared to controls, though no diﬀerence in [PCr] in subjects with BD type I [154]. In a related study, left ventrolateral PFC [PCr] was reduced in euthymic BD subjects compared to controls [155]. It has also been shown that [PCr] is reduced in the whole brain as well as right hemisphere GM in bipolar subjects irrespective of mood state [156]. Other studies indicate that there are no signiﬁcant diﬀerences between bipolar subjects’ [PCr] and those of healthy controls [157–164]. A major limitation of these studies, however, is that subjects were often in diﬀerent mood states, and bioenergetics markers could vary signiﬁcantly between mania, euthymia, and depression. Table 1. Studies reporting phosphocreatine and total creatine levels in major depressive disorder. 2.3. Altered Brain Bioenergetics in Human Depression When the authors compared GM and WM, however, they found that [tNTP] was reduced in WM but not in GM before treatment. In a study of older subjects with MDD, again with tissue segmentation, increased WM [β-NTP] and increased GM [PCr] were positively associated with executive function [137]. In a larger study encompassing 50 subjects with MDD, [PCr] was signiﬁcantly elevated in GM in depression but reduced in WM, while depression ratings were correlated with GM [PCr], but not with WM [PCr] [138]. One reason for these negative findings, however, may be a failure to segment the brain regions studied into gray matter (GM) and white matter (WM). When segmentation is used, differences in brain energy storage that are intrinsic to the different metabolic properties of GM and WM may be revealed. When patients with MDD were compared to healthy controls and whole brain metabolites were segmented into GM and WM, it was observed that total tissue (GM+WM) [β-NTP] and [tNTP] were lower in depressed subjects and that [tNTP] decreased after 12 weeks of treatment with sertraline [136]. When the authors compared GM and WM, however, they found that [tNTP] was d d i WM b i GM b f I d f ld bj i h MDD i i h These ﬁndings may indicate that increased [PCr] is associated with depression but is also a marker of antidepressant response-readiness, implying that eﬀorts to increase [PCr] could increase the likelihood of antidepressant response in some patients. This is consistent with a study by Iosifescu et al. [134], which found that subjects who responded to triiodothyronine (T3) exhibited increased [tNTP] but reduced [PCr], while elevated baseline [PCr] predicted response. There are also several reports that [PCr] can increase with antidepressant treatment. Treatment with acetyl-L-carnitine was associated with an antidepressant response and [PCr] in the prefrontal cortex (PFC) increased in 8 of 25 Biomolecules 2019, 9, 406 tandem with improvements in depression severity [139]. Similarly, adolescent females treated with ﬂuoxetine and adjunctive creatine exhibited increases in [PCr] [133]. Spectroscopic evidence for altered bioenergetics in BD has previously been reviewed [140]. BD has been associated with changes in [tCr]. 2.3. Altered Brain Bioenergetics in Human Depression Study Condition/Control Brain Region Change Compared to Controls 31P-MRS Studies reporting phosphocreatine levels in MDD and Bipolar Disorder Kato 1992 [129] MDD-D/MDD-E 30mm frontal axial slice None Kato 1994 [154] BDII/HC 30mm frontal axial slice ↓ BDI/HC 30mm F axial slice None Murashita 2000 [161] BD/HC (+ PS) OCC ↓after PS in BD except in lithium responders Pettegrew 2002 [139] MDD/HC PFC ↑after treatment associated with AD response Iosifescu 2008 [134] MDD/HC 20 mm-thick axial slice None overall but ↓baseline PCr in those who responded to T3 Sikoglu 2013 [164] BD/HC FL None Weber 2013 [155] BD-E/HC L VLP FC ↓ Yuksel 2015 [163] BD/HC (+ PS) OCC ↓after PS in HC but not BD; no diﬀerence in PCr at baseline Dudley 2016 [156] BD/HC WB WM ↓ Harper 2016 [137] MDD/HC WB WM, WB GM ↑ Harper 2017 [138] MDD/HC WB WM, WB GM ↑in GM, ↓in WM Table 1. Studies reporting phosphocreatine and total creatine levels in major depressive disorder. 9 of 25 Biomolecules 2019, 9, 406 Table 1. Cont. 2.3. Altered Brain Bioenergetics in Human Depression Study Condition/Control Brain Region Change Compared to Controls 1H-MRS Studies Reporting total creatine levels in MDD and Bipolar Disorder Hamakawa 1999 [141] BD-D/BD-E L FL ↓ Auer 2000 [113] MDD/HC B A CC None Farchione 2000 [114] MDD/HC B DL PFC None Kumar 2002 [115] MDD/HC DL WM, ACC None Cecil 2003 [142] BD-D/HC CV ↓ Deicken 2003 [143] BD- E/HC B Hippo ↓ Gruber 2003 [123] MDD/HC L PF WM ↑ Michael 2003 [122] MDD/HC L AMG None Pﬂeiderer 2003 [117] MDD/HC L A CC None Dager 2004 [147] BD/HC FL WM, BG, Thal tCr inversely correlated with depression severity Brambilla 2005 [150] BD/HC L DL PFC None Mirza 2006 [120] MDD/HC B Thal None Frye 2007 [150] BD-D/HC A CC, M CC, M PFC ↑ Gabbay 2007 [124] MDD/HC L CN ↑ Moore 2007 [152] BD/HC B ACC None Olvera 2007 [151] BD/HC L DLPFC None Patel 2008 [149] BD-D/HC B VL PFC ↑ Port 2008 [144] BD-D/HC R CN ↓ Nery 2009 [125] MDD/HC L DL PFC ↑in women ↓in men Öngür 2009 [153] BD-M/HC B ACC and POC None Venkatraman 2009 [128] MDD/HC M PFC ↓ Caetano 2011 [147] BD/HC R M PFC, L DL PFC WM ↓ Portella 2011 [118] MDD/HC B VM PFC None McEwen 2012 [116] PPD/HC B M PFC None Özdel 2012 [145] BD-E/HC B M PFC ↓ Bradley 2016 [121] MDD/HC B CN, B Put, B Thal None Li 2016 [126] MDD/HC P CC ↓ Njau 2017 [127] MDD/HC SG ACC, D ACC ↑ Rosa 2017 [119] PPD/HC B A CC, L DL PFC None A: anterior; AMG: amygdala; B: bilateral; BD: bipolar disorder; BD D: bipolar depressed state; BD E: bipolar Condition/Control Brain Region A: anterior; AMG: amygdala; B: bilateral; BD: bipolar disorder; BD-D: bipolar, depressed state; BD-E: bipolar, euthymic state; BDI: bipolar disorder type I; BDII: bipolar disorder type II; BD-M: bipolar, manic or mixed state; BG: basal ganglia; CC: cingulate cortex; CV: cerebellar vermis; D: dorsal; DL: dorsolateral; FL: frontal lobes; GM: gray matter; HC: healthy controls; Hippo: hippocampus; Ins: insula; L: left; M: medial; MCC: middle cingulate cortex; MDE: major depressive episode; MDD: major depressive disorder; MDD-D: major depressive disorder, depressed state; MDD-E; major depressive disorder, euthymic state; M: medial; OCC: occipital cortex; OFC: orbitofrontal cortex; OL: occipital lobe; P: posterior; PCr: phosphocreatine; PFC: prefrontal cortex; POC: parieto-occipital cortex; PPD: post-partum depression; PS: photic stimulation; Put: putamen; R: right; SG: subgenual; tCr: total creatine (phosphocreatine + creatine); Thal: thalamus; TL: temporal lobes; T3: triiodothyronine; V: ventral; VL: ventrolateral; WB: whole brain; WM: white matter; ↑: signiﬁcantly increased/higher; ↓: signiﬁcantly reduced/lower. 2.3. Altered Brain Bioenergetics in Human Depression A: anterior; AMG: amygdala; B: bilateral; BD: bipolar disorder; BD-D: bipolar, depressed state; BD-E: bipolar, euthymic state; BDI: bipolar disorder type I; BDII: bipolar disorder type II; BD-M: bipolar, manic or mixed state; BG: basal ganglia; CC: cingulate cortex; CV: cerebellar vermis; D: dorsal; DL: dorsolateral; FL: frontal lobes; GM: gray matter; HC: healthy controls; Hippo: hippocampus; Ins: insula; L: left; M: medial; MCC: middle cingulate cortex; MDE: major depressive episode; MDD: major depressive disorder; MDD-D: major depressive disorder, depressed state; MDD-E; major depressive disorder, euthymic state; M: medial; OCC: occipital cortex; OFC: orbitofrontal cortex; OL: occipital lobe; P: posterior; PCr: phosphocreatine; PFC: prefrontal cortex; POC: parieto-occipital cortex; PPD: post-partum depression; PS: photic stimulation; Put: putamen; R: right; SG: subgenual; tCr: total creatine (phosphocreatine + creatine); Thal: thalamus; TL: temporal lobes; T3: triiodothyronine; V: ventral; VL: ventrolateral; WB: whole brain; WM: white matter; ↑: signiﬁcantly increased/higher; ↓: signiﬁcantly reduced/lower. Several studies have suggested dynamic abnormalities in PCr synthesis in BD. In subjects with BD who were treated with lithium, [PCr] fell after photic stimulation (a method of increasing visual Several studies have suggested dynamic abnormalities in PCr synthesis in BD. In subjects with BD who were treated with lithium, [PCr] fell after photic stimulation (a method of increasing visual 10 of 25 Biomolecules 2019, 9, 406 cortex activity) in subjects who did not respond to lithium but remained stable in lithium-responsive subjects and controls [161]. This suggested that subjects with BD have a deﬁcit in PCr synthesis that is ameliorated by lithium. In a similar study [163], however, [PCr] fell in response to photic stimulation in controls, but not in bipolar subjects, even though PCr/ATP ratios were reduced in BD, and [ATP] fell in BD in response to photic stimulation. A study using magnetization transfer to estimate the rate constant for the creatine kinase reaction in BD found that it did not diﬀer signiﬁcantly between euthymic or depressed bipolar subjects and controls [162]. In contrast, subjects with a ﬁrst episode of bipolar depression (BD) or mania and psychotic features exhibited a 13% reduction in the rate constant for the creatine kinase reaction [159]. The diﬀerence between these studies may be due to the absence of psychosis among the subjects in the ﬁrst study or, again, to the diﬀerence in mood states, as many of the subjects in the second study were manic. 2.4. Biochemical Eﬀects of Creatine Supplementation Evidence that brain creatine and phosphocreatine metabolism are altered in depression has suggested that they have promise as antidepressant treatments. Creatine monohydrate, the most common commercially-available form of creatine, has the ability to alter brain creatine levels. The ingestion of 20 g per day of creatine for a month increased brain creatine levels measured by 1H MRS by, on average, 4.7% in gray matter and 11.5% in cerebral white matter, though there was signiﬁcant inter-subject variability that appeared to be related to both gender and body mass [165]. In a placebo controlled study, Lyoo et al. found that supplementation with 0.3 g/kg/d for one week, followed by 0.03 g/kg/d for one week, increased brain [tCr]/[n-acetyl aspartate] ratios by 8.1%, and brain [tCr]/[choline] ratios by 9.3%, as measured by 1H MRS. Similar changes were not observed in a placebo control group. The researchers also found, using 31P MRS, that creatine supplementation reduced [β-NTP] signiﬁcantly and produced a trend toward increased [PCr] [166]. The overall eﬀect of creatine supplementation on [PCr] is unclear, but in a study of creatine supplementation in a variety of tissues in several animal species, it appeared that, while the ratio of [PCr] to [Cr] did not change, total [PCr] and [Cr] both increased [167]. This appears to be consistent with ﬁndings in human skeletal muscle [168,169]. It was also shown that creatine supplementation at 20g/day × 5 days followed by 5 g/day × 2 days in healthy subjects reduced the fMRI BOLD signal in the V1 region in a visual stimulation paradigm compared to placebo [170]. Creatine supplementation also signiﬁcantly improved performance on the backward digit span, a test of cognition. The authors speculated that creatine reduces the BOLD signal by increasing local energy stores and thereby reducing the metabolic stimulus for cortical blood ﬂow, or, instead, promoting an increase in the eﬃciency of O2 uptake with associated attenuation of the BOLD signal. 2.5. Clinical Studies in Conditions Related to Depression Subjects who received 20 g of creatine per day exhibited signiﬁcantly less decline in cognitive performance, motor performance and mood state than subjects who had received placebo after 24 h of sleep deprivation [177]; in a subsequent study, creatine supplementation improved performance on central executive tasks after 36h of sleep deprivation, though it did not aﬀect mood [178]. Another small study of healthy young adults who were not sleep-deprived found no eﬀect of creatine on cognitive performance compared to placebo over 6 weeks [179]. In the elderly, however, creatine supplementation at 20 g per day for two weeks appears to improve a broad array of cognitive measures [180]. Inspired by ﬁndings like those above, Kaptsan and colleagues [181] examined whether creatine could improve neurocognitive and other symptoms in schizophrenia. The investigators randomized 12 patients with schizophrenia to creatine 3 g or 5 g per day or placebo for 3 months in a double-blind, crossover fashion. They found that there was no signiﬁcant diﬀerence between the groups with respect to improvements in neurocognitive function or on study measures such as the Positive and Negative Symptoms Scale (PANSS) or the Clinical Global Impression-Improvement scale, though there were no signiﬁcant adverse eﬀects. The study did not speciﬁcally assay for improvements in mood related to creatine, though no signiﬁcant diﬀerence were observed in the PANSS Negative Symptom or PANSS General Psychopathology subscales, which might indirectly capture depressive symptoms. Amital et al. (2006) provided oral creatine to subjects with PTSD who were taking SSRIs or SNRIs, with or without comorbid depression. Subjects received 3 g/day of creatine for 1 week, then 5 gm/day for three weeks. The authors found that subjects exhibited signiﬁcant improvements in the HAM-D, HAM-A, Sheehan Disability Scale, and the Clinician Assessment of PTSD Symptoms, and that improvements were greater in the six subjects who had comorbid MDD [182]. Fibromyalgia is a rheumatological condition that is often associated with depression [183]. Creatine was thought to have potential as a treatment for ﬁbromyalgia because of its beneﬁts for muscle strength and pain [184]. Amital et al. [185] ﬁrst reported the potential antidepressant beneﬁt of creatine in persons with ﬁbromyalgia based on the experiences of one of the participants in the PTSD study cited above [182]; the patient was a 52-year-old who was treated with creatine in addition to citalopram for 4 weeks. 2.5. Clinical Studies in Conditions Related to Depression In human trials, creatine has been studied extensively for the treatment of neuropsychiatric conditions other than depression—especially neurodegenerative illnesses such as Parkinson disease (PD) and Huntington disease (HD), as well as in other neuromuscular disorders. The results of many of these studies are reviewed elsewhere in this issue, but we touch on some of this work here because it provided preliminary clinical evidence that creatine supplementation could improve mood. A 2-year study of the eﬀect of creatine supplementation on the progression of Parkinson disease randomized 60 subjects between placebo or three stages of creatine dosing: 20 g/day for 6 days, 2 g/day for 6 months, and 4 g/day for 18 months. Although there was no signiﬁcant eﬀect on primary PD symptoms, the investigators observed a signiﬁcant improvement, relative to placebo, in the “mentation, behavior, mood” subscale of the Uniﬁed Parkinson Disease Rating Scale (UPDRS) [171]. Unfortunately, a subsequent multisite study involving 1741 subjects randomized between creatine monohydrate 10 g/day and placebo for 5 years found no diﬀerence between groups on the UPDRS mental subscale, nor on the Beck Depression Inventory [172]. 11 of 25 Biomolecules 2019, 9, 406 A separate study also failed to ﬁnd evidence of a beneﬁcial eﬀect of creatine on mood in a trial of creatine and strength training in older women (who were without clinical depression at baseline). The women were between the ages of 60 and 80, and were randomized between four arms: creatine alone, placebo alone, creatine plus strength training, and placebo plus strength training. The investigators found that mood changes in the creatine groups did not diﬀer from those in the placebo groups, but that strength training, whether added to creatine or placebo, was associated with improvement in mood. p Persons with depression may exhibit mental fatigue and there are phenomenological similarities between mental fatigue and depression [173,174]. Kato et al. found, in healthy subjects, that the rate of occipital cortex phosphocreatine depletion in response to photic stimulation is associated with the rate of improvement after rest during the Uchida-Kraepelin test (UKT), a paradigm for measuring mental fatigue [175]. In a follow-up to this study, Watanabe et al. discovered that supplementation with 8 g of creatine per day for 5 days reduced mental fatigue on the UKT compared to placebo [176]. Creatine has also been shown to improve cognitive performance in persons subjected to sleep deprivation. 2.6. Clinical Trials of Creatine for Depression Creatine monohydrate has, to this point, been studied only in small clinical trials for the treatment of MDD, BD, and depression associated with methamphetamine use disorder (see Table 2). With a few exceptions, most trials to date have been positive. To our knowledge, the ﬁrst trial to examine creatine for the treatment of depression was conducted by Roitman and colleagues [187]. They examined eight patients with MDD and two patients with BDU and treated them with open-label creatine at 3–5 g/day for four weeks, as an add-on to their existing antidepressants or mood stabilizers. Although both of the patients with BD developed mania/hypomania and were withdrawn from the study, seven out of the eight patients with MDD exhibited signiﬁcant improvement while receiving creatine. The adverse events reported in the MDD group were mild and transient. Subsequently, Kondo et al. [133] conducted an open-label trial of creatine supplementation in female adolescents with MDD who had not responded adequately to SSRIs. The study included ﬁve girls who had taken ﬂuoxetine for at least 8 weeks but who continued to have clinically signiﬁcant depressive symptoms, as evidence by a score on the Children’s Depression Rating Scale-Revised (CDRS-R) of at least 40. The subjects were treated with 4g of creatine per day for 8 weeks. The mean CDRS-R score fell by 50.6% between baseline and week 8. The baseline CDRS-R score was correlated with pH as measured by 31P MRS, and inversely correlated with [β-NTP]. Creatine-treated subjects exhibited a signiﬁcant increase in whole brain [PCr] compared to controls. In a follow up to this study, Kondo and colleagues randomized 34 adolescent and young-adult women with MDD who had not responded to an SSRI to placebo or creatine monohydrate in doses of 2 g, 4 g, or 10 g/day for 8 weeks. They found that creatine increased [PCr] in the frontal cortex compared to placebo, and that higher [PCr] were associated with lower depression scores. There was not a signiﬁcant diﬀerence between creatine groups with respect to the change in [PCr] [170]. 2.5. Clinical Studies in Conditions Related to Depression She exhibited improvements in both her Hamilton Depression Rating Scale (HAM-D) scores (which fell from 24 to 16) and symptoms of ﬁbromyalgia. In a randomized, placebo-controlled trial for ﬁbromyalgia lasting 16 weeks, supplementation with creatine at 20 g per day for 5 days, followed by 5 g per day for 15 weeks, produced signiﬁcant improvements in mental health scores on the Short-Form 36 disability rating scale compared to placebo, though no signiﬁcant diﬀerence was observed in the depression or fatigue subscales of the Fibromyalgia Impact Questionnaire [186]. 12 of 25 Biomolecules 2019, 9, 406 12 of 25 2.6. Clinical Trials of Creatine for Depression In a later study, 52 adult women up to age 65 with depression (HAM-D > 16 and conﬁrmation by the Structured Clinical Interview for DSM-IV) were randomized 1:1 between escitalopram (10 mg per day for one week and then 20 mg per day for seven weeks) plus creatine (3 g per day for one week, then 5 g per day for seven weeks) or matched placebo, to ascertain whether creatine enhanced response to SSRIs. The subjects were otherwise unmedicated before the trial and most (78.8%) were medication-naïve. The creatine-treated group exhibited a superior antidepressant response, compared to the placebo group, as early as week 2, which continued for the 8 weeks of the study; the mean reduction in HAM-D score for the creatine group at week 8 was 79.7%, while in the placebo group it was 62.5%. The creatine group did not experience signiﬁcantly more adverse eﬀects than the placebo group [188]. Yoon and colleagues later reported neuroimaging results from the women participating in the study above. They hypothesized that creatine administration would increase structural connectivity between rich-club hub network connections, as these connections are energy-demanding. Using 1H MRS and structural connectivity imaging, they found that prefrontal N-acetylaspartate levels increased with creatine compared to placebo, and also that rich-club hub network connections in the creatine group increased signiﬁcantly more than in the placebo group or in healthy controls. There was, however, no evidence that changes in rich-club connectivity were associated with the degree of antidepressant response. 13 of 25 Biomolecules 2019, 9, 406 Table 2. Clinical trials involving creatine for the treatment of depression. 2.6. Clinical Trials of Creatine for Depression Study Population (n) Design Creatine Dose Duration Eﬀect Signiﬁcant Adverse Eﬀects Related to Creatine Roitman 2007 [186] MDD-D (n = 8); BD-D (n = 2) Open-label, adjunctive 3–5 g/day 4 weeks Average HAM-D scores declined from 23.1 at baseline to 12.6 at week 4 Both bipolar subjects developed hypomania/mania Kondo 2011 [133] Adolescent girls with MDD-D (n = 5) Open-label, adjunctive 4 g/day 8 weeks The mean CDRS-R score fell by 50.6% None Kondo 2016 [170] Adolescent and young-adult women with MDD-D (n = 34) Open-label, adjunctive, dose-ranging 2 g, 4 g, or 10 g/day 8 weeks Creatine increased frontal cortical phosphocreatine levels in a fashion associated with lower depression ratings None Lyoo 2012 [188] Women with MDD-D (n = 52) Randomized, double-blind, placebo-controlled, adjunctive 3 g/day × 1 week then 5 g/day × 7 weeks 8 weeks HAM-D scores in the creatine group fell by 79.7% by week 8, compared to 62.5% in the placebo group None Nemets 2013 [189] MDD-D (n = 18) Randomized, double-blind, placebo-controlled, adjunctive 5 g/day or 10 g/day 4 weeks No signiﬁcant diﬀerence between creatine and placebo in HAM-D scores None Hellem 2015 [193] Methamphetamine dependence with depression (n = 14) Open-label, monotherapy 5 g/day 8 weeks Mean HAM-D scores fell to 10.4 by week 2, representing response Gastrointestinal symptoms (n = 5) and muscle cramps (n = 2) Kious 2017 [190] Women with MDD-D (n = 15) Open-label, adjunctive 5 g/day (with 5-HTP 200 mg twice daily) 8 weeks HAM-D scores improved by ~60% by week 8 None Toniolo 2017 [191] BD-D (n = 18) Randomized, double-blind, placebo-controlled, adjunctive 6 g/day 6 weeks Signiﬁcant improvement in verbal ﬂuency but no signiﬁcant changes in other measures reported None Toniolo 2018 [192] BD-D (n = 53) Randomized, double-blind, placebo-controlled, adjunctive 6 g/day 6 weeks No signiﬁcant diﬀerence in MADRS scores between groups, but MADRS remission rate was signiﬁcantly greater in creatine group (52.9% vs. 11.1%) Two participants in creatine group developed hypomania/mania BD-D: bipolar disorder, depressed state; CDRS-R: Children’s Depression Rating Scale, Revised; HAM-D; 17-item Hamilton Depression Rating Scale; MADRS: Montgomery-Asberg Depression Rating Scale MDD-D: major depressive disorder, depressed state. Table 2. Clinical trials involving creatine for the treatment of depression. 14 of 25 Biomolecules 2019, 9, 406 The only negative trial of creatine in MDD to our knowledge was conducted by Nemets and Levine [189]. 2.6. Clinical Trials of Creatine for Depression Strikingly, however, they did ﬁnd a signiﬁcant diﬀerence in the likelihood of MADRS remission (score <= 12) between the two groups; using intention-to-treat analysis, they found 52.9% remission in the creatine group, with only 11.1% remission in the placebo group. Two patients in the creatine group switched to mania/hypomania early in the study, but no other signiﬁcant adverse eﬀects were observed. Although creatine may increase the risk of developing hypomania or mania in persons with bipolar depression [186], it has been investigated in two trials for persons with bipolar depression. In the ﬁrst trial, Toniolo and colleagues [191] randomized 18 patients with bipolar depression to 6 g of creatine or placebo as augmentation to existing mood-stabilizers or antipsychotics daily for 6 weeks, and assessed the eﬀect on several measures of cognition. They found that subjects taking creatine had a signiﬁcant improvement in verbal ﬂuency but no signiﬁcant change in the other measures included. They did not report any eﬀects on the measures of mood included in the study, which included the HAM-D, Montgomery-Asberg Depression Rating Scale (MADRS), and the Young Mania Rating Scale. , g y g p g ( ), g g In a more recent study, Toniolo and colleagues [192] randomized 53 patients with BD type I or II who were currently in a depressive episode to 6g of creatine daily or matched placebo for 6 weeks, as an adjunct to their existing medications. The researchers did not identify any signiﬁcant diﬀerence between creatine and placebo on the primary endpoint, change in the MADRS after 6 weeks. Strikingly, however, they did ﬁnd a signiﬁcant diﬀerence in the likelihood of MADRS remission (score <= 12) between the two groups; using intention-to-treat analysis, they found 52.9% remission in the creatine group, with only 11.1% remission in the placebo group. Two patients in the creatine group switched to mania/hypomania early in the study, but no other signiﬁcant adverse eﬀects were observed. Hellem et al. (2015) studied the eﬀects of supplementation of 5 g of creatine per day used as monotherapy (i.e., without concomitant antidepressants) on depressive symptoms over 8 weeks in persons with methamphetamine dependence. In this open-label trial involving 14 subjects, the authors found that HAM-D scores were signiﬁcantly reduced by as soon as 2 weeks after the start of creatine supplementation. 2.6. Clinical Trials of Creatine for Depression They enrolled 18 subjects (14 women) who had received an SSRI, SNRI, or noradrenergic and speciﬁc serotonergic antidepressant (NaSSA) for at least three weeks and who continued to be depressed in a 4-week trial of adjunctive creatine at 5 g or 10 g per day, or placebo. Although two women receiving in the trial showed an early > 50% reduction in HAM-D scores, overall there was no signiﬁcant diﬀerence between either creatine dose or placebo. The authors concluded that creatine may not be eﬀective for the treatment of depression as an augmenting agent, though it is noteworthy that the period of supplementation in this study was much shorter than in the other, positive, trials. More recently, our group conducted an open-label trial involving 15 adult women who had failed to respond to adequate trials of at least one SSRI or SNRI, who were treated with 5 g of creatine daily in combination with the serotonin precursor 5-hydroxytryptophan (5-HTP) at 200 mg twice daily for 8 weeks. We found that subjects exhibited signiﬁcant improvements in HAM-D scores compared to baseline, with an average decrease of approximately 60%. There were no signiﬁcant adverse events [190]. Although creatine may increase the risk of developing hypomania or mania in persons with bipolar depression [186], it has been investigated in two trials for persons with bipolar depression. In the ﬁrst trial, Toniolo and colleagues [191] randomized 18 patients with bipolar depression to 6 g of creatine or placebo as augmentation to existing mood-stabilizers or antipsychotics daily for 6 weeks, and assessed the eﬀect on several measures of cognition. They found that subjects taking creatine had a signiﬁcant improvement in verbal ﬂuency but no signiﬁcant change in the other measures included. They did not report any eﬀects on the measures of mood included in the study, which included the HAM-D, Montgomery-Asberg Depression Rating Scale (MADRS), and the Young Mania Rating Scale. In a more recent study, Toniolo and colleagues [192] randomized 53 patients with BD type I or II who were currently in a depressive episode to 6g of creatine daily or matched placebo for 6 weeks, as an adjunct to their existing medications. The researchers did not identify any signiﬁcant diﬀerence between creatine and placebo on the primary endpoint, change in the MADRS after 6 weeks. 2.6. Clinical Trials of Creatine for Depression They also found that Beck Anxiety Inventory scores were signiﬁcantly reduced, and that brain [PCr], measured by 31P MRS, were signiﬁcantly increased after 8 weeks. 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X-linked mental retardation with seizures and carrier manifestations is caused by a mutation in the creatine-transporter gene (SLC6A8) located in Xq28. Am. J. Hum. Genet. 2002, 70, 1349–1356. 3. Conclusions Creatine is a naturally-occurring organic acid that serves as an energy buﬀer and energy shuttle in tissues, such as brain and skeletal muscle, that exhibit dynamic energy requirements. Evidence, deriving from a variety of scientiﬁc domains, that brain bioenergetics are altered in depression and related disorders is growing. Clinical studies in neurological conditions such as PD have indicated that creatine might have an antidepressant eﬀect, and early clinical studies in depressive disorders—especially MDD—indicate that creatine may have an important antidepressant eﬀect. Future work should, we think, involve larger clinical trials of creatine when used as an adjunctive treatment in MDD, extend to encompass trials of creatine as monotherapy, examine the potential eﬃcacy of creatine as an augmenting agent when combined with neurostimulation techniques such as ECT and TMS, 15 of 25 Biomolecules 2019, 9, 406 and better characterize the neurochemical and network-level eﬀects of creatine and their correlations with antidepressant response. and better characterize the neurochemical and network-level eﬀects of creatine and their correlations with antidepressant response. Author Contributions: Conceptualization, B.M.K., D.G.K., P.F.R.; Writing—Original Draft Preparation, B.M.K.; Writing—Review & Editing, B.M.K., D.G.K., P.F.R.; Supervision, P.F.R. Funding: This work was supported by the U.S. Department of Veterans Aﬀairs (VA) Rocky Mountain VISN 19 Mental Illness Research, Education and Clinical Center (MIRECC), as well as VA grant I01-CX000812 to Dr. Renshaw and VA grant I01-CX001611 to Dr. Kondo. The views in this paper are those of the authors and do not necessarily represent the oﬃcial policy or position of the Department of Veterans Aﬀairs or the United States Government. Dr. Kious was supported by a National Alliance for Research on Schizophrenia and Depression Young Investigator Grant (2016). Acknowledgments: Xian-Feng Shi provided the example 31P-MRS spectrum included in Figure 1. Conﬂicts of Interest: The authors declare no conﬂicts of interest. Conﬂicts of Interest: The authors declare no conﬂicts of interest. References [CrossRef] 10. Salomons, G.S.; van Dooren, S.J.; Verhoeven, N.M.; Cecil, K.M.; Ball, W.S.; Degrauw, T.J.; Jakobs, C. 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https://openalex.org/W4298874644	https://www.scielo.br/j/rsp/a/fyTSjqS3vyQn5hDYn6SnrRQ/?lang=pt&format=pdf	Portuguese	null	Hipovitaminose A em crianças de áreas rurais do semi-árido baiano	Revista de saúde pública/Revista de Saúde Pública	1,995	cc-by	4,007	Separatas/Reprints: Matildes da Silva Prado - Escola de Nutrição da Universidade Federal da Bahia - Rua Araújo Pinho, 32 - 40110- 150 - Canela - Salvador, BA - Brasil - Fax: (071) 245.0587 Recebido em 29.3.1994. Reapresentado em 23.4. 1995. Aprovado em 27. 6. 1995. Matildes da Silva Prado, Ana Marlúcia Oliveira Assis, Maisa Cruz Martins, Maria da Purificação Araújo Nazaré, Ioná F. Bonfim Rezende, Maria Ester Pereira Conceição Matildes da Silva Prado, Ana Marlúcia Oliveira Assis, Maisa Cruz Martins, Maria da Purificação Araújo Nazaré, Ioná F. Bonfim Rezende, Maria Ester Pereira Conceição Escola de Nutrição da Universidade Federal da Bahia- Brasil (M. S. P., A. M. O. A.), Secretaria de Saúde do Estado - Salvador - Brasil (M. C. M., M. P. A. N.), Conselho Nacional de Desenvolvimento Tecnológico/CNPq-Brasília, D. F. - Brasil (I. F. B. R., M. E. P. C. - bolsistas) Objetivou-se avaliar a distribuição e a magnitude da deficiência de vitamina A e o consumo dietético de 161 crianças de 6 a 72 meses de idade, de áreas rurais do Município de Cansanção-Bahia, Brasil. Os níveis de retinol sérico foram medidos pelo método espectrofotométrico (Bessey-Lowry modificado por Araújo e Flores, 1978). A média do retinol sérico distribuiu- se homogeneamente entre as diferentes faixas etárias. Níveis inadequados de retinol sérico (deficiente <10,0 mg/dl e baixos <20,0 mg/dl) foram detectados em 44,7% das crianças, caracterizando a deficiência como problema de saúde pública. Os níveis de retinol sérico não mostraram associação estatisticamente significante com sexo e idade das crianças, contudo as menores de 24 meses apresentaram prevalência mais alta de níveis inadequados. A principal fonte de vitamina A, disponível para essas crianças, é representada pelos carotenóides, em especial beta-caroteno. Foi observada maior diversificação no consumo dos alimentos de conteúdos moderado e baixo em vitamina A no grupo de 24 a 72 meses de idade, sem contudo assegurar níveis adequados de retinol sérico para este grupo etário. Deficiência de Vitamina A, epidemiologia. Inquéritos sobre dieta. População rural. Sommer e col.30, 1986; West e col.33, 1991; Rahmathullah e col.25, 1990; Milton e col.18, 1987; Barreto e col. 4,1994). Sommer e col.30, 1986; West e col.33, 1991; Rahmathullah e col.25, 1990; Milton e col.18, 1987; Barreto e col. 4,1994). Introdução A vitamina A é tradicionalmente conhecida como um micronutriente essencial na manutenção da inte- gridade do sistema ocular (Sommer e col.30, 1986; Olson22, 1986; WHO16, 1982), sistema imune (Nauss21,1986), divisão e diferenciação celular (De Luca e col.9, 1989). Essa deficiência geralmente apresenta-se acompa- nhada de outras carências nutricionais, e a desnutrição energético protéica é um exemplo (Venkataswamy e col.31 , 1977). É conhecido também que o processo metabólico, que envolve a vitamina A e seus precurso- res, depende da biodisponibilidade de outros nutrien- tes; a diminuição das vitaminas C e E na dieta contribui com a oxidação da vitamina A na luz intestinal e a carência de lipídeos compromete a absorção desse micronutriente em especial dos carotenóides. O avanço no conhecimento sobre esse micronu- triente permitiu estabelecer, também, a sua associ- ação com a morbi-mortabilidade infantil; esta asso- ciação tem sido demonstrada em estudos observaci- onais (Sommer e col.29,1983; Milton e col.18,1987; El Bushra e col.10,1992). Estudos de intervenção, controlados, envolvendo crianças menores de seis anos de idade de áreas onde existe a deficiência nas formas clínicas ou subclínicas, têm confirmado a redução na morbi-mortalidade para o grupo suplementado quando comparado com o controle (Muhilal e col.19, 1988; Daulaire e col.8,1992; p Estudos desenvolvidos em vários países têm demonstrado que a deficiência está ainda associada com o desmame precoce e o consumo inadequado de alimentos fontes de vitamina A preformada ou carotenóides (Mele e col.17,1991; Newman20,1993). Esses fatores podem explicar a ampla difusão da deficiência de vitamina A nos países periféricos, onde se estima que 43 milhões das crianças menores de cinco anos de idade apresentam deficiência desse micronutriente (Vital32, 1991). às escolas das localidades, com as crianças em jejum. Compareceram 223 crianças para a coleta de sangue; entretanto, não foi possível dosar o retinol em 62 delas, porque a quantidade de sangue foi insuficiente ou a criança não colaborou. Assim, o estudo foi realizado com 161 crianças. Existem evidências de que se a renda e o nível educacional elevam-se, a dieta ingerida torna-se mais diversificada e as manisfestações da deficiência A diminuem e/ou não aparecem, (Beaton e col.6,1992). Para o estudo da ingestão dietética, foi constitu- ída uma subamostra aleatória, em relação à popula- ção de estudo, representando 76,8% das crianças com informações da dosagem do retinol. * Dra. Leonor Maria Pacheco Santos - Escola de Nutrição da Universidade Federal da Bahia (Comunicação pessoal) Avaliação Bioquímica g O presente estudo teve como objetivo avaliar a distribuição e magnitude da deficiência de vitamina A e o consumo dietético de crianças de seis a setenta e dois meses de idade, residentes em áreas rurais do Município de Cansanção-Bahia. De cada criança, em jejum, foram coletados 5ml de sangue por venopunção, utilizando-se material descartável. As amostras de sangue foram colocadas em tubos de vacutener, devidamente protegidas da luz. Após a retração do coágulo (cerca de 20 min após a coleta), este material foi centrifugado a 7.000 rpm durante 10 min, para separação do soro; e imediata- mente congelado a -15 °C. As amostras foram trans- portadas sob refrigeração em caixas de isopor, cuida- dosamente lacradas, até o Laboratório de Bioquímica da Nutrição da UFBA, para análise. O método bioquímico espectrofotométrico de Bessey-Lowry, modificado por Araújo e Flores2 (1978), foi adotado para determinar os níveis de retinol sérico. As amos- tras foram dosadas em duplicata. População Integra o presente estudo a população infantil de 6 a 72 meses de idade, cadastradas pelas Associa- ções de Pequenos Agricultores, vinculadas ao Pro- jeto Cansanção (Gaudenzi e col.13, 1982), oriundas de famílias de pequenos produtores rurais das loca- lidades do Município de Cansanção-Bahia: Caeta- no, Capoeira, Lagoa das Moças e Lage de Gameleira. Após conhecer os objetivos da investigação e os procedimentos para o diagnóstico da deficiência de vitamina A, o voluntário responsável pela criança assinou um termo de consentimento e compareceu Devido à baixa prevalência de nível de retinol sérico deficiente, optou-se por agregar as classifi- cações "baixa" e "deficiente" na categoria de ní- veis retinol sérico "inadequado", para fins de aná- lise estatística. Introdução Os dados foram coletados por professores e estudantes da Escola de Nutrição da UFBA, no período de dezem- bro de 1992 a janeiro de 1993, época de safra de alimentos fontes de vitamina A. Não existem dados que caracterizem a distribui- ção e severidade da deficiência de vitamina A para toda a população brasileira; contudo, estudos obser- vacionais isolados registram para a região Nordeste inadequação dos níveis séricos de retinol, variando de 14,7 a 54,7%* (Batista Filho e Torres5, 1982); sinais clínicos foram detectados na década de 80 para essa mesma região (Santos e col.28, 1983). Caracterização da Área O Município de Cansanção está situado no Nor- deste do Estado da Bahia, numa das regiões mais secas e quentes do trópico semi-árido, a uma distân- cia de 350 km da capital, com superfície de 1.317 km2. Conforme o Censo Demográfico de 1991, apresenta uma população de aproximadamente 30.800 habitantes, sendo 23.900 residentes na zona rural e 6.900 na área urbana (IBGE12, 1991). Para interpretação dos resultados utilizou-se os critérios propostos pelo Interdepartmental Committee on Nutrition for National Defense (ICNND14, 1963) que classifica os níveis de retinol em 4 categorias: alto (> 50,0 mg/dl), aceitável (20,0 a 49,9 mg/dl), baixo (10,0 a 19,9 mg/dl) e deficiente (< 10 mg/dl). A caracterização da deficiência de vitamina A como um problema de saúde pública foi baseada nos critérios recomendados pela Pan Ame- rican Health Organization/ World Health Organization (PAHO/WHO23, 1970). Segundo es- sas instituições, a deficiência de vitamina A consti- tui problema de saúde pública quando >5,0% ou 15,0% ou mais da população apresentarem níveis sérico de retinol < 10,0 mg/dl ou < 20 mg/dl, respec- tivamente. A economia do município tem como sustentácu- lo as atividades agrícolas baseadas no cultivo de mandioca, milho, feijão e sisal; a atividade secundá- ria e complementar é representada pela ovinocultura. A pecuária é exercida de forma extensiva, por pe- quena parcela de médios e grandes proprietários existentes na área (IBGE12, 1991). Inquérito Dietético O método recordatório de 24h foi usado para estimar a ingestão de macro e micronutrientes con- sumidos pelas crianças. A mãe ou o responsável pela criança foi entrevistada em sua residência por nutricionistas e estudantes devidamente treinados. Como recurso para ajudar à mãe a recordar as porções de alimentos servidos e aumentar a confia- bilidade das informações (Witschi34, 1990), foi uti- lizado um álbum com desenhos de alimentos e suas dimensões e medidas-padrão de líquidos (Araújo e col.3,1993). A freqüência alimentar foi outro méto- do de inquérito dietético adotado para estimar o consumo mensal, semanal e diário de alimentos fontes de vitamina A. Utilizou-se a proposta do International Vitamin A Consultative Group (IVACG15, 1989) que classifica os alimentos con- forme o conteúdo de retinol equivalente, em alto, moderado e baixo, para caracterizar o padrão de consumo de vitamina A. pela criança foi entrevistada em sua residência por nutricionistas e estudantes devidamente treinados. Como recurso para ajudar à mãe a recordar as porções de alimentos servidos e aumentar a confia- bilidade das informações (Witschi34, 1990), foi uti- lizado um álbum com desenhos de alimentos e suas dimensões e medidas-padrão de líquidos (Araújo e col.3,1993). A freqüência alimentar foi outro méto- do de inquérito dietético adotado para estimar o consumo mensal, semanal e diário de alimentos fontes de vitamina A. Utilizou-se a proposta do International Vitamin A Consultative Group (IVACG15, 1989) que classifica os alimentos con- forme o conteúdo de retinol equivalente, em alto, moderado e baixo, para caracterizar o padrão de consumo de vitamina A. Infelizmente a falta de informação nas tabelas de composição de alimentos do país impossibilita quantificar a contribuição específica dos carotenóides na dieta da população estudada. Contudo, os con- sultores do IVACG, baseados em informações da FAO, agruparam os alimentos vegetais segundo a percetagem de beta-caroteno, em três categorias: alta, moderada e baixa, permitindo caracterizar, ainda que grosseiramente, o padrão de consumo deste micronutriente. A distribuição dos níveis séricos de retinol é apresentada na Tabela 2. Níveis séricos de retinol considerados deficientes e baixos foram detectados em 4,3 e 40,4% das crianças respectivamente, totalizando 44,7% dos níveis considerados inade- quados. Não foi identificado nenhum caso de nível de retinol sérico considerado alto. Análise Estatística A análise quali-quantitativa da dieta foi realiza- da através dos "softwares" Sistema de Apoio em Nutrição, versão 1.0, desenvolvido pela Escola Paulista de Medicina, e do Epi-Info. Os resultados foram comparados à ingestão diária recomendada pela Organização das Nações Unidas para a Agri- cultura e Alimentos e Organização Mundial de Saúde (FAO/OMS11, 1991). Os resultados do inquérito dietético (recordatório de 24 h) estão apresentados na Tabela 5. O consumo de energia, expressa em percentagem das recomen- dações dietéticas permitidas, apresentou valor abai- xo do recomendado para todas as faixas etárias; ressalte-se que a energia proveniente do consumo de coco de ouricuri não foi computada por ser este alimento consumido "ad libitum", o que pode ter subestimado este cálculo. O consumo da proteína ultrapassou a recomendação para as crianças de 24 a 72 meses. A ingestão média de ferro foi inadequa- da para as crianças menores de 36 meses de idade. A mais baixa inadequação do consumo de vitamina As análises estatísticas foram realizadas utilizan- do-se o programa SPSS-PC+ e DEPID versão 2.12, para cálculo do intervalo de confiança. Devido a distribuição não homogênea dos níveis de retinol sérico, optou-se em utilizar a média geométrica para este indicador bioquímico. A prevalência e a média foram as estatísticas utilizadas para detectar a ocor- rência do evento, e o teste de qui-quadrado e a análise de variância foram adotados, respectivamente, para testar a diferença entre as proporções e as várias médias, a um nível de significância de 5,0%. Inquérito Dietético A adequação dos níveis séricos de retinol, se- gundo a faixa etária, não mostrou associação com a idade das crianças (p=0,06); contudo aquelas maio- res de 60 meses apresentaram a menor taxa de inadequação (Tabela 3). A deficiência da vitamina A distribui-se homogeneamente entre os meninos e meninas (p=0,93) (Tabela 4). Resultados A média e o desvio-padrão dos níveis séricos do retinol, segundo a faixa etária, estão apresentados na Tabela l. Os menores valores de retinol sérico foram encontrados para os menores de 12 meses de idade, contudo nenhuma diferença estatisticamente significante foi detectada entre as médias (p=0,31). vitamina A na dieta dos pré-escolares é representa- do pelo fígado; pequena proporção (7,1%) das cri- anças menores de dois anos consome este alimento urna vez por semana. Para as maiores de dois anos, este percentual eleva-se para 24,2%. Dos alimentos de conteúdo moderado em vita- mina A, somente a batata doce e a manga atingem maior percentual de consumo alimentar no grupo de crianças menores de dois anos de idade. Para as crianças de 24 a 72 meses de idade, verifica-se maior diversificação no consumo destes alimentos (Tabela 6 e 7 ). Os alimentos que compõem a lista de baixo conteúdo em vitamina A são consumidos pelas crianças dos dois grupos etários, contudo a maior freqüência foi observada para o grupo de 24 a 72 meses de idade. Comentários Segundo a definição do PAHO/WHO23 (1970), a deficiência da vitamina A, para as crianças rurais das localidades do semi-árido estudado, constitui proble- ma de saúde pública, desde quando foi detectado, C foi observada para os menores de 12 meses. A adequação do consumo deste micronutriente au- menta com a idade, sem contudo atingir os valores recomendados (Tabela 4). As Tabelas 6 e 7 ilustram a freqüência do consu- mo de alimentos fontes de vitamina A, cuja principal fonte é representada pelos carotenóides, em especial o beta-caroteno. O alimento de alto conteúdo em contribuíram para uma melhor utilização da vitami- na A, sem contudo assegurar os níveis adequados de retinol sérico para este grupo etário. Maiores percentuais de níveis de retinol sérico inadequados foram detectados em crianças menores de 24 meses de idade. O padrão alimentar dessas crianças é caracterizado por dietas lácteas, altamen- te diluídas e elevados percentuais de hidrato de carbono (Prado e col.24, 1975). Este padrão de con- sumo, aliado à supressão precoce do leite materno (Assis e col.1, 1994) e consumo reduzido de alimen- tos fontes de carotenóides, podem explicar os mai- ores níveis de inadequação de retinol sérico. A alta prevalência de parasitose intestinal observa- da neste grupo populacional (Gaudenzi e col.13,1992) é outro fator que pode estar comprometendo a absor- ção e a utilização da vitamina A ingerida. É conhecido que crianças portadoras de doenças infecciosas e para- sitárias, mesmo nas suas formas subclínicas, apresen- tam menores níveis de retinol sérico. Se durante o período de safra, quando há aumen- to da disponibilidade de alimentos fontes de vitami- na A, forem detectados percentuais de níveis de retinol sérico que colocam a deficiência de vitamina A como problema de saúde pública, efeitos dramá- ticos poderão ser esperados durante a entressafra, quando há escassez desses e de outros alimentos. Os resultados desta investigação evidenciam o risco a que está exposto esse grupo de crianças, uma vez que a vitamina A tem função específica na redução da morbi-mortalidade infantil. A curto prazo recomenda-se a suplementação em massa com vitamina A para a população estuda- da, em especial em época de seca e entressafra. A adoção de política agrícola que assegurem a produ- ção, aumento do consumo de alimentos ricos neste micronutriente e melhoria das condições básicas de vida devem ser priorizadas como medidas que a médio prazo serão capazes de erradicar a deficiên- cia nutricional na área. * Dra. Leonor Maria Pacheco Santos, Escola de Nutrição da Universidade Federal da Bahia (Comunicação pessoal). Comentários nesta população, um percentual maior do que 15,0% de níveis séricos de retinol considerados inadequa- dos (<20,0 mg/dl). g Este percentual é mais baixo do que o observado por Santos* (54,7%), para pré-escolares de áreas urbanas do semi-árido baiano, e inferior também aos 51,4% detectados por Roncada e col. 27(1978), em crianças migrantes em trânsito pela capital do Estado de São Paulo. Referências Bibliográficas Interrelationship between diarrhea and vitamin A deficiency a risk factor for diarrhea? Pediat. Infect. Dis., 11: 380-4, 1992. 24. PRADO, M. S. et al. Padrão e seleção de alimentos comple- mentares e secedâneos do leite materno em comunidades rurais do semi-árido baiano. Rev. Nutr. PUCCAMP, 8: 47-64,1995, f 11. FAO/OMS. Necesidades de vitamin A, hierro folato y vitamin B12. Roma, 1991. (Estudios FAO/Alimentación y Nutrición No 23). , , 25. RAHMATHULLAR, L. M. B. et al. Reduced mortality among children in Southern India receiving a small weekly dose of vitamin A. J. Med., 323: 929-35, 1990. 12. FUNDAÇÃO IBGE. Censo demográfico Bahia 1991. Rio de Janeiro, 1992. 13. GAUDENZIE, N. et al. Projeto Cansanção: uma vivência da universidade no sertão da Bahia. Salvador, Coordena- ção Central de Extensão, 1992. 26. REUNION CONSULTIVA CONJUNTA FAO/OMS/UNU DE EXPERTOS EN NECESIDADES DE ENERGIA Y DE PROTEINAS, Roma, 1981. Informe. Ginebra, OMS, 1985 (OMS - Ser. Inf. Téc., 724). ç 14. INTERDEPARTAMENTAL COMMITTEE ON NUTRITION FOR NATIONAL DEFENSE. Manual for nutrition survey. Washington, D. C., Governement Printing Office, 1963. 27. RONCADA, M. J. et al. Hipovitaminose A em filhos de migrantes nacionais em trânsito pela capital do Estado de São Paulo, Brasil: estudo clínico-bioquímico. Rev.Saúde Pública, 12: 345-50, 1978. 15. INTERNATIONAL VITAMIN A CONSULTIVE GROUP. Guidelines for the development of a simplified dietary assessment to identify groups at risk for inadequate intake of vitamin A: report of the international vitamin A Consultative Group. Washington, 1989. 28. SANTOS, L. M. P. et al. Xerophthalmia in the state of Paraiba Northeast of Brazil: clinical findings. Am. J. Clin. Nutr., 38: 139-44, 1983. 16. JOINT WHO/UNICEF/USAID/Helen Keller International/ IVAGG. Meeting on the Control of Vitamin A Deficiency and Xerophthalmia, Jakarta, 1980. Report. Geneva, 1982. (WHO-Tech. Rep. Ser., 672). 29. SOMMER, A. et al. Increased mortality in children with mild vitamin A deficiency. Lancet, 2:585-8, 1983. 30. SOMMER, A. et al. Impact of vitamin A supplementation on childhood mortality. Lancet, 1: 1169-91, 1986. p 17. MELE, L. et al. Nutritional and household risk factors for xerophthalmia in Aceh, Indonesia: a case-control study. Am. J. Clin. Nutr., 53:1460-5, 1991. 31.VENKATASWANY, G. et al. Retinol-binding protein in serum of xerophthalmic, malnourished children before and after treatment at a nutrition center. Am. J. Clin. Nutr., 30: 1968-73, 1977. 18. MILTON, R.C. et al. Mild vitamin A deficiency and childhood morbidity-an Indian experience. Am. J. Clin. Nutr., 46: 287-9, 1987. 32. Referências Bibliográficas Para a população estudada, as principais fontes de vitamina A são constituídas basicamente por carotenóides cuja conversão é de aproximadamente 20-50% da quantidade ingerida (Blomhoff e col.7, 1992). A maior freqüência no consumo de alimen- tos ricos em beta-caroteno, tais como: manga, ma- mão, batata doce entre outros, talvez explique os menores percentuais de níveis séricos de retinol considerados inadequados para as crianças de 24 a 72 meses de idade. A presença de vitamina C na dieta e a gordura oriunda do coco de ouricuri, além de uma boa adequação de ferro dietético, seguramente 1. ASSIS, A. M. O. et al. Prática do aleitamento materno em comunidades rurais do semi-árido baiano. Rev. Saúde Pública, 28: 308-4, 1994. 2. ARAÚJO, C. R. C. & FLORES, H. Improved spectrophoto- metric vitamin A assay. Clin. Chem., 24: 386, 1978. Ú 3. ARAÚJO, M. P. N. et al. Dimensionamento de medidas caseiras. Salvador, Departamento das Ciências da Nutri- ção/Escola de Nutrição-UFBA, 1993. 4. BARRETO, M. L. et al. Effect of vitamin A supplementation on diarrhoea and acute lower-respiratory-tract infection in young children in Brazil. Lancet, 344: 228-31,1994. y g 5. BATISTA FILHO, M. & TORRES, M. A. A. Acesso a terra e situação nutricional em populações do smi-árido nor- destino. Rev. Pernambucana Desenv., 9: 101-19, 1982. 6. BEATON, G. H. et al. Effectiveness of vitamin A supplementa- tion in the control of young child morbidity and mortality in developing countries. Toronto, International Nutrition Program, 1992. Nutrition, 1993. 7. BLOMHOFF, R. et al. Vitamin A: physiological and bioche- mical processing. Ann. Rev. Nutr., 12: 37-57, 1992. 21. NAUSS, M. K. Influence of vitamin a status on the immune system. In: Bauernfeind, J. C. Vitamin A deficiency and its control. Orlando, Academic Press, 1986. p. 207-36. g 8. DAULAIRE, N. M. P. et al. Childhood mortality after a high dose of vitamin A in a high risk population. BMJ (London), 304: 207-10, 1992. 22. OSLON, J. A. Phisiologiccal and metabolic basis of myor sigs of vitamin A deficiency. In: Bauernfeind, J. C. Vitamin A deficiency and its control. Orlando, Academic Press, 1986. p. 350-84. , 9. DELUCA, L. M. & McDOWELL, L. M. Effects of vitamin A status on hamster tracheal epithelium in vivo and vitro. Food Nutr.BulL, 11: 20-4, 1989. p 23. PAN AMERICAN HEALTH ORGANIZATION. Hypovita- minosis A in the Americas. Washington, 1970. 10. EL BUSHRA, H. E. et al. Referências Bibliográficas VITAMIN A FIELD SUPPORT PROJECT (VITAL). Inter- national Science and Technology Institute, Vital nutrients. Arlington. 1991. 19. MUHIAL, P. et al. Vitamin A-fortified monosodium glutamate and health, gowth and survival of children: a controlled field trial. Am. J. Clin. Nutr., 48: 1271-4, 1988. g field trial. Am. J. Clin. Nutr., 48: 1271-4, 1988. 33. WEST, K. P. et al. Efficacy of vitamin A in reducing pre-school child mortality in Nepal. Lancet, 338: 67-71, 1991. 20. NEWMAN, V. Vitamin A and breastfeeding: a comparison of data from developed and developing countries. San Diego, Cooperative Agreement, United Satates Agency for International Development Office of 34. WITSCHI, J. C. Short-term dietary and recording methods. In: Willett, W. Nutritional epidemiology. Oxford, Oxford University, 1990. p. 52-65. The distribution and magnitude of vitamin A deficiency and dietary consumption of 161 children at 6 to 72 months of age in rural zones in Cansanção-Bahia-Brazil were evaluated. The serum retinol levels were measured by the spectrophotome- tric method (Bassey-Lowry modified by Araújo and Flores). The serum retinol average was found to be distributed homogeneously throughout the different age groups. Inadequate serum retinol levels (<20,0 mg/dl) were detected in 44.7% of the children, which characterized the deficiency as constituting a public health problem. The serum retinol levels showed no statistically significant association as between the sex and age of the children; however the chilfren of less than 24 months showed a higher prevalence of inadequate serum retinol levels. The main available source of vitamin A for these children was represented by carotenoids, especially beta-carotene. Foods regarded as being rich in vitamin A were consumed by all age groups. The greatest diversification of consumption of foordstuffs with moderate and low vitamin A content was observed in the group of children of from 24 to 72 months of age, through this was no guarantee of adequate serum retinol levels in this group however. Vitamin A deficiency, epidemiology. Diet survays. Rural population. Abstract The distribution and magnitude of vitamin A deficiency and dietary consumption of 161 children at 6 to 72 months of age in rural zones in Cansanção-Bahia-Brazil were evaluated. The serum retinol levels were measured by the spectrophotome- tric method (Bassey-Lowry modified by Araújo and Flores). The serum retinol average was found to be distributed homogeneously throughout the different age groups. Inadequate serum retinol levels (<20,0 mg/dl) were detected in 44.7% of the children, which characterized the deficiency as constituting a public health problem. The serum retinol levels showed no statistically significant association as between the sex and age of the children; however the chilfren of less than 24 months showed a higher prevalence of inadequate serum retinol levels. The main available source of vitamin A for these children was represented by carotenoids, especially beta-carotene. Foods regarded as being rich in vitamin A were consumed by all age groups. The greatest diversification of consumption of foordstuffs with moderate and low vitamin A content was observed in the group of children of from 24 to 72 months of age, through this was no guarantee of adequate serum retinol levels in this group however. Vitamin A deficiency, epidemiology. Diet survays. Rural population.
https://openalex.org/W4379412825	https://jurnal.plb.ac.id/index.php/atrabis/article/download/565/372	Indonesian	null	Strategi Marketing Menggunakan Instagram	Atrabis: Jurnal Administrasi Bisnis	2,021	cc-by	3,959	ATRABIS: Jurnal Administrasi Bisnis ATRABIS: Jurnal Administrasi Bisnis Vol. 7, No. 1 Juni 2021 Strategi Marketing Menggunakan Instagram (Studi kasus: Sapinesia) Asye Rachmawaty Program Studi Manajemen Informatika Politeknik LP3I e-mail: asyerachmawaty@plb.ac.id Abstrak: Tingginya pengguna media sosial di Indonesia, membuat para pelaku bisnis/UMKM mengembangkan strategi marketingnya dengan cara memanfaatan media sosial sebagai salah satu media promosi. Sudah banyak diantara pelaku bisnis memanfaatkan media sosial untuk memperkenalkan bisnis dan produk-produk yang dipasarkannya. Namun bagaimana memaksimalkan fitur-fitur dari media sosial yang digunakan, ternyata masih banyak pelaku bisnis yang kurang memahami sehingga strategi marketing dengan sosial medianya kurang memberikan keuntungan. Penulis, melalui penelitian ini ingin mengetahui bagaimana perkembangan sebuah bisnis/UMKM yang menggunakan sosial media dalam menjalankan strategi marketingnya, yaitu memperkenalkan produknya dengan memanfaatkan iklan berbayar pada salah satu media sosial. Metode yang digunakan dalam penelitian ini adalah metode deskriptif kualitatif, dimana peneliti melakukan pengumpulan data dengan observasi partisipan. Hasil yang ditemukan dalam penelitian ini, bahwa dengan memanfaatkan fitur-fitur yang tersedia pada sebuah media sosial, membuat konten-konten yang menarik secara berkelanjutan, memaksimalkan akses link yang pada berbagai akun, dan memiliki teknik closing yang baik, dapat menjadi salah satu strategi marketing yang sangat mutakhir di era digital. Kata Kunci : Media Sosial, Instagram, Strategi Marketing. PENDAHULUAN Adapun tujuan dari penelitian ini adalah untuk mengetahui keberhasilan sapinesia dalam menjalankan strategi marketing untuk menaikkan jumlah followers melalui Instagram Ads. pada enam akun resminya. Dengan jumlah followers sebanyak itu, lima akun resmi lainnya yang juga aktif dalam iklan berbayar, penulis tertarik untuk meneliti sejauh mana keberhasilan strategi marketing menggunakan Instagram Ads. yang selama ini mereka lakukan. Adapun tujuan dari penelitian ini adalah untuk mengetahui keberhasilan sapinesia dalam menjalankan strategi marketing untuk menaikkan jumlah followers melalui Instagram Ads. pada enam akun resminya. PENDAHULUAN Media sosial sebagai sarana pemasaran digital, saat ini, menjadi sangat penting kehadirannya bagi para pelaku bisnis dalam memperkenalkan produknya. Beragam fitur yang ditawarkan media sosial juga memudahkan dalam melakukan promosi. A.A. Manik Pratiwi (2020:75): “Pemasaran media sosial adalah bentuk mempromosikan beragam konten bisnis dalam berbagai cara kepada pengguna media sosial. Kegiatan ini dilakukan untuk menemukan formula yang tepat dalam menyebarkan informasi yang dibutuhkan untuk mengarahkan tujuan bisnis dan peningkatan penjualan.” Dewi Utari, Dewi Endah Fajariana (2018). Terdapat banyak media sosial yang menjadi kegemaran warganet di Indonesia, salah satunya adalah Instagram. Rakha fahreza (2020) dalam tulisannya menyebutkan bahwa dari 150 juta pengguna media sosial di Indonesia, 80% diantaranya adalah pengguna Instagram yaitu sekitar 120 juta orang. Itu yang mengakibatkan banyaknya pelaku bisnis mulai merambah Instagram sebagai media pemasaran produk mereka. Salah satu produk yang diperkenalkan dan dipasarksn melalui akun media sosial Instagram adalah sei sapi. Dikutip dalam sebuah tulisan karya Syifa Nuri Khairunnisa (2020), bahwa sei merupakan salah satu sajian khas Provinsi Nusa Tenggara Timur tepatnya Kabupaten Rote Ndao. Daging ini diiris tipis-tipis memanjang kemudian diasapi dengan bara api hingga matang. 39 ATRABIS: Jurnal Administrasi Bisnis Vol. 7, No. 1 Juni 2021 Karena nikmatnya, daging sei ini terkenal di seluruh NTT hingga akhirnya sebuah UMKM berani memperkenalkan daging sei ini secara online melalui media sosial Instagram. Tak hanya satu akun akun saja, sapinesia, hingga saat ini memiliki enam akun resmi dan kerap mempromosikan produknya menggunakan iklan berbayar dengan fitur Instagram Ads. Tak hanya iklan berbayar dalam memperkenalkan produk sei sapinya, sapinesia juga memberikan promosi gratis biaya kirim se-Jawa. Berikut adalah akun utama dari sapinesia dengan jumlah followers lebih dari 15 ribu akun. Gambar 1. Akun Instagram @sapinesia Gambar 1. Akun Instagram @sapinesia Dengan jumlah followers sebanyak itu, lima akun resmi lainnya yang juga aktif dalam iklan berbayar, penulis tertarik untuk meneliti sejauh mana keberhasilan strategi marketing menggunakan Instagram Ads. yang selama ini mereka lakukan. Adapun tujuan dari penelitian ini adalah untuk mengetahui keberhasilan sapinesia dalam menjalankan strategi marketing untuk menaikkan jumlah followers melalui Instagram Ads. pada enam akun resminya. Gambar 1. Akun Instagram @sapinesia Gambar 1. Akun Instagram @sapinesia Gambar 1. Akun Instagram @sapinesia Dengan jumlah followers sebanyak itu, lima akun resmi lainnya yang juga aktif dalam iklan berbayar, penulis tertarik untuk meneliti sejauh mana keberhasilan strategi marketing menggunakan Instagram Ads. yang selama ini mereka lakukan. Strategi Marketing Online g g Menurut Assauri (2014:168), “strategi pemasaran adalah serangkaian tujuan dan sasaran, kebijakan dan aturan yang memberi arah kepada usaha-usaha pemasaran perusahaan dari waktu ke waktu, pada masing-masing tingkatan dan acuan serta aloksinya, terutama sebagai tanggapan perusahaan dalam menghadapi lingkungan dan keadaan persaingan yang selalu berubah”. Strategi pemasaran memberikan arah dalam kaitannya dengan segmentasi pasar, identifikasi pasar sasaran, positioning dan bauran pemasaran. Moh Rusdi (2019). Strategi pemasaran menurut Dewi U. dan Dewi E. (2018) merupakan rangkaian suatu kegiatan yang terarah untuk mencapai sasaran dan dengan pola berpikir yang inovatif dan kreatif, untuk menghadapi kecenderungan yang terjadi di dalam perusahaan maupun di luar perusahaan, yang akan berpengaruh terhadap kepentingan maupun masa depan perusahaan sendiri. Berdasarkan beberapa teori di atas, dapat disimpulkan bahwa strategi pemasaran adalah suatu kegiatan yang terarah berdasarkan tujuan dan aturan, dilakukan secara kreatif dan inovatif sesuai perkembangan jaman untuk menghasilkan sesuatu yang baik bagi perusahaan. Untuk mencapai kesuksesan dalam menjalankan strategi marketing, salah 40 ATRABIS: Jurnal Administrasi Bisnis Vol. 7, No. 1 Juni 2021 satu unsur pentingnya adalah bauran pemasaran atau marketing mix. Menurut Kotler (2001: 264-312) bauran pemasaran memiliki lima poin, yaitu: 1) Advertising atau periklanan; 2) Sales Promotion; 3) Public Relation and Publisity; 4) Personal Selling; dan 5) Direct Marketing. Masing-masing poin bauran pemasaran memiliki manfaat yang berbeda dengan tujuan yang sama, yaitu kesuksesan berbisnis. satu unsur pentingnya adalah bauran pemasaran atau marketing mix. Menurut Kotler (2001: 264-312) bauran pemasaran memiliki lima poin, yaitu: 1) Advertising atau periklanan; 2) Sales Promotion; 3) Public Relation and Publisity; 4) Personal Selling; dan 5) Direct Marketing. Masing-masing poin bauran pemasaran memiliki manfaat yang berbeda dengan tujuan yang sama, yaitu kesuksesan berbisnis. Dalam penelitian ini, akan dibahas bauran pemasaran yang pertama, yaitu advertising atau periklanan menggunakan media sosial. Permatasari (2016:5) mengungkapkan karakteristik melakukan promosi melalui media sosial, yaitu: 1) Promosi dan pemasaran dilakukan kapan saja selama tersambung dengan jaringan internet; 2) Jangkauan luas dan tak terbatas; 3) Dapat memilih beragam sosial media yang ada; 4) Penyebaran informasi cepat; 5) Akses konsumen mudah; 6) Waktu promosi 24 jam; 7) Rawan akan resiko; dan 8) Biaya promosi rendah karena tidak perlu mencetak poster atau memasang iklan di media (contoh : televisi atau radio). Media Sosial Media sosial menurut Caleb T. Carr dan Rebecca A. Hayes (2015) adalah media berbasis Internet yang memungkinkan pengguna berkesempatan untuk berinteraksi dan mempresentasikan diri, baik secara seketika ataupun tertunda, dengan khalayak luas maupun tidak yang mendorong nilai dari user-generated content dan persepsi interaksi dengan orang lain. Kotler dan Keller (2016) mengatakan bahwa media sosial adalah media yang digunakan oleh konsumen untuk berbagi teks, gambar, suara, dan video informasi baik dengan orang lain maupun perusahaan dan vice versa. Strategi Marketing Online Penelitian sebelumnya, Zahrah Latifah (2018), menyimpulkan bahwa penggunaan media sosial sebagai media promosi dapat digunakan untuk menyampaikan informasi, menerima feedback dari konsumen, serta untuk menyampaikan pesan dalam berbagai jenis konten yang dapat membuat promosi menjadi semakin menarik untuk dilihat oleh konsumen Instagram Anugerah Ayu (2019) dalam artikelnya mengatakan bahwa ”Instagram adalah sebuah aplikasi berbagi foto dan video yang memungkinkan pengguna mengambil foto, mengambil video, menerapkan filter digital, dan aktivitas berjejaring lainnya. Nama Instagram berasal dari Kata ‘Instan’ dan ‘telegram’. Kata ‘instan’ yang mendasari penamaan ‘insta’ dimaksudkan seperti kamera polaroid yang pada masanya lebih dikenal dengan sebutan "foto instan". Dengan makna ini Instagram juga dapat menampilkan foto- foto secara instan, seperti polaroid di dalam tampilannya. Sedangkan kata telegram merujuk pada sebuah alat yang bekerja mengirimkan informasi kepada orang lain dengan cepat.” Terdapat beberapa fitur canggih dalam aplikasi Instagram, diantaranya adalah sebagai berikut: 1) Berbagi foto dan video adalah fitur utama dari Instagram; 2) Komentar dan like, fitur yang sama dengan aplikasi lainnya yaitu Facebook; 3) Explore yaitu fitur pencarian, menampilkan foto atau video terpopuler, dan lokasi terdekat; 4) Instagram Story yaitu fitur pengambilan foto atau video untuk kemudian ditampilkan di kilas cerita Instagram; dan 5) IGTV yaitu fitur pemutaran video berdurasi hingga 60 menit dengan ukuran file hingga 5.4 GB. Fitur lainnya yang dimiliki Instagram sebagai media promosi atau periklanan adalah Instagram Ads. Dimana pemilik akun dapat menayangkan iklan 41 ATRABIS: Jurnal Administrasi Bisnis Vol. 7, No. 1 Juni 2021 dalam bentuk foto ataupun video dengan jangkauan yang lebih luas, sehingga tujuan promosi dapat tercapai. dalam bentuk foto ataupun video dengan jangkauan yang lebih luas, sehingga tujuan promosi dapat tercapai. METODE PENELITIAN Teknik pengumpulan data dilakukan dengan cara observasi lapangan. Metode kualitatif menjadi pilihan penulis dalam menentukan cara mencari, mengumpulkan, mengolah, dan menganalisis data hasil penelitian. Penelitan yang digunakan adalah penelitian kualitatif deskriptif. Penelitian deskriptif kualitatif adalah pengumpulan data suatu latar alamiah dengan maksud menafsirkan fenomena yang terjadi dimana peneliti adalah sebagai instrument kunci, hasil penelitian lebih menekankan makna dari pada generalisasi. (Albi Anggito, Johan Setiawan:2018) PEMBAHASAN Sapinesia adalah salah satu jenis usaha yang bergerak dibidang kuliner, yaitu olahan daging sapi, tepatnya sei sapi. Sei sapi ini dijual dalam bentuk frozen atau makanan beku, dipasarkan secara online melalui beberapa media sosial, website, dan juga marketplace. Terdapat tiga daerah pengiriman di pulau Jawa, yaitu Jawa Barat, Jawa Tengah, dan Jawa Timur. Sei sapi ini diperkenalkan oleh sapinesia melalui media sosial Instagram dengan fasilitas iklan berbayar, lalu kemudian calon konsumen digiring untuk mempelajari lebih lanjut mengenai sei sapi olahan mereka melalui website yang berisi berbagai informasi produk, testimoni, dan juga formulir pembelian yang tahapan berikutnya adalah pemberian link WhatsApp admin dari sapinesia. Dalam melakukan promosinya, sapinesia mempunyai beberapa akun aktif di Instagram dengan nama akun yang berbeda namun hampir sama dan tampilan profil juga feeds yang sama di setiap akun. Berikut profil dari beberapa akun resmi sapinesia. 42 ATRABIS: Jurnal Administrasi Bisnis Vol. 7, No. 1 Juni 2021 Gambar 2. Profil @Sapinesia Gambar 3. Profil @Sapinesia.id Gambar 4. Profil Frozenseisapinesia Gambar 5. Profil @Seisapinesiafrozen Gambar 3. Profil @Sapinesia.id Gambar 2. Profil @Sapinesia p Gambar 4. Profil Frozenseisapinesia Gambar 5. Profil @Seisapinesiafrozen 43 ATRABIS: Jurnal Administrasi Bisnis Vol. 7, No. 1 Juni 2021 Gambar 6. Profil @Seisapinesiafrozenku Gambar 7. Profil @Seisapinesiaku Setidaknya, hingga penulis melakukan penelitian, terdapat enam akun resmi sapinesia. Dapat dilihat pada masing-masing profilnya, cara penulisan hingga link yang mereka tampilkan adalah sama. Penulis melakukan penelusuran pada masing-masing akun, dan ternyata baik link marketplace, contact person, dan website mereka adalah sama. Berikut adalah tampilan dari marketplace dan website sapinesia: Gambar 7. Profil @Seisapinesiaku Gambar 6. Profil @Seisapinesiafrozenku Gambar 7. Profil @Seisapinesiaku Gambar 6. Profil @Seisapinesiafrozenku Gambar 6. Profil @Seisapinesiafrozenku Setidaknya, hingga penulis melakukan penelitian, terdapat enam akun resmi sapinesia. Dapat dilihat pada masing-masing profilnya, cara penulisan hingga link yang mereka tampilkan adalah sama. Penulis melakukan penelusuran pada masing-masing akun, dan ternyata baik link marketplace, contact person, dan website mereka adalah sama. Berikut adalah tampilan dari marketplace dan website sapinesia: Setidaknya, hingga penulis melakukan penelitian, terdapat enam akun resmi sapinesia. Dapat dilihat pada masing-masing profilnya, cara penulisan hingga link yang mereka tampilkan adalah sama. Penulis melakukan penelusuran pada masing-masing akun, dan ternyata baik link marketplace, contact person, dan website mereka adalah sama. Berikut adalah tampilan dari marketplace dan website sapinesia: Gambar 9. Website Sapinesia Gambar 8. Sapinesia on marketplace Gambar 9. Website Sapinesia Gambar 8. Sapinesia on marketplace Gambar 8. Sapinesia on marketplace Gambar 9. Website Sapinesia Gambar 9. PEMBAHASAN Website Sapinesia 44 ATRABIS: Jurnal Administrasi Bisnis Vol. 7, No. 1 Juni 2021 Pada marketplace tersebut hanya ada satu akun sapinesia yang pengirimannya berasal dari kota Sidoarjo, dengan jumlah pengikut 2600 akun dan penilaian sebesar 4,6 dari 5,0. Dapat dilihat juga bahwa paket produk mereka sudah banyak yang membelinya melalui marketplace tersebut. Sedangkan untuk website, ada banyak sub-link yang dibagikan sesuai dengan nama akun Instagram seperti pada gambar berikut. Penulis masuk dengan link dari akun Instagram @sapinesia yaitu www.sapinesia.com/frozen2 yang menampilkan banyak informasi produk, promo, testimoni pembeli, hingga formulir untuk calon konsumen. Berdasarkan teori mengenai karakteristik pemasaran atau promosi melalui media sosial oleh Permatasari (2016:5) serta hasil penelitian yang dilakukan oleh penulis, dapat dibahas beberapa karakteristik sebagai berikut: Promosi dan pemasaran dilakukan kapan saja selama tersambung dengan jaringa internet Media sosial Instagram sebagai sarana melakukan promosi dan pemasaran, memudahkan pengguna yaitu pelaku bisnis untuk memasarkan produknya kapan saja selama adanya jaringan internet. Hal itupun yang dilakukan oleh Sapinesia dalam memasarkan produk sei sapinya. p y Pada awalnya penulis mengenal sapinesia melalui jaringan internet, yaitu media sosial Instagram yang langsung terlihat pada laman Instagram tanpa penulis follow terlebih dahulu. Sapinesia dengan keenam akunnya menggunakan fitur Instagran Advertising atau biasa dikenal dengan sebutan Instagram Ads., yaitu fitur iklan berbayar dengan jangkauan lebih luas sehingga promosi dapat dilihat oleh lebih banyak akun bahkan yang belum mengikuti akun yang melakukan promosi tersebut, dalam hal ini sapinesia. Siapapun, kapanpun, dan dimanapun selama lokasi akunnya terjangkau oleh promosi berbayar tersebut, maka dapat melihat iklan bersponsor dari Sapinesia. Dengan iklan yang menarik, berupa video testimoni seseorang yang lahap menikmati hidangan sei sapi, tentu saja banyak viewers yang tertarik untuk mengetahui lebih lanjut mengenai hidangan sei sapi tersebut. Berikut salah satu iklan bersponsor dari Sapinesia. 45 ATRABIS: Jurnal Administrasi Bisnis Vol. 7, No. 1 Juni 2021 Gambar 10. Iklan berbayar Sapinesia melalui akun @seisapinesiaku Terlihat dari gambar tersebut, bahwa akun @seisapinesiaku melakukan iklan bersponsor dengan 455,414 views nya. Sudah ratusan ribu akun yang menyaksikan video tersebut. Gambar 10. Iklan berbayar Sapinesia melalui akun @seisapinesiaku Terlihat dari gambar tersebut, bahwa akun @seisapinesiaku melakukan iklan bersponsor dengan 455,414 views nya. Sudah ratusan ribu akun yang menyaksikan video tersebut. Terlihat dari gambar tersebut, bahwa akun @seisapinesiaku melakukan iklan bersponsor dengan 455,414 views nya. Sudah ratusan ribu akun yang menyaksikan video tersebut. Jangkauan luas dan tak terbatas Berikut media sosial yang menjadi aplikasi pemasaran: Instagram (akun resmi), Facebook (Fanpage), Youtube (testimoni), dan WhatsApp (customer service). p g y g Rakha F. (2020) dalam artikelnya menyebutkan bahwa terdapat 10 media sosial yang paling banyak digunakan, yaitu: YouTube; WhatsApp; Facebook; Instagram; TikTok; Line; Twitter; Reddit; Pinterest; dan Tumblr. Setidaknya, Sapinesia menggunakan empat dari 10 media sosial sebagai media pemasarannya. Berikut media sosial yang menjadi aplikasi pemasaran: Instagram (akun resmi), Facebook (Fanpage), Youtube (testimoni), dan WhatsApp (customer service). Jangkauan luas dan tak terbatas Sapinesia memiliki enam akun resmi pada media sosial Instagram dan seringkali aktif memperkenalkan produk sei sapinya dengan fitur Instagram Ads. Selain jangkauan iklan tersebut sangat luas, sesuai dengan biaya yang dianggarkan, hal ini berpotensi untuk menarik lebih banyak followers karena kaingintahuan dari calon konsumen yang secara tidak sengaja menyaksikan iklan berbayar tersebut yang tampil di feed Instagram mereka. Berikut adalah profil akun @seisapinesiaku pada tanggal 5 Maret 2021 dengan 336 followers, namun pada Gambar 7 yang diambil tanggal 27 Mei 2021, jumlah followers dari akun @seisapinesiku sudah mencapai 4043 followers. Terbukti dengan mengaktifkan iklan berbayar, akun @seisapinesia mendapatkan peningkatan followers sebanyak 3707 dalam waktu dua bulan saja. Terlihat pada gambar sebelumnya, dalam satu tayangan iklan saja ternyata jumlah viewers-nya mencapai ratusan ribu. Dapat dikatakan bahwa jangkauan iklan berbayar ini sangat luas, hal ini dapat dilihat melalui menu insight yang terdapat pada akun profesional Instagram. 46 ATRABIS: Jurnal Administrasi Bisnis Vol. 7, No. 1 Juni 2021 Gambar 11. Jumlah followers akun @seisapinesiaku pada 5 Maret 2021 Dapat memilih beragam sosial media yang ada Rakha F. (2020) dalam artikelnya menyebutkan bahwa terdapat 10 media sosial yang paling banyak digunakan, yaitu: YouTube; WhatsApp; Facebook; Instagram; TikTok; Line; Twitter; Reddit; Pinterest; dan Tumblr. Setidaknya, Sapinesia menggunakan empat dari 10 media sosial sebagai media pemasarannya. Berikut media sosial yang menjadi aplikasi pemasaran: Instagram (akun resmi), Facebook (Fanpage), Youtube (testimoni), dan WhatsApp (customer service). Gambar 11. Jumlah followers akun @seisapinesiaku pada 5 Maret 2021 Gambar 11. Jumlah followers akun @seisapinesiaku pada 5 Maret 2021 Gambar 11. Jumlah followers akun @seisapinesiaku pada 5 Maret 2021 Gambar 11. Jumlah followers akun @seisapinesiaku pada 5 Maret 2021 Dapat memilih beragam sosial media yang ada Dapat memilih beragam sosial media yang ada Rakha F. (2020) dalam artikelnya menyebutkan bahwa terdapat 10 media sosial yang paling banyak digunakan, yaitu: YouTube; WhatsApp; Facebook; Instagram; TikTok; Line; Twitter; Reddit; Pinterest; dan Tumblr. Setidaknya, Sapinesia menggunakan empat dari 10 media sosial sebagai media pemasarannya. Berikut media sosial yang menjadi aplikasi pemasaran: Instagram (akun resmi), Facebook (Fanpage), Youtube (testimoni), dan WhatsApp (customer service). Dapat memilih beragam sosial media yang ada Rakha F. (2020) dalam artikelnya menyebutkan bahwa terdapat 10 media sosial yang paling banyak digunakan, yaitu: YouTube; WhatsApp; Facebook; Instagram; TikTok; Line; Twitter; Reddit; Pinterest; dan Tumblr. Setidaknya, Sapinesia menggunakan empat dari 10 media sosial sebagai media pemasarannya. Penyebaran informasi cepat Melalui media sosial, informasi yang disampaikan dapat menyebar dengan sangat cepat. Hal ini dikemukakan juga oleh Staf Ahli Menteri Bidang Komunikasi dan Media Massa Kementerian Komunikasi dan Informasi (Kemenkominfo) RI, Drs. Gun Gun Siswadi, M.Si. (2016) bahwa Pesatnya teknologi informasi memungkinkan publik berinteraksi lebih cepat dalam mengabarkan peristiwa dibanding media arus utama. Hal ini kemudian memunculkan istilah citizen journalism, dimana informasi dari masyarakat dapat langsung disebarluaskan melalui jagat maya. Konsep ini lebih mengedepankan kecepatan penyebaran informasi. Ini sudah dibuktikan oleh Sapinesia, ketika jumlah followers-nya bertambah banyak dalam waktu dua bulan saja. Testimoni Sapinesia juga banyak ditayangkan melalui channel Youtube oleh para Youtuber dengan jumlah views ribuan. Tentu saja hal ini diharapkan dapat mempengaruhi tingkat penjualan sei sapi Sapinesia, diharapkan viewers akan semakin penasaran setelah melihat testimoni dari para Youtuber yang berpengalaman di bidang kuliner. Biasanya, calon pembeli yang sudah melihat iklan sebuah produk di media sosial seperti Instagram ataupun Facebook, akan mencari tahu lebih lanjut melalui review dari beberapa Youtuber terpercaya, baru setelah itu mereka akan memutuskan untuk membelinya atau tidak. Berikut beberapa testimoninya. 47 ATRABIS: Jurnal Administrasi Bisnis Vol. 7, No. 1 Juni 2021 Gambar 12. Reviews Sapinesia di Youtube Gambar 12. Reviews Sapinesia di Youtube Gambar 12. Reviews Sapinesia di Youtube Gambar 12. Reviews Sapinesia di Youtube Gambar 12. Reviews Sapinesia di Youtube Gambar 12. Reviews Sapinesia di Youtube Rawan akan resiko Era digital, dimana teknologi informasi menjadi yang paling banyak digunakan dalam berbagai kegiatan, sangat memungkinkan para penjahat internet atau dikenal dengan hacker untuk melakukan banyak tindakan kejahatan. Salah satu kejahatannya adalah memperjual-belikan database konsumen demi meraup keuntungan pribadi. Hal ini bukan hanya menjadi kekhawatiran pelau usaha kecil ataupun menengah, tapi juga kehawatiran pelaku usaha yang database konsumennya sudah sangat banyak. Yang perlu dilakukan untuk mengatasi kejahatan siber adalah: 1) menggunakan pelatihan dan aktivitas yang akan mendidik karyawan tentang dasar-dasar keamanan siber, misalnya, untuk tidak membuka atau menyimpan file dari email atau situs web yang tidak dikenal karena dapat membahayakan seluruh perusahaan; 2) mengingatkan karyawan secara rutin tentang cara menangani data sensitif, misalnya, untuk menyimpan hanya di layanan cloud tepercaya dengan autentikasi diaktifkan, jangan membagikannya dengan pihak ketiga yang tidak tepercaya; 3) menerapkan penggunaan perangkat lunak yang sah, diunduh dari sumber resmi;4) membuat cadangan data penting dan perbarui peralatan serta aplikasi TI secara teratur untuk menghindari kerentanan yang belum ditambal yang dapat menjadi penyebab kebocoran; dan 5) menggunakan produk titik akhir khusus yang menuntut manajemen minimum yang memungkinkan karyawan melakukan pekerjaan utama mereka, namun tetap terlindung dari malware, ransomware, pengambilalihan akun, penipuan online, dan penipuan. Stephan Neumeier dalam Rafki Fahrizal (2020). iaya promosi rendah karena tidak perlu mencetak poster atau memasang iklan di edia Sapinesia yang memiliki enam akun resmi di Instagram, aktif membuat iklan berbayar. Tentu saja ada biaya untuk melakukan promosi tersebut, tapi jika dibandingkan dengan menayangkan iklan di media massa seperti majalah, koran, ataupun televisi, dimana harga tayang iklan televisi per 30 detiknya bisa mencapai puluhan bahkan ratusan juta rupiah, maka bagi pelaku usaha menengah ke bawah jauh lebih efisien menayangkan iklan di media sosial yang biayanya jauh lebih terjangkau. Cukup dengan puluhan ribu per hari saja, pelaku usaha dapat menayangkan iklannya di media sosial dengan ribuan cakupan akun aktif. Sapinesia membuat promo gratis biaya kirim se-Jawa, kemungkinan jangkauan iklan yang ditargetkan Sapinesia tertuju pada Pulau Jawa. Akses konsumen mudah Akses konsumen mudah Media sosial dengan akses tak terbatas, menjadi penghubung antara pelaku usaha dengan konsumennya. Untuk mendapatkan berbagai informasi yang dibutuhkan, pengguna media sosial dapat melakukan pencarian bukan hanya informasi yang ada di dalam negeri saja, namun informasi yang berada di luan negeri pun didapatkan dengan mudah. Hal ini pun berlaku pada dunia bisnis, dimana konsumen mendapatkan kemudahan dalam mengakses produk apapun yang ingin dicarinya. Sapinesia menyertakan beberapa link pada profil akun Instagramnya untuk memudahkan calon konsumen mengaksesnya, termasuk dalam melakukan transaksi. Gambar 13. Kemudahan Akses Konsumen Pada gambar tersebut di atas, Sapinesia memberian kemudahan bagi calon konsumennya untuk mendapatkan informasi lebih lanjut mengenai usaha dan produknya. Pada gambar tersebut di atas, Sapinesia memberian kemudahan bagi calon konsumennya untuk mendapatkan informasi lebih lanjut mengenai usaha dan produknya. 48 ATRABIS: Jurnal Administrasi Bisnis Vol. 7, No. 1 Juni 2021 Waktu promosi 24 jam Dunia internet tak terbatas oleh waktu, kapanpun pengguna dapat mengaksesnya tanpa kecuali selama terhubung dengan jaringan internet. Oleh sebab itu, meskipun beberapa pakar periklanan online mengatakan adanya waktu-waktu khusus dimana banyak pengguna mengakses internet atau media sosial, namun para pelaku usaha tetap dapat melakukan promosi selama 24 jam pada jam yang diinginkan. KESIMPULAN Berdasarkan penelitian yang dilakukan penulis secara online, dengan menjadi instrumen kunci, dimana penulis memperhatikan dan mengalaminya sebagai konsumen secara langsung, dapat disimpulkan bahwa media sosial saat ini adalah sarana paling penting bagi Sapinesia dalam mempromosikan produknya. Hal ini dibuktikan dengan perkembangan dari masing-masing akun resmi Sapinesia di Instagram yang jumlah followers-nya meningkat tajam dalam waktu relatif singkat. Meningkatnya followers juga 49 ATRABIS: Jurnal Administrasi Bisnis Vol. 7, No. 1 Juni 2021 membuktikan bahwa semakin banyak konsumen yang tertarik pada produk olahan daging sapi dari Sapinesia tersebut. Keterbatasan yang dimiliki penulis adalah tidak dapatnya mengetahui perkembangan penjualan produk dari Sapinesia, dan insight dari iklan yang ditayangkan, sehingga penulis tidak dapat membahas mengenai hal tersebut. Penulis berharap dapat melakukan penelitian lebih lanjut agar dapat menampilkan informasi yang lebih dalam lagi. Serta tidak menutup kemungkinan bagi peneliti lainnya jika tertarik untuk melanjutkan penelitian ini. Daftar Pustaka [1] Albi Anggito, Johan Setiawan. (2018). Metodologi Penelitian Kualitatif. CV Jejak. https://books.google.co.id/books?hl=id&lr=&id=59V8DwAAQBAJ&oi=fnd& pg=PP1&dq=penelitian+deskriptif+kualitatif+menurut+para+ahli&ots=5Haxs DhrEq&sig=- IyZobJmjXJczoqwevitBzE0n1M&redir_esc=y#v=onepage&q=penelitian%20d eskriptif%20kualitatif%20menurut%20para%20ahli&f=false [2] Anugerah, A. (2019). Instagram Adalah Platform Berbagi Foto Dan Video, Ini Deretan Fitur Canggihnya. Diakses 29 Mei 2021. https://www.liputan6.com/tekno/read/3906736/instagram-adalah-platform- berbagi-foto-dan-video-ini-deretan-fitur-canggihnya [3] Arif, M. (2016). Media Massa Semakin Tergerus Perkembangan Teknologi Informasi. Diakses 31 Mei 2021. https://www.unpad.ac.id/2016/12/media-massa-semakin-tergerus- perkembangan-teknologi-informasi/ [4] Assauri, S. (2014). Manajemen Pemasaran. Jakarta: Rajawali Pers. [5] Carr, Caleb T. and Hayes, Rebecca A. (2015). Sosial Media: Defining, Developing, and Divining, Atlantic Journal of Communication. [6] Dewi, U., Dewi, E F. (2018). Strategi Pemasaran Melalui Media Sosial Instagram. Jurnal Widya Cipta, Vol 2, No 2. 271-278. http://ejournal.bsi.ac.id/ejurnal/index.php/widyacipta [7] Feibe, K. dkk. (2018). Analisis Strategi Pemasaran Dalam Menigkatkan Penjualan Motor Yamaha Mio Pada PT. Hasjrat Abadi Outlet Yamaha Sam Ratulangi. Jurnal EMBA, Vol. 6, No. 2, 968-977. https://ejournal.unsrat.ac.id/index.php/emba/article/viewFile/20024/20294 [8] Kotler, Philip dan Gary Asmtrong. (2001). PrinsipPrinsip Pemasaran. (Terj) Damos Shihombing. Jakarta: Erlangga [9] Kotler, Philip and Kevin Lane Keller, 2016. Marketing Managemen, 15th Edition, Pearson Education,Inc. [10] Moh Rusdi. (2019). Strategi Pemasaran Untuk Meningkatkan Volume Penjualan Pada Perusahaan Genting Ud. Berkah Jaya. Jurnal Trunojoyo, Vol. 6 (2), 49-54. [11] Permatasari, G. (2016). Efektivitas Media Sosial Instagram sebagai Media Promosi Produk Olahan Pertanian Yogurt Cimory. Institut Pertanian Bogor. [10] Moh Rusdi. (2019). Strategi Pemasaran Untuk Meningkatkan Volume Penjualan Pada Perusahaan Genting Ud. Berkah Jaya. Jurnal Trunojoyo, Vol. 6 (2), 49-54. [11] Permatasari, G. (2016). Efektivitas Media Sosial Instagram sebagai Media Promosi Produk Olahan Pertanian Yogurt Cimory. Institut Pertanian Bogor. [12] Rakha F. (2020). 10 Macam Media Sosial yang Paling Sering Digunakan Oleh Orang Indonesia. https://www.merdeka.com/jatim/10-macam-media-sosial-yang-paling-sering- digunakan-oleh-orang-indonesia-kln.html?page=1 50 ATRABIS: Jurnal Administrasi Bisnis Vol. 7, No. 1 Juni 2021 [13] Rafki, F. (2020). Tips Bagi Perusahaan Agar Terhindar dari Insiden Kebocoran Data. Diakses 31 Mei 2021. https://infokomputer.grid.id/read/122411078/tips-bagi-perusahaan-agar- terhindar-dari-insiden-kebocoran-data [14] Syifa, N K. (2020). Apa Itu Sei Sapi? Daging Asap Khas NTT Yang Sedang Naik Daun. Diterbitkan 24 Juli 2020. https://www.kompas.com/food/read/2020/07/24/080800275/apa-itu-sei-daging- asap-khas-ntt-yang-sedang-naik-daun?page=all [15] Zahrah, L., Djuara, P. (2018). Hubungan Penggunaan Media Sosial Dengan Perkembangan Usaha Kecil Dan Menengah Kuliner Wilayah Solo Raya. Jurnal IPB, Vol. 16 No. 1, 75-88. https://Instagram.com/sapinesia https://Instagram.com/sapinesiaku https://Instagram.com/sapinesia.id https://Instagram.com/frozenseisapinesia https://Instagram.com/seisapinesiafrozen https://Instagram.com/seisapinesiafrozenku [13] Rafki, F. (2020). Tips Bagi Perusahaan Agar Terhindar dari Insiden Kebocoran Data. Diakses 31 Mei 2021. https://infokomputer.grid.id/read/122411078/tips-bagi-perusahaan-agar- terhindar-dari-insiden-kebocoran-data 51 51 51
https://openalex.org/W4289477692	https://www.e3s-conferences.org/10.1051/e3sconf/202234202003/pdf	English	null	InSAR assets in ground movements survey on abandoned coalfields	Springer Link (Chiba Institute of Technology)	2,022	cc-by	4,366	© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 342, 02003 (2022) Journées Scientifiques AGAP Qualité 2022 E3S Web of Conferences 342, 02003 (2022) Journées Scientifiques AGAP Qualité 2022 https://doi.org/10.1051/e3sconf/202234202003 * Corresponding author: j.morel@brgm.fr Atouts de l'InSAR pour le suivi des mouvements de terrain dans les zones minières abandonnées Jacques Morel1,, Daniel Raucoules1 , Michael Foumelis1 and Sandrine Lemal1 1BRGM, French Geological Survey, 3 avenue Claude Guillemin, 45060 Orléans, France Abstract. Rising groundwater in abandoned mines may result in ground movements at the surface overlying underground works. Feedbacks on several abandoned coalfields show that ground uplift is generally observed, reaching in some cases several centimetres or more. Although such ground movements, slow and associated with slight slopes, are not expected to generate surface damages, the survey of potential ground movements on abandoned coalfield is necessary, especially to confirm the end of mining- induced subsidence. The most common used method for this mission is levelling. A retro-analysis of ground movements, based on Interferometric Synthetic Aperture Radar (InSAR) technique, covering more than 20 years after mine activity ceasing, has been realised on the French abandoned coalfield of Nord-Pas de Calais. This analysis aimed to extend ground movements detection capabilities in areas not covered by levelling as well as compare InSAR analysis to levelling data for evaluating the robustness of satellite-based displacement measurements. InSAR analysis was able to highlight ground movements of a few millimetres per year, with the same order of precision than classical levelling methods. The observed ground displacement patterns expand well beyond the coalfield, indicating the influence of non-mining-induced phenomena, such as local geology and surface morphology. Our findings underline the operational capability of space-borne InSAR for monitoring abandoned coalfields. Résumé. La remontée des eaux souterraines dans les mines abandonnées peut entraîner des mouvements de terrain à la surface recouvrant les travaux souterrains. Les retours d'expérience sur plusieurs bassins houillers abandonnés montrent qu’un soulèvement est généralement observé, atteignant dans certains cas plusieurs centimètres ou plus. Bien que ces mouvements lents et à très grand rayon de courbure ne soient pas susceptibles de générer des dommages en surface, la surveillance de ces mouvements de sol reste nécessaire, notamment pour confirmer la fin des affaissements d’origine minière. La méthode la plus couramment utilisée pour cette mission est le nivellement. Une rétro-analyse des mouvements du sol, basée sur la technique InSAR (Interferometric Synthetic Aperture Corresponding author: j.morel@brgm.fr https://doi.org/10.1051/e3sconf/202234202003 E3S Web of Conferences 342, 02003 (2022) Journées Scientifiques AGAP Qualité 2022 Radar), couvrant plus de 20 ans après la cessation de l'activité minière, a été réalisée sur le bassin houiller abandonné du Nord-Pas de Calais. Atouts de l'InSAR pour le suivi des mouvements de terrain dans les zones minières abandonnées Cette analyse visait à étendre les capacités de détection des mouvements du sol dans les zones non couvertes par le nivellement, ainsi qu'à comparer l'analyse InSAR aux données de nivellement pour évaluer la robustesse des mesures de déplacement par satellite. L'analyse InSAR a permis de mettre en évidence des mouvements du sol de quelques millimètres par an, avec le même ordre de précision que les méthodes classiques de nivellement. Les déplacements du sol observés s'étendent bien au-delà du bassin houiller, indiquant l'influence de facteurs non induits par l'exploitation minière, tels que la géologie locale et la morphologie de surface. Nos conclusions soulignent la capacité opérationnelle de l'InSAR spatial pour la surveillance des bassins houillers abandonnés. 1 Introduction The Nord-Pas de Calais coal basin is located in the North of France. It extends from East to West on approximately 100 km for a width of 10 to 20 km (figure 1). The basin was exploited between 1750 and 1990, for a total production of 2400 Mt. Coal extraction, down to more than 1000 m deep, and along superimposed seams, has generated subsidence that, in some places, reached cumulatively more than 20 m. Fig. 1. Location and configuration of the Nord-Pas de Calais coalfield (underground mining works in grey) Fig. 1. Location and configuration of the Nord-Pas de Calais coalfield (underground mining works in grey) At the end of the exploitation, the termination of pumping operations initiated a progressive flooding of underground works, which will last in the case of the Nord-Pas de Calais basin more than one century. This flooding process may result in ground movements at the surface overlying underground works. After a transition phase, lasting a few years, 2 https://doi.org/10.1051/e3sconf/202234202003 E3S Web of Conferences 342, 02003 (2022) Journées Scientifiques AGAP Qualité 2022 during which residual subsidence occurs, ground uplift is generally observed [1, 2]. This uplift finds its origin in two mechanisms: during which residual subsidence occurs, ground uplift is generally observed [1, 2]. This uplift finds its origin in two mechanisms: 1. The dilation of underground caved works under pore pressure rise: this mechanism constitutes the main source of the uplift phenomenon, and will be related to the volume of underground works; 2. The swelling of clay material present in the overburden: the contribution of this second mechanism is much smaller and will concern essentially the first hundreds of meters below the surface. Feedback on several European abandoned coalfields [3] shows that this uplift can reach several centimetres or more. Largest uplifts are generally observed on places where largest subsidence occurred during exploitation. Their kinetic is directly related to the one of rising groundwater. In some cases (as observed for the Lorraine Coal basin in France), a small reactivation of subsidence might be observed before heave, due to the rearrangement of equilibrium found after residual subsidence. Although of much smaller intensity than subsidence during exploitation, these ground movements induced by water recovery are not negligible. g g In the framework of its missions, the Post-mining Department of the French Geological Survey (BRGM) has the mandate of surveying these potential ground movements. 1 Introduction The most common used monitoring technique is levelling, performed at a given frequency. On the Nord-Pas de Calais coal basin, five levelling lines of several kilometres long, which were implemented during exploitation for the monitoring of subsidence, are used for the post-mining survey and measured on a yearly basis. The levelling lines provide high precision (of a few millimetres) measurements of ground movement. However, this technique stays spatially limited and does not offer the opportunity to detect ground movements at the coalfield scale. Mining-induced ground movement might occur also at places not covered by levelling. Mine water recovery is a process that can last several tens of years. With the goal to optimise the long-term survey, an InSAR retro-analysis of ground movements, covering more than 20 years after mine activity ceasing, has been realised on the Nord-Pas de Calais coalfield. Our work aims to: 1. Extend ground movements detection capabilities in areas not covered by levelling; 2. Compare InSAR analysis to levelling data to evaluate robustness of displacement measurements by spaceborne geodetic techniques; 3. Investigate the impact of non-mining-induced factors on measured ground movements. 2 Methodology Spaceborne InSAR is a technique for processing of SAR images acquired from Earth Observation satellites. The interferometric processing of a sequence of SAR acquisitions imaged from approximately the same location at different dates allows the measurement of ground displacements, with a precision of a few millimetres. Such multi-temporal InSAR analysis is applicable for monitoring wide areas, while permitting the retro-analysis using already archived SAR data. The above facts make spaceborne InSAR a relevant technique to detect and monitor mine induced ground deformation. In the current study, we aim at detecting motion rate typically of the order of several millimetres per year (5 mm/yr or less, as expected by levelling campaigns), expanded over 3 E3S Web of Conferences 342, 02003 (2022) Journées Scientifiques AGAP Qualité 2022 https://doi.org/10.1051/e3sconf/202234202003 several kilometre distances. For this purpose, the entire archive of the European Space Agency’s (ESA) historical ERS and ENVISAT as well as the Copernicus Sentinel-1 SAR missions (in descending acquisition geometries) were used, covering an area of approximately 5500 km2: 1. ERS mission – 246 images covering the period 1995-2000; three adjacent tracks were necessary to cover the entire coalfield; 2. ENVISAT mission – 141 images covering the period 2002-2010; three adjacent tracks were necessary to cover the entire coalfield; 3. Sentinel-1 mission – 164 IW TOPSAR images covering the period 2015-2018; a single track was enough to cover the entire coalfield. Noteworthy is the fact that the repeat cycle of Sentinel 1 mission is 6-12 days (compared to 35 days for ERS and ENVISAT). For a given time span, Sentinel 1 provides a temporally denser archive resulting into more robust InSAR results. The InSAR processing implemented is the interferometric stacking, which is based on the estimation of the average displacement rates by examining a set of successfully unwrapped differential interferograms [4, 5, and 6]. Processing was performed using the GAMMA software packages [7]. Considering the large amount of data that had to be managed, calculations were implemented on a Virtual Machine (VM) of the European Space Agency (ESA) Grid Processing on Demand (G-POD) service (https://gpod.eo.esa.int/). ( p gp ) For the co-registration of SAR scenes an iteration process applying initially image matching followed by Enhanced Spectral Diversity approach [8, 9]. The ALOS Global Digital Surface Model (AW3D30) (http://eorc.jaxa.jp/ALOS/en/aw3d30/) was used to compensated for the topographic component. Multi-looking factors both in range and azimuth were considered leading to interferometric outputs at 60m spatial resolution. 3 Results Displacements are measured along the radar beam’s Line-of-Sight (LOS). It is assumed that ground motion is occurring essentially along the vertical direction, and given that the area is practically flat, the vertical motion can therefore be reliably deduced from the LOS measurements. Slight deviations could be introduced due to differences in the incident angle between ERS/ENVISAT and Sentinel-1 observations. The displacement rates are expressed in millimetres per year over the considered period, with local reference point at the town of Lille, far enough from the coalfield influence. As expected, the density of InSAR results is much higher in urban and suburban areas compared to vegetated lands (forest and/or agricultural fields), mainly due to temporal decorrelation effects. 2 Methodology More details on the interferometric methods used can be found in [10], [11], [12] and [13]. 3.1 ERS and ENVISAT observation periods (1995-2000 and 2002-2010) A significant subsidence reaching approximately 4 mm/year can be noticed in the central region of the coalfield during the ERS observation period (1995-2000) (figure 2). This phenomenon occurs a few years after the end of the exploitation and might be attributed to residual subsidence. During the following period, ENVISAT observation (2002-2010), the subsidence in this central region is lower but still present (at 2 mm/year), despite the fact that residual subsidence is expected to end few months to few years after exploitation [14]. 4 E3S Web of Conferences 342, 02003 (2022) Journées Scientifiques AGAP Qualité 2022 https://doi.org/10.1051/e3sconf/202234202003 An increase in subsidence rates can be observed on some isolated areas during the ENVISAT period, e.g. more than 12 years after the end of coal exploitation. This observation points out a clear and significant impact of non-mining-induced phenomena on measured ground movements. This result is developed in paragraph 3.3. Fig. 2. InSAR ground displacement rates during ERS (1995-2000) (up) and ENVISAT (2002-2010) (down) observation periods. Selected local reference point shown as square. In background a hillshade based on the AW3D30 DSM heights. Black polygons correspond to mining concessions (see Figure 1). Fig. 2. InSAR ground displacement rates during ERS (1995-2000) (up) and ENVISAT (2002-2010) (down) observation periods. Selected local reference point shown as square. In background a hillshade based on the AW3D30 DSM heights. Black polygons correspond to mining concessions (see Figure 1). 3.2 Sentinel-1 observation period (2015-2018) Subsidence is still observed over the main part of the region reaching in some places up to 5 mm/year (figure 3). At the eastern part of the coal basin subsidence is still ongoing, while extending much farer than the mining works influence, especially towards the southeast. Other areas with no relation to the coalfield show also significant subsidence, however, they cannot be attributed to mining-induced effects. Specific natural areas, such as riverbeds, are characterised by high subsidence rates, while a very localised uplift pattern is recognised within the urban area of Valenciennes (figure 4). 5 5 E3S Web of Conferences 342, 02003 (2022) Journées Scientifiques AGAP Qualité 2022 https://doi.org/10.1051/e3sconf/202234202003 Journées Scientifiques AGAP Qualité 2022 All these observations point out the influence of natural and/or non-mining-induced anthropogenic phenomena. These phenomena should be taken into account when interpreting the origin of ground movements in the coal basin. All these observations point out the influence of natural and/or non-mining-induced anthropogenic phenomena. These phenomena should be taken into account when interpreting the origin of ground movements in the coal basin. Fig. 3. Sentinel-1 InSAR ground displacement rates for the period 2015-2018. Selected local reference point shown as square. In background a hillshade based on the AW3D30 DSM heights. Black polygons correspond to mining concessions (see Figure 1). Fig. 3. Sentinel-1 InSAR ground displacement rates for the period 2015-2018. Selected lo reference point shown as square. In background a hillshade based on the AW3D30 DSM heig Black polygons correspond to mining concessions (see Figure 1). Fig. 3. Sentinel-1 InSAR ground displacement rates for the period 2015-2018. Selected local reference point shown as square. In background a hillshade based on the AW3D30 DSM heights. Black polygons correspond to mining concessions (see Figure 1). Fig. 4. Subsidence along a channelled river and localised uplift in urban area of Valenciennes, as shown by Sentinel-1 InSAR results (period 2015-2018). Satellite imagery layer in background (source ESRI basemaps). Fig. 4. Subsidence along a channelled river and localised uplift in urban area of Valenciennes, as shown by Sentinel-1 InSAR results (period 2015-2018). Satellite imagery layer in background (source ESRI basemaps). 3.3 Influence of non-mining-induced phenomena 7 7 E3S Web of Conferences 342, 02003 (2022) Journées Scientifiques AGAP Qualité 2022 https://doi.org/10.1051/e3sconf/202234202003 3.3 Influence of non-mining-induced phenomena Large and long-term geological processes, such as tectonic activity or post-glacial rebound, might generate ground movements reaching up to 1 cm/year in the Nord-Pas de Calais region [15]. However, the most important natural factor remains local geology. Figure 5 shows the geology characterizing the Nord-Pas de Calais region. The coalfield is located close to the boundary between upper Cretaceous (green colour, mainly at the SW of the 6 6 6 E3S Web of Conferences 342, 02003 (2022) Journées Scientifiques AGAP Qualité 2022 https://doi.org/10.1051/e3sconf/202234202003 coal basin) and lower Eocene (orange colour, dominating the north-eastern part of the coal basin). The eastern part of the coal basin is also characterized by an important amount of alluvial deposits (light grey colour). Highest subsidence rates are essentially observed in this part of the coal basin during the Sentinel-1 observation period (cf. Figure 3), one of the most remarkable example being along the channelled Sensée riverbed. coal basin) and lower Eocene (orange colour, dominating the north-eastern part of the coal basin). The eastern part of the coal basin is also characterized by an important amount of alluvial deposits (light grey colour). Highest subsidence rates are essentially observed in this part of the coal basin during the Sentinel-1 observation period (cf. Figure 3), one of the most remarkable example being along the channelled Sensée riverbed. No further attempts were made to interpret these naturally induced (non-mining related) signals, given the focus of the current work. Fig. 5. Geology of the Nord-Pas de Calais coalfield area (BRGM 1/1.000.000 scale geological map, source http://infoterre.brgm.fr). Mining concessions are shown as dark red polygons. Fig. 5. Geology of the Nord-Pas de Calais coalfield area (BRGM 1/1.000.000 scale geological map, source http://infoterre.brgm.fr). Mining concessions are shown as dark red polygons. Anthropogenic activities might also result in significant ground movements. Figure 6 shows a detail view of the central part of the coal basin, where the highest ground displacement rates are identified on open pit quarries, often associated with old coal extraction waste heaps. Other anthropogenic factors such as ground water pumping for domestic use or forgotten underground stone quarries might also induce ground movements. movements. Fig. 6. Correlation between highest observed displacement rates (period 2015-2018) and anthropogenic activities (quarries, red rectangles) in the study area. Fig. 6. Correlation between highest observed displacement rates (period 2015-2018) and anthropogenic activities (quarries, red rectangles) in the study area. 4 Intercomparison between InSAR and levelling The location of the five levelling lines used for ground movement surveys on the coal basin is shown in figure 7. As previously mentioned, they do not allow for a complete coverage of the coalfield, whereas some of them are located within the underground works influence zone. Each levelling line measurement is referred to benchmarks located at both ends of the line. Measurements are carried out once per year, with precision at the order of a few millimetres. Two of the levelling lines indicate a continuous subsidence since the beginning of the measurements (examples of the Billy-Montigny and Lens levelling lines, figure 7), at a rate of 1 to 4 mm/year. Two others do not show clear trend, with lower displacement rates (Bruay and Estevelles-Courrières levelling lines). The last one shows apparent continuous uplift since the beginning of the measurements (Wallers-Arenberg levelling line). p g g ( g g ) Fig. 7. Location of the five levelling line in the Nord-Pas de Calais coal basin and the corresponding vertical displacement measurements for campaigns between 2011 and 2018. The inter-comparison between InSAR displacement rates and levelling has been done for the period 2015-2018 covered by Sentinel-1 mission data. Fig. 7. Location of the five levelling line in the Nord-Pas de Calais coal basin and the corresponding vertical displacement measurements for campaigns between 2011 and 2018. The inter-comparison between InSAR displacement rates and levelling has been done for the period 2015-2018 covered by Sentinel-1 mission data. The inter-comparison between InSAR displacement rates and levelling has been done for the period 2015-2018 covered by Sentinel-1 mission data. 8 8 https://doi.org/10.1051/e3sconf/202234202003 E3S Web of Conferences 342, 02003 (2022) Journées Scientifiques AGAP Qualité 2022 The results presented in the former paragraph, particularly through figures 3 and 5, show the strong influence of non-mining-induced factors, especially geology, on the origin of measured ground movements. Hence, the potential mining-induced contribution (that could be associated with rising groundwater) to these already very small movements, cannot be distinguished from that associated with non-mining-induced phenomena. The following proposed interpretation considers then the measured ground movements as the sum of all factors likely to generate ground movements, without being able at this stage to distinguish them. The impact of non-mining-induced phenomena, however, appears to be preponderant. Figure 8 presents InSAR displacement rates in the surroundings of the Wallers levelling line. 4 Intercomparison between InSAR and levelling Alluvial deposits are particularly presents in the sector where this levelling line is implemented. Figure 8 shows that the benchmarks at both ends of the levelling line are also affected by ground movements during the observation period. Subsidence rates at the levelling benchmarks up to 4.3 mm/year are measured. This relative movement of the levelling benchmarks generates an offset, which must be corrected when interpreting levelling data. Moreover, the middle part of the Wallers levelling line appears to undergo slower subsidence than its ends, with displacement rates less than 3 mm/yr. Therefore, what was interpreted as uplift based on levelling (Figure 7) is in fact the result of subsidence being faster at its ends where the reference benchmarks are located (Figure 8). Fig. 8. InSAR ground displacement rates in the Wallers levelling line area, Sentinel-1 period (2015- 2018). World topographic map in background (source ESRI basemaps). Fig. 8. InSAR ground displacement rates in the Wallers levelling line area, Sentinel-1 period (2015- 2018). World topographic map in background (source ESRI basemaps). Fig. 8. InSAR ground displacement rates in the Wallers levelling line area, Sentinel-1 period (2015- 2018). World topographic map in background (source ESRI basemaps). This intercomparison points out that differential motion between benchmarks at both ends of a levelling line (i.e. one exhibits a relative motion respect to the other), shall introduce not only an offset but also a distortion in the produced displacement profile. This This intercomparison points out that differential motion between benchmarks at both ends of a levelling line (i.e. one exhibits a relative motion respect to the other), shall introduce not only an offset but also a distortion in the produced displacement profile. This 9 9 E3S Web of Conferences 342, 02003 (2022) Journées Scientifiques AGAP Qualité 2022 https://doi.org/10.1051/e3sconf/202234202003 is clearly visible on the Bruay levelling line (the westernmost line in the coalmine basin; see figure 7) for which a difference of about 1 mm/yr is observed between both ends (figure 9). is clearly visible on the Bruay levelling line (the westernmost line in the coalmine basin; see figure 7) for which a difference of about 1 mm/yr is observed between both ends (figure 9). Fig. 9. Displacement rate differences along the Bruay levelling line, after adjustment on one of the levelling benchmarks. Fig. 9. Displacement rate differences along the Bruay levelling line, after adjustment on one of the levelling benchmarks. 4 Intercomparison between InSAR and levelling Following the adjustment of the InSAR displacement rates to compensate for the referencing differences generated by the movement of levelling benchmarks, displacements rates calculated with both methods fit remarkably well (figure 10). This result shows that InSAR is a perfectly adapted tool to monitor ground displacements with a precision equivalent to that of levelling method. 10 E3S Web of Conferences 342, 02003 (2022) Journées Scientifiques AGAP Qualité 2022 https://doi.org/10.1051/e3sconf/202234202003 Fig. 10. Comparison of the displacements calculated from Sentinel-1 InSAR and levelling for (a) Billy-Montigny, (b) Estevelles-Courrières, (c) Lens and (d) Wallers-Arenberg levelling lines (see Figure 7). Fig. 10. Comparison of the displacements calculated from Sentinel-1 InSAR and levelling for (a) Billy-Montigny, (b) Estevelles-Courrières, (c) Lens and (d) Wallers-Arenberg levelling lines (see Figure 7). Fig. 10. Comparison of the displacements calculated from Sentinel-1 InSAR and levelling for (a) Billy-Montigny, (b) Estevelles-Courrières, (c) Lens and (d) Wallers-Arenberg levelling lines (see Figure 7). 5 Conclusion This study demonstrated that the effective monitoring of mining-induced ground movements at the scale of a coalfield requires techniques able to operate at large spatio- temporal scales as well as properly designed to facilitate identification of non-mining related deformation mechanisms, particularly the influence of local geological conditions. Spaceborne InSAR techniques offers the opportunity to examine historical data archives, while covering much larger regions compared to standard levelling methods. By achieving comparable level of precision, InSAR has been proven well suited for revealing ground movements not detectable by local levelling campaigns. InSAR appears also as a complementary tool to emphasize the impact of non-mining- induced phenomena on levelling measurements, especially on the potential movement of levelling benchmarks. These results highlight the necessity of being cautious when interpreting the origin of damages caused by ground movements. Our findings underline the capability of satellite InSAR for the operational surveying of ground movements on abandoned coalfields. The ability to cover wide area, the frequency of data availability, the accuracy of the measured displacements and the opportunity to perform retro analysis are strong assets in post-mining monitoring context. The authors would like to thank the French Ministry for the Ecological and Inclusive Transition, for financial support and the ESA Research and Service Support (RSS) for the provisioning of the employed computer resources. The Hauts de France Regional Directorate for Environment, Development and Housing (DREAL) is also gratefully acknowledged. 11 https://doi.org/10.1051/e3sconf/202234202003 E3S Web of Conferences 342, 02003 (2022) Journées Scientifiques AGAP Qualité 2022 E3S Web of Conferences 342, 02003 (2022) References 1. C. Banton, L. Bateson, H. McCormack, R. Holley, I.A. Watson, R. Burren, D. Lawrence, F. Cigna, Proceedings of the Mine Closure International conference, 97– 108 (M Tibbett, AB Fourie & C Digby (eds), 2013) 2. X. Devleeschouwer, P.Y. Declercq, B. Flamion, J. Brixko, A. Timmermans, J. Vanneste, Proceedings of the Post-Mining Symposium (2008) 3. C. Herrero, A. Munoz, J.C. Catalina, F. Hadj-Hassen, R. Kuchenbecker, V. Spreckels, J. Juzwa, S. Bennett, M. Purvis, D. 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Colas, M. de Michele, Proceedings of the International Geoscience and Remote Sensing Symposium (2019) (2007) 13. M. Foumelis, D. Raucoules, B. Colas, M. de Michele, Proceedings of the International Geoscience and Remote Sensing Symposium (2019) ( ) 13. M. Foumelis, D. Raucoules, B. Colas, M. de Michele, Proceedings of the International Geoscience and Remote Sensing Symposium (2019) 14. References B.N. Whittaker, D.J. Reddish, Subsidence. Occurrence, Prediction and Control, (Elsevier, 1989) 15. H. Van Vliet-Lanoë, Evolution morphotectonique récente du bassin houiller Nord-Pas de Calais dans le cadre de l’Europe de l’Ouest, (Specific report for Charbonnages de France, 1999) 12
https://openalex.org/W3215890699	https://bmcpublichealth.biomedcentral.com/track/pdf/10.1186/s12889-021-12211-8	English	null	Association of early-life undernutrition and risk of dyslipidemia in adulthood: a population-based cohort study	BMC public health	2,021	cc-by	7,033	Association of early-life undernutrition and risk of dyslipidemia in adulthood: a population-based cohort study Minmin Wang1†, Mengfei Liu1†, Chuanhai Guo1, Fenglei Li2, Zhen Liu1, Yaqi Pan1, Fangfang Liu1, Ying Liu1, Huanyu Bao1, Zhe Hu1, Hong Cai1, Zhonghu He1* and Yang Ke1* Minmin Wang1†, Mengfei Liu1†, Chuanhai Guo1, Fenglei Li2, Zhen Liu1, Yaqi Pan1, Fangfang Liu1, Ying Liu1, Huanyu Bao1, Zhe Hu1, Hong Cai1, Zhonghu He1* and Yang Ke1* Wang et al. BMC Public Health (2021) 21:2129 https://doi.org/10.1186/s12889-021-12211-8 Wang et al. BMC Public Health (2021) 21:2129 https://doi.org/10.1186/s12889-021-12211-8 Abstract 1 Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Laboratory of Genetics, Peking University Cancer Hospital & Institute, #52 Fucheng Rd, Haidian District, 100142 Beijing, China Correspondence: zhonghuhe@foxmail.com; keyang@bjmu.edu.cn †Minmin Wang and Mengfei Liu contributed equally to this work. Background Mortality associated with cardiovascular disease (CVD) has risen in China over the last decade, and CVD accounted for over 40% of deaths from all causes in 2014 [1]. Lipid abnormalities are a principal Full list of author information is available at the end of the article Abstract Background: The association of early-life undernutrition and dyslipidemia found in previous studies may be con- founded by the uncontrolled age difference between exposed and unexposed participants. The study aimed to investigate the association of early-life undernutrition and the risk of dyslipidemia in adulthood with good control of the age variable. Methods: We took the Great Chinese Famine (1959–1961) as a natural experiment of severe undernutrition. This study was based on the baseline investigation of a population-based cohort in rural China. Undernutrition in early life was defined as being exposed to famine at younger than 3 years of age. Three approaches including Adjustment, Restriction, and Matching were applied to control the confounding effect of age. Logistic regression models were applied to evaluate the association between early-life famine and the presence of dyslipidemia. Stratified analysis by gender was also performed, and potential effect modification was tested by adding the interaction term of the fam- ine exposure variable and gender into the model. Results: Undernutrition in early life was associated with increased risk of borderline high and above (BHA) levels of total cholesterol (TC, ORAdjustment = 1.61; ORRestriction = 1.56; ORMatching = 1.87), triglycerides (TG, ORAdjustment = 1.33; ORRestriction = 1.30; ORMatching = 1.34), low-density lipoprotein cholesterol (LDL-C, ORAdjustment = 1.75; ORRestriction = 1.53; ORMatching = 1.77) and dyslipidemia (ORAdjustment = 1.52; ORRestriction = 1.45; ORMatching = 1.60), as well as high levels of TC, TG, LDL-C and dyslipidemia. An inverse association of undernutrition and risk of low high-density lipoprotein cholesterol (HDL-C) was found. Female participants with undernutrition experience had an increased risk of BHA TG and LDL-C (TG: ORAdjustment, female = 1.45; ORRestriction, female = 1.39; ORMatching, female = 1.51; LDL-C: ORAdjustment, female = 2.11; ORRestriction, female = 1.80; ORMatching, female = 2.15), but this association was not found in males. Conclusion: Early-life undernutrition increased the risk of TC, TG, LDL-C, and dyslipidemia. Gender would significantly modify this effect for TG and LDL-C. These results emphasize the importance of nutritional conditions in the early stages of life to long-term health consequences. Keywords: Undernutrition, Great Chinese Famine, Dyslipidemia, Confounding effect Correspondence: zhonghuhe@foxmail.com; keyang@bjmu.edu.cn †Minmin Wang and Mengfei Liu contributed equally to this work. Methods P l ti contributor to CVD, and the prevalence of dyslipidemia is over 30% in rural residents in China [2–5]. Dyslipi- demia is a long-term result of the combined effects of genetic factors and environmental exposure (such as unhealthy diet and lifestyle). Exposure to undernutri- tion in early life will likely have a long-term influence on lipid metabolism mode [6–8]. This study was conducted in Hua County of Henan Prov- ince, China, which covered an area of 700 mile2. This area is predominantly rural and 90% of the total population is involved in agriculture. Henan province suffered severe food shortages during the Great Famine. The mortal- ity in 1960 reached 39.6‰, which was two-fold higher than the average level in 1956–58 [9, 13]. The population sizes of individuals of 51–53 years (born in 1959–1961) decreased about 50% compared with the population of 54–56 years (born in 1956–1958) according to the popu- lation statistics in 2012 [14]. China experienced a serious famine in 1959–1961, which caused millions of excess deaths with an annual mortality of over 3.0% during the years of famine [9– 11]. The Great Chinese Famine was characterized by long duration, a large affected geographic area, and the serious consequences it engendered. In the current era, most middle-aged Chinese were exposed to this famine at some point in their early life.h The study enrolled participants aged 45–69 years from 668 randomly selected villages in Hua County based on the baseline investigation of Endoscopic Screening for Esophageal Cancer in China (ESECC) randomized con- trolled trial (ClinicalTrials.gov identifier: NCT01688908). The design and preliminary results of the “ESECC” trial can be found elsewhere [15]. Briefly, 668 out of 846 vil- lages in rural Hua County with population size ranging from 500 to 3000 were randomly selected and permanent residents aged 45–69 in each of the target villages were invited to participate in the trial on a voluntary basis. A total of 35,772 permanent residents were recruited dur- ing the period December 2011–September 2016. There have been several studies that have taken the 1959–61 Chinese famine as a natural experiment to investigate the association of severe undernutrition and the onset of dyslipidemia in adulthood. Methods P l ti As it is difficult to establish an “unexposed control” in paral- lel with the famine-exposed cohort, previous stud- ies typically applied a “birth cohort” design, where the control cohorts are selected from individuals of post- famine birth who are at least 3–5 years younger than the exposed subjects. The association of undernutri- tion and the risk of dyslipidemia is then evaluated by comparing the prevalence of dyslipidemia in these two cohorts. However, under this study design, it is of con- cern that the estimated undernutrition effect might be confounded by age, as age is strongly associated with undernutrition exposure as well as with the risk of dyslipidemia [4]. Given the fact there is almost no overlap in the age range in the two birth cohorts, using multivariable models in traditional statistical meth- ods may not be sufficient to diminish the confound- ing effect of age. One meta-analysis [12] suggested that most of the associations observed in undernutri- tion and metabolic syndrome would turn null if age was additionally matched when selecting the control cohorts. This revealed that uncontrolled age differences might explain some aspects of the effect attributed to undernutrition in previous studies. As a result, age dif- ferences in undernutrition exposed and unexposed cohorts should be controlled as the priority in studies investigating the association between early-life under- nutrition and long-term health risk. g p p All participants in this study were measured for height and weight at enrollment. Participants’ seated blood pressure was measured after 5 min of rest with a mer- cury sphygmomanometer. A computer-aided one-on-one questionnaire was completed by well-trained interview- ers to collect information on personal characteristics, socioeconomic status, lifestyle factors and health sta- tus. A fasting blood sample of ~ 5 mL was then collected from each participant in a heparin sodium anticoagulant tube. These tubes were then centrifuged at 1000 rpm for 5 min and the supernatants were sent within 4 h for measurement of total cholesterol (TC), low-density lipo- protein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG) at the clini- cal laboratory of Hua County People’s Hospital. Lipid measurements were conducted using a HITACHI7600 automatic biochemistry analyzer (Hitachi High Technol- ogies Co., Tokyo, Japan) with commercially available rea- gents (Autobio Diagnostics Co., Ltd., Beijing, China). All experiments were performed in accordance with relevant guidelines and regulations. Detailed information on the measurement of lipids can be found elsewhere [4]. Methods P l ti In this study, we applied three approaches for analy- sis, namely Adjustment, Restriction, and Matching to control for the confounding effect of age, and then investigated the association of early-life undernutrition exposure and risk of dyslipidemia in adulthood based on the baseline investigation of a population-based cohort in rural China. © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wang et al. BMC Public Health (2021) 21:2129 Page 2 of 9 Page 2 of 9 Definition of dyslipidemia According to the guidelines for the prevention and treatment of dyslipidemia in Chinese adults (2016 ver- sion), borderline high TC was defined as ≥200 mg/dL (5.2 mmol/L) and <240 mg/dL (6.2 mmol/L); high TC as ≥240 mg/dL (6.2 mmol/L); borderline high TG as ≥150 mg/dL (1.7 mmol/L) and <200 mg/dL (2.3 mmol/L); high TG as ≥200 mg/dL (2.3 mmol/L); borderline high LDL-C as ≥130 mg/dL (3.4 mmol/L) and <160 mg/dL (4.1 mmol/L); high LDL-C as ≥160 mg/dL (4.1 mmol/L); reduced HDL-C as <40 mg/dL (1.0 mmol/L). In view of discrepancies in age distribution [4] and in the iden- tified association of HDL-C and other lipid indicators Statistical analysis was performed using STATA (Ver- sion 13.1; Stata Corp LLC, TX, USA). All tests were two- sided and P values < 0.05 were considered statistically significant. Covariates Risk factors for dyslipidemia identified in the previous study were collected as covariates [4], including age, occupation (physical or nonphysical worker), body mass index (BMI: normal, overweight, or obesity), blood pres- sure (normal, or hypertension), cigarette smoking (no smoking, moderate-smoker, and heavy-smoker), alcohol drinking (no drinking, moderate-drinker, and heavy- drinker), fried food intake (seldom intake, occasional or often intake), salty food intake (seldom intake, occasional or often intake), spicy food intake (seldom intake, occa- sional or often intake), heartburn and regurgitation, and self-reported history of diabetes. Detailed information on the definitions and coding forms of the covariates were listed in Supplementary Table 1. Distinct from other cross-sectional studies, the base- line investigation of the “ESECC” continued from 2011 to 2016. Due to the five-year enrollment process, par- ticipants in both the undernutrition-exposed and non- exposed cohorts had a relatively wide age range at enrollment where individuals were at 50–60 years and 43–53 years in the exposed and non-exposed cohort, respectively. The overlap in age at enrollment provided an opportunity to control the confounding effect of age through three analytical approaches. First, under the Adjustment Approach, all participants who fulfilled the eligibility criteria were included, and a multivariable model was adopted to adjust the age variable as in previ- ous studies [16, 17]. Secondly, the Restriction Approach included only participants aged 50–53 years (the over- lapped age range for undernutrition-exposed cohort and non-exposed cohort) in order to decrease the age differ- ence between cohorts before statistical adjustment. The multivariable model was also applied to further diminish the confounding effect of age. Lastly, under the Matching Approach, individual-level matching was applied to com- pletely rule out the confounding effect of the age variable where each subject in the undernutrition-exposed cohort was matched to a subject from the non-exposed cohort by age and gender. These three approaches showed an increasingly strong capacity to control age differences between cohorts despite decreased sample size (Fig. 1). Study design Undernutrition exposure was defined based on partici- pants’ date of birth. Individuals who were under the age of 3 during the 1959–1961 Great Chinese Famine (i.e., Wang et al. BMC Public Health (2021) 21:2129 Page 3 of 9 born between 1 January 1956 and 31 December 1961) were defined as having early-life undernutrition. The non-exposed cohort was defined as participants born between 1 January 1963 and 31 December 1968 with one year after the famine as a washout period to minimize misclassification. born between 1 January 1956 and 31 December 1961) were defined as having early-life undernutrition. The non-exposed cohort was defined as participants born between 1 January 1963 and 31 December 1968 with one year after the famine as a washout period to minimize misclassification. (Fig. 2, Supplementary Fig. 1), dyslipidemia was defined based only on TC, TG and LDL-C. Two sets of defini- tions for dyslipidemia were used in this study, namely borderline high and above (BHA) dyslipidemia, and high dyslipidemia. BHA dyslipidemia was defined as the pres- ence of a high level or borderline high level of any factor including TC, TG and LDL-C, and high dyslipidemia was defined as the presence of a high level of any one of these three lipid molecules in a given study subject. i Eligibility criteria for the current study included: 1) “ESECC” cohort member; 2) member of the defined undernutrition exposed or non-exposed cohorts; 3) signed informed consent for participation; 4) question- naire completed; 5) blood samples provided, with valid test results for blood lipids. Statistical analysis Th Chi The Chi-square test and Student t-test were used to com- pare demographic characteristics and behavioral factors in exposed and non-exposed cohorts. Logistic regres- sion models were applied to evaluate the association of early-life undernutrition experience and the presence of dyslipidemia. In multivariable models, covariates of risk factors for dyslipidemia were adjusted. The identified associations were further validated in subgroup analy- sis stratified by BMI groups given the strong association of BMI and dyslipidemia. Considering the gender het- erogeneity in dyslipidemia prevalence, trends with age and associated factors [4], we estimated the association in stratification of gender. Potential interaction between undernutrition exposure and gender was tested by add- ing an interaction term of the undernutrition variable and gender into the model. Results From the baseline investigation of the ESECC trial, 7474 participants in the exposed cohort and 4666 in the non-exposed cohort were eligible for the current study. All 12,140 eligible individuals were enrolled into the Wang et al. BMC Public Health (2021) 21:2129 Page 4 of 9 Fig. 1 Flowchart for participants enrolled in three analytic approaches Fig. 1 Flowchart for participants enrolled in three analytic approaches matching, the mean age was 51.85 years for both cohorts. The exposed and non-exposed cohorts showed significant differences in BMI, prevalence of hyper- tension, and intake of spicy food. These two groups of participants did not show marked differences in other behavioral or lifestyle variables. Regarding the distri- bution of lipid indicators, undernutrition exposed sub- jects had a significantly higher level of TC and HDL-C compared with non-exposed participants across these three analytic approaches. The undernutrition exposure was also associated with a significantly higher level of TG under the Restriction and Matching Approach. For LDL-C, the Adjustment Approach demonstrated a sig- nificant difference in these two groups but not in the other two approaches. Adjustment Approach, and from this group, 3201 indi- viduals aged 50–53 years (2477 from the undernutrition exposed cohort and 724 from the non-exposed cohort) were enrolled into the Restriction Approach; and 531 age- and gender-matched pairs (1062 individuals) were included in the Matching Approach (Fig. 1). Key demographic characteristics and behavior vari- ables were compared in the exposed and non-exposed cohorts using three separate analytic approaches (Table 1, Supplementary Table 2). In the Adjust- ment Approach, the mean age of enrolled partici- pants was 48.78 years for the non-exposed cohort and 55.86 years for the exposed cohort. In the Restriction Approach, the mean age was 50.94 and 52.16 years for non-exposed and exposed subjects. In individual-level Wang et al. BMC Public Health (2021) 21:2129 Page 5 of 9 Fig. 2 Estimates of association of early-life undernutrition and risk of dyslipidemia in three analytic approaches. A Association of early-life undernutrition and risk of borderline high and above (BHA) status of dyslipidemia in adulthood. B Association of early-life undernutrition and risk of high status of dyslipidemia in adulthood. ORs were adjusted for age, occupation, BMI, blood pressure, cigarette smoking, alcohol drinking, fried food intake, salty food intake, spicy food intake, heartburn and regurgitation, and self-reported history of diabetes Fig. Results TG and BHA LDL-C in females (TG: ORAdjustment, female = 1.45, 95% CI: 1.17–1.81; ORRestriction, female = 1.39, 95% CI: 1.04–1.86; ORMatching, female = 1.51, 95% CI: 1.04– 2.20; LDL-C: ORAdjustment, female = 2.11, 95% CI: 1.47–3.02; ORRestriction, female = 1.80, 95% CI: 1.15–2.81; ORMatching, female = 2.15, 95% CI: 1.19–3.88). However, this associa- tion was not identified among males. The gender specific association patterns of undernutrition and TC, HDL-C, and dyslipidemia were not consistent across the three analytic approaches (Fig. 3, Supplementary Fig. 1). TG and BHA LDL-C in females (TG: ORAdjustment, female = 1.45, 95% CI: 1.17–1.81; ORRestriction, female = 1.39, 95% CI: 1.04–1.86; ORMatching, female = 1.51, 95% CI: 1.04– 2.20; LDL-C: ORAdjustment, female = 2.11, 95% CI: 1.47–3.02; ORRestriction, female = 1.80, 95% CI: 1.15–2.81; ORMatching, female = 2.15, 95% CI: 1.19–3.88). However, this associa- tion was not identified among males. The gender specific association patterns of undernutrition and TC, HDL-C, and dyslipidemia were not consistent across the three analytic approaches (Fig. 3, Supplementary Fig. 1). TG and BHA LDL-C in females (TG: ORAdjustment, female = 1.45, 95% CI: 1.17–1.81; ORRestriction, female = 1.39, 95% CI: 1.04–1.86; ORMatching, female = 1.51, 95% CI: 1.04– 2.20; LDL-C: ORAdjustment, female = 2.11, 95% CI: 1.47–3.02; ORRestriction, female = 1.80, 95% CI: 1.15–2.81; ORMatching, female = 2.15, 95% CI: 1.19–3.88). However, this associa- tion was not identified among males. The gender specific association patterns of undernutrition and TC, HDL-C, and dyslipidemia were not consistent across the three analytic approaches (Fig. 3, Supplementary Fig. 1). Results 2 Estimates of association of early-life undernutrition and risk of dyslipidemia in three analytic approaches. A Association of early-life undernutrition and risk of borderline high and above (BHA) status of dyslipidemia in adulthood. B Association of early-life undernutrition and risk of high status of dyslipidemia in adulthood. ORs were adjusted for age, occupation, BMI, blood pressure, cigarette smoking, alcohol drinking, fried food intake, salty food intake, spicy food intake, heartburn and regurgitation, and self-reported history of diabetes To evaluate the effect of early-life undernutrition on long-term risk of dyslipidemia, we compared the prevalent risk of dyslipidemia in the exposed cohort to that of the non-exposed cohort. As shown in Fig. 2A, undernutrition increased the risk of having BHA TC (ORAdjustment = 1.61, 95% CI: 1.37–1.89; ORRestriction = 1.56, 95% CI: 1.27–1.93; ORMatching = 1.87, 95% CI: 1.42– 2.47), TG (ORAdjustment = 1.33, 95% CI: 1.13–1.55; ORRestriction = 1.30, 95% CI: 1.05–1.60; ORMatching = 1.34, 95% CI: 1.02–1.76), LDL-C (ORAdjustment = 1.75, 95% CI: 1.35–2.27; ORRestriction = 1.53, 95% CI: 1.10–2.12; ORMatching = 1.77, 95% CI: 1.15–2.73), and dyslipidemia (ORAdjustment = 1.52, 95% CI: 1.31–1.76; ORRestriction = 1.45, 95% CI: 1.19–1.77; ORMatching = 1.60, 95% CI: 1.24–2.06), and these associations were statistically significant over all three approaches. When the outcome was set as high levels of TC, TG, LDL-C, and dyslipidemia (Fig. 2B), similar patterns of association were observed. Regard- ing HDL-C, an inverse association in undernutrition and risk of low HDL-C was identified (ORAdjustment = 0.34, 95% CI: 0.26–0.43; ORRestriction = 0.27, 95% CI: 0.18–0.42; ORMatching = 0.30, 95% CI: 0.18–0.51, Supplementary Fig. 1). The identified association of undernutrition expo- sure and risk of dyslipidemia remained in subgroup anal- ysis stratified by BMI groups, especially in participants with BMI ≤ 24 kg/m2 (Supplementary Fig. 2).i TG and BHA LDL-C in females (TG: ORAdjustment, female = 1.45, 95% CI: 1.17–1.81; ORRestriction, female = 1.39, 95% CI: 1.04–1.86; ORMatching, female = 1.51, 95% CI: 1.04– 2.20; LDL-C: ORAdjustment, female = 2.11, 95% CI: 1.47–3.02; ORRestriction, female = 1.80, 95% CI: 1.15–2.81; ORMatching, female = 2.15, 95% CI: 1.19–3.88). However, this associa- tion was not identified among males. The gender specific association patterns of undernutrition and TC, HDL-C, and dyslipidemia were not consistent across the three analytic approaches (Fig. 3, Supplementary Fig. 1). Discussion Nutrition is vital to the developmental processes of humans, and undernutrition in early-stage of develop- ment may have significant long-term health effects [6–8]. In the current study, we took the Great Chinese Fam- ine as a model for the investigation of the association of severe under-nutrition and risk of dyslipidemia during adulthood. After carefully controlling the confounding effect of age, results of this study confirmed that exposure to undernutrition in early life (being exposed to the Great Famine under the age of 3) increases the prevalent risk of dyslipidemia in adulthood. A strength of this study lies in the design and ana- lytic approaches. In previous studies, there were mainly two methods used to control the confounding effect of the age variable, namely adjustment and combination [12, 16–19]. The principle of adjustment is to report the age-adjusted prevalence of dyslipidemia by using a In the subgroup analysis stratified by gender (Fig. 3, Supplementary Fig. 1, Supplementary Fig. 3), under- nutrition was associated with an increased risk of BHA Wang et al. Discussion BMC Public Health (2021) 21:2129 Page 6 of 9 Table 1 Distribution of age, gender and four lipid indicators in three analytic approaches a Adjustment Approach enrolled 12,140 individuals met eligibility criteria b Restriction Approach enrolled 3201 individuals aged 50–53 years c Matching Approach enrolled 531 age- and gender-matched pairs (1062 individuals) d The Chi-square test and Student’s t test were used to compare demographic characteristics and behavioral factors in the undernutrition exposed and non-exposed Adjustment Approacha Restriction Approachb Matching Approachc Non- exposed cohort (N = 7474) Undernutrition exposed cohort (N = 4666) P valued Non- exposed cohort (N = 2477) Undernutrition exposed cohort (N = 724) P valued Non- exposed cohort (N = 531) Undernutrition exposed cohort (N = 531) P valued Age Mean (SD) 48.78 (2.12) 55.86 (2.23) < 0.001 50.94 (0.92) 52.16 (0.95) < 0.001 51.85 (0.94) 51.85 (0.94) – Gender Male 3422 (45.79) 2213 (47.43) 0.077 1149 (46.39) 320 (44.20) 0.299 241 (45.39) 241 (45.39) – Female 4052 (54.21) 2453 (52.57) 1328 (53.61) 404 (55.80) 290 (54.61) 290 (54.61) TC level (mmol/L) Mean (SD) 4.69 (0.88) 4.86 (0.91) < 0.001 4.68 (0.88) 4.91 (0.91) < 0.001 4.61 (0.85) 4.92 (0.93) < 0.001 TC categories Ideal 5545 (74.19) 3103 (66.50) < 0.001 1842 (73.64) 475 (65.61) < 0.001 410 (77.21) 347 (65.35) < 0.001 Borderline High 1533 (20.51) 1200 (25.72) 518 (20.91) 190 (26.24) 94 (17.70) 139 (26.18) High 396 (5.30) 363 (7.78) 135 (5.45) 59 (8.15) 27 (5.08) 45 (8.47) TG level (mmol/L) Mean (SD) 1.62 (1.50) 1.57 (1.16) 0.099 1.56 (1.20) 1.67 (1.42) 0.039 1.50 (0.85) 1.72 (1.56) 0.004 TG categories Ideal 5171 (69.16) 3218 (68.97) 0.309 1718 (69.36) 481 (66.44) 0.307 365 (68.74) 346 (65.16) 0.017 Borderline High 1168 (15.63) 771 (16.52) 410 (16.55) 128 (17.68) 106 (19.96) 93 (17.51) High 1135 (15.19) 677 (14.51) 349 (14.09) 115 (15.88) 60 (11.30) 92 (17.33) LDL-C level (mmol/L) Mean (SD) 2.45 (0.62) 2.54 (0.66) < 0.001 2.48 (0.59) 2.53 (0.71) 0.107 2.49 (0.60) 2.52 (0.74) 0.402 LDL-C categories Ideal 6943 (92.90) 4215 (90.33) < 0.001 2298 (92.77) 646 (89.23) 0.001 492 (92.66) 469 (88.32) 0.032 Borderline High 444 (5.94) 362 (7.76) 153 (6.18) 59 (8.15) 32 (6.03) 45 (8.47) High 87 (1.16) 89 (1.91) 26 (1.05) 19 (2.62) 7 (1.32) 17 (3.20) HDL-C level (mmol/L) Mean (SD) 1.33 (0.36) 1.38 (0.38) < 0.001 1.25 (0.32) 1.50 (0.35) < 0.001 1.24 (0.26) 1.52 (0.35) < 0.001 HDL-C categories Normal 6694 (89.56) 4266 (91.43) 0.001 2149 (86.76) 697 (96.27) < 0.001 465 (87.57) 511 (96.23) < 0.001 Low 780 (10.44) 400 (8.57) 328 (13.24) 27 (3.73) 66 (12.43) 20 (3.77) Table 1 Distribution of age, gender and four lipid indicators in three analytic approaches individuals (who have been exposed to famine) were assigned into the control group and led to potentially biased estimation. Discussion In this study, the baseline investiga- tion was of 5 years duration for the ESECC trial, creating a unique situation where the age range was lengthened (50–60 years in the exposed cohort and 43–53 years in the non-exposed cohort) and overlapped at 50–53 years in the two birth cohorts. This age overlap helps strengthen the power of age adjustment in multivariable multivariable model. However, adjustment was usually not been strong enough, given the fact that there was an “age gap” in the conventional birth cohorts under a cross- sectional design. As such, residual confounding still had the potential to bias the magnitude or even the direc- tion of the observed association. The second method employed an age-appropriate control by combining post- and pre-famine cohorts together. This method may result in exposure contamination that pre-famine cohort Wang et al. BMC Public Health (2021) 21:2129 Page 7 of 9 Fig. 3 Association of undernutrition and risk of borderline high and above (BHA) dyslipidemia status stratified by gender. Interaction between early-life undernutrition and gender was tested by adding the interaction term of the undernutrition variable and gender into the model. ORs were adjusted for age, occupation, BMI, blood pressure, cigarette smoking, alcohol drinking, fried food intake, salty food intake, spicy food intake, heartburn and regurgitation, and self-reported history of diabetes Fig. 3 Association of undernutrition and risk of borderline high and above (BHA) dyslipidemia status stratified by gender. Interaction between early-life undernutrition and gender was tested by adding the interaction term of the undernutrition variable and gender into the model. ORs were adjusted for age, occupation, BMI, blood pressure, cigarette smoking, alcohol drinking, fried food intake, salty food intake, spicy food intake, heartburn and regurgitation, and self-reported history of diabetes survival (e.g. catch-up growth) but may increase the risk of metabolic disorders especially during a period of ade- quate or plentiful nutrition in growth. 2) The epigenetic dysregulation theory proposes that epigenetic change is one of the molecular mechanisms behind these epidemi- ological associations of early-life undernutrition and the risk of metabolic disorders. Exposure to undernutrition in sensitive stages of development may lead to epigenetic changes, which in turn may generate the unique birth phenotype and result in dysregulation in physiological functions. models under the Adjustment Approach. Discussion Moreover, we made use of two other analytical approaches (Restriction and Matching) to reduce the age difference and to make the exposed and non-exposed cohorts more compara- ble based on this unique “overlapped age range” in the ESECC cohort members. Due to these measurements, for the first time we can efficiently reduce the potential con- founding effects brought by age. f In this study, we found that early-life undernutri- tion increased the risk of having BHA and high levels of TC, TG, LDL-C, and dyslipidemia during adulthood, and this confirmed results in several previous studies in China [10, 16, 17, 20]. Hypotheses have been proposed to explain this phenomenon including the thrifty phenotype hypothesis [21–23] and epigenetic dysregulation theory [24, 25]: 1) The “thrifty phenotype” hypothesis proposes that fetal and infant undernutrition would set in train mechanisms of nutritional thrift, which has a differential impact on the growth of different organs, with selective protection of brain growth. The adaptive responses to the nutritional thrift permanently change the structure and function of the body which could be beneficial for early When stratified by gender, female participants with severe undernutrition experience had an increased risk of BHA TG and LDL-C, but this association was not identi- fied in males. Potential explanations for this gender spe- cific association pattern in undernutrition and BHA TG and LDL-C included: 1) males usually have higher mor- tality rates under the influence of nutritional deficiency, and as such male survivors tend to be stronger and healthier and less vulnerable to health problems during adulthood [26]. 2) Chinese traditional culture has shown that male offspring may get preferential attention and Wang et al. BMC Public Health (2021) 21:2129 Page 8 of 9 LDL-C, and dyslipidemia, and decreased the risk of hav- ing reduced HDL-C in adulthood. The effect of under- nutrition on TG and LDL-C abnormalities was evident among females but not observed in males. These results emphasize the importance of nutrition conditionals in early-stage of life to the long-term health consequences. suffer less severe food shortages as compared with female offspring [27]. For TC, although the P value for interac- tion was < 0.05 in the Adjustment Approach, the absolute difference in estimated ORs between females and males was small (1.58 vs. 1.60) with little biological meaning; and neither of the P values of interaction was significant in Restriction and Matching Approaches. Abbreviations BHA B d l h Abbreviations BHA: Borderline high and above; BMI: Body mass index; CVD: Cardiovascular disease; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; TC: Total cholesterol; TG: Triglycerides. Heterogeneity of the association pattern was detected in HDL-C and the other three lipid indicators. That is, undernutrition exposure in early life decreased the risk of having reduced HDL-C but increased the risk of high TC, TG, and LDL-C during adulthood. This association of undernutrition and HDL-C has rarely been reported in previous studies. To date, only one study suggested that women with famine experience in adolescence had a significantly decreased prevalence of low HDL-C as compared with non-exposed controls [28]. Previously, we reported prevalence and trends with age were notably different between HDL-C and three other lipid indica- tors in the ESECC population [4]. This revealed HDL-C may have a unique biologic role in the process of lipid metabolism. Conclusions The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. In summary, we carefully evaluated the association of undernutrition in early life and the risk of dyslipidemia, based on the Great Chinese Famine of 1959–61. Rigor- ous study design and analytic approaches were applied to control the confounding effect of age. Results from this study demonstrated that undernutrition exposure in early life increased the risk of having high levels of TC, TG, Acknowledgements We would like to thank the Government of Hua County, Health and Family Planning Commission of Hua County, Henan Province, and all the participants of ESECC trial. We also would like to thank Dr. Michael A. McNutt for editing and correction of this manuscript. Funding This work was supported by Charity Project of National Ministry of Health [grant number 201202014], Natural Science Foundation of China [grant number 82073626, 81773501], the national key R&D program of China [grant number 2016YFC0901404], the National Science & Technology Fundamental Resources Investigation Program of China [grant number 2019FY101102], the Beijing-Tianjin-Hebei basic research cooperation project [grant number J200016], the Digestive Medical Coordinated Development Center of Beijing Hospitals Authority (XXZ0204), Beijing Natural Science Foundation [grant number 7182033], Science Foundation of Peking University Cancer Hospital [grant number 2020–7]. Authors’ contributions Yang Ke and Zhonghu He designed research; Zhonghu He, Mengfei Liu, Chuanhai Guo, Fenglei Li, Zhen Liu, Yaqi Pan, Fangfang Liu, Ying Liu, Huanyu Bao, Zhe Hu, Hong Cai conducted research; Minmin Wang, Mengfei Liu, Zhonghu He analyzed data or performed statistical analysis; Minmin Wang, Mengfei Liu, Zhonghu He and Yang Ke wrote paper; Yang Ke and Zhonghu He had primary responsibility for final content. All authors read and approved the final manuscript. The study also has limitations. First, this is a single- center study in Henan Province. Although this is an agri- cultural-dominant area that suffered severe food shortage in 1959–61, results of the current study still require fur- ther verification in other populations. Second, the non- exposed cohort defined in this study may not be ideally clean even with a one-year washout period, as recovery from famine is usually slow-paced. Third, unmeasured confounding might still exist although several lifestyle factors were collected and accounted for in this study. The online version contains supplementary material available at https://doi. org/10.1186/s12889-021-12211-8. Additional file 1: Supplement Table 1. Definitions and coding forms of risk factors of dyslipidemia investigated in ESECC trial from rural Hua County, China. Supplement Table 2. Selected demographic and behavioral characteristics in three analytical approaches. Supplementary Figure 1. Estimates of association of undernutrition and risk of low HDL-C in pooled analysis and subgroup analysis. Supplementary Figure 2. Association of undernutrition and dyslipidemia stratified by BMI groups in three analytic approaches. Supplementary Figure 3. Association of undernutrition and high status of dyslipidemia stratified by gender in three analytic approaches*. Findings from this study have practical implications. 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Author details 1 1 Key Laboratory of Carcinogenesis and Translational Research (Ministry of Edu- cation/Beijing), Laboratory of Genetics, Peking University Cancer Hospital & Institute, #52 Fucheng Rd, Haidian District, 100142 Beijing, China. 2 Hua County People’s Hospital, Anyang, Henan Province, China. 17. Wang Z, Li C, Yang Z, Ma J, Zou Z. Fetal and infant exposure to severe Chinese famine increases the risk of adult dyslipidemia: results from the China health and retirement longitudinal study. BMC Public Health. 2017;17(1):488. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 11. Song S, Wang W, Hu P. Famine, death, and madness: schizophrenia in early adulthood after prenatal exposure to the Chinese great leap for- ward famine. Soc Sci Med. 2009;68(7):1315–21. 11. Song S, Wang W, Hu P. Famine, death, and madness: schizophrenia in early adulthood after prenatal exposure to the Chinese great leap for- ward famine. Soc Sci Med. 2009;68(7):1315–21. 12. Li C, Lumey LH. Exposure to the Chinese famine of 1959-61 in early life and long-term health conditions: a systematic review and meta-analysis. Int J Epidemiol. 2017;46(4):1157–70. 12. Li C, Lumey LH. Exposure to the Chinese famine of 1959-61 in early life and long-term health conditions: a systematic review and meta-analysis. Int J Epidemiol. 2017;46(4):1157–70. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. 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https://openalex.org/W3134567456	https://digital.csic.es/bitstream/10261/239058/1/vaccines-09-00243.pdf	English	null	Emerging SARS-CoV-2 Variants and Impact in Global Vaccination Programs against SARS-CoV-2/COVID-19	Vaccines	2,021	cc-by	9,376	Keywords: SARS-CoV-2; COVID-19; variant; spike protein; mutation; lineage; vaccine efﬁcacy; neutralizing antibodies Citation: Gómez, C.E.; Perdiguero, B.; Esteban, M. Emerging SARS-CoV-2 Variants and Impact in Global Vaccination Programs against SARS-CoV-2/COVID-19. Vaccines 2021, 9, 243. https://doi.org/ 10.3390/vaccines9030243 Carmen Elena Gómez * , Beatriz Perdiguero and Mariano Esteban * Carmen Elena Gómez * Centro Nacional de Biotecnología, Department of Molecular and Cellular Biology, Consejo Superior de Investigaciones Cientíﬁcas (CNB-CSIC), Campus de Cantoblanco, 28049 Madrid, Spain; perdigue@cnb.csic.es * Correspondence: cegomez@cnb.csic.es (C.E.G.); mesteban@cnb.csic.es (M.E.); Tel.: +34-915854560 (C.E.G.); +34-915854553 (M.E.) Abstract: The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants in different continents is causing a major concern in human global health. These variants have in common a higher transmissibility, becoming dominant within populations in a short time, and an accumulation of a high number of mutations in the spike (S) protein, especially within the amino terminal domain (NTD) and the receptor binding domain (RBD). These mutations have direct implications on virus infection rates through higher afﬁnity of S RBD for the cellular angiotensin- converting enzyme-2 (ACE-2) receptor. There are also signs of enhanced virulence, re-infection frequency, and increased resistance to the action of monoclonal and polyclonal antibodies from convalescence sera and in vaccinated individuals in regions where the variants spread dominantly. In this review, we describe the different SARS-CoV-2 variants that have thus far been identiﬁed in various parts of the world with mutational changes and biological properties as well as their impact in medical countermeasures and human health. Review Emerging SARS-CoV-2 Variants and Impact in Global Vaccination Programs against SARS-CoV-2/COVID-19 * , Beatriz Perdiguero and Mariano Esteban * Citation: Gómez, C.E.; Perdiguero, B.; Esteban, M. Emerging SARS-CoV-2 Variants and Impact in Global Vaccination Programs against SARS-CoV-2/COVID-19. Vaccines 2021, 9, 243. https://doi.org/ 10.3390/vaccines9030243 1. Introduction The four SARS-CoV-2 vaccines licensed at present by the regulatory agencies are based on nucleic acid or non-replicating viral vectored platforms. The two vaccines based on messenger ribonucleic acid (mRNA) have been developed by Moderna (mRNA-1273) and Pﬁzer/BioNTech (BNT162b2) pharmaceutical companies and contain the genetic information for the synthesis of the stabilized pre-fusion form of the SARS-CoV-2 spike (S) protein encapsulated in a lipid nanoparticle (LNP) vector that enhances uptake by host immune cells. These vaccines used host cell transcription and translation machinery to produce the viral S protein, that is afterwards processed and recognized by speciﬁc B and T cells eliciting both humoral and cellular adaptive immune responses able to confer protection against COVID-19 illness, including severe disease. The reported efﬁcacy of a two-dose regimen of the mRNA-1273 or the BNT162b2 vaccines is 94.1% [4] or 95% [5], respectively. The two other licensed vaccines have been developed by Oxford University/AstraZeneca (AZD1222) and Janssen (Ad26.COV2.S) pharmaceutical companies and are based on two different modiﬁed non-replicating adenoviruses. The AstraZeneca candidate is a mono- valent vaccine composed of a single recombinant, replication-deﬁcient chimpanzee ade- novirus (ChAdOx1) vector encoding the S glycoprotein of SARS-CoV-2. The S protein is expressed in the trimeric pre-fusion conformation. Following administration, the S glycoprotein of SARS-CoV-2 is expressed locally, stimulating neutralizing antibody and cellular immune responses, which may contribute to protection against COVID-19. The AZD1222 vaccine has an efﬁcacy of 63.09% against symptomatic SARS-CoV-2 infection. Vaccine efﬁcacy was 62.6% in participants receiving two recommended doses with any dose interval (ranging from 3 to 23 weeks) [6]. The Janssen vaccine is based on the adenovirus serotype 26 (Ad26) which expresses the stabilized pre-fusion SARS-CoV-2 S protein. As opposed to the ubiquitous Ad5 serotype, very few people have been exposed to the rare Ad26 serotype; therefore, pre-existing immunity against the vector reducing this candi- date’s immunogenicity is not expected to be a major concern. A phase 3 randomized and placebo-controlled trial of the single-dose Ad26.COV2.S in approximately 40,000 partici- pants is currently ongoing. The primary analysis of 39,321 participants using a data cut-off date of 22 January 2021 demonstrated a vaccine efﬁcacy of 66.9%. The ﬁfth vaccine waiting for approval has been developed by Novavax Company (NVX-CoV2373). It is a protein subunit vaccine constructed from the full-length, stabi- lized, pre-fusion SARS-CoV-2 S glycoprotein, produced in the established Sf9 insect cell expression system and adjuvanted by saponin-based Matrix M1 [7]. 1. Introduction Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causal agent of the worldwide coronavirus disease 2019 (COVID-19) pandemic, which is causing major health as well as social and economic burden with unprecedented consequences. Records show 116 million infection cases worldwide with a global death of 2.6 million people early March 2021. At the beginning of the SARS-CoV-2 pandemic, there were only modest levels of genetic evolution mainly because of two factors: (i) the global absence of immunity against this new pathogen; and (ii) the low mutation rates of the coronaviruses which encode an enzyme with proofreading function that increases the ﬁdelity of the replication process [1]. In early March 2020, a new variant was detected with a single D614G mutation in the spike (S) glycoprotein of SARS-CoV-2 that spread to global dominance over the next month due to increased transmissibility and virus replication [2,3]. Since December 2020, novel SARS-CoV-2 variants that accumulate a high number of mutations, mainly in the S protein, have been detected in some geographical regions. These variants have been considered by the World Health Organization (WHO) as variants of concern (VOC) because of their potential risk to human health. The changes observed in the viral mutation rate during the course of the pandemic indicate a tendency towards a rapid antigenic variation and, hence, it is important to strengthen surveillance systems to control the emergence and the dissemination of new variants, looking over their impact on disease transmission and severity and on the efﬁcacy of vaccines and treatments used globally. Academic Editor: Steven B. Bradfute Received: 22 February 2021 Accepted: 9 March 2021 Published: 11 March 2021 Received: 22 February 2021 Accepted: 9 March 2021 Published: 11 March 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). The COVID-19 pandemic has required rapid action and development of vaccines in an unprecedented timeframe. According to WHO (https://www.who.int/publications/ m/item/draft-landscape-of-covid-19-candidate-vaccines (accessed on 9 March 2021)), https://www.mdpi.com/journal/vaccines Vaccines 2021, 9, 243. https://doi.org/10.3390/vaccines9030243 Vaccines 2021, 9, 243 2 of 13 76 vaccine candidates based on several different platforms are being currently evaluated in human clinical trials, while 182 candidates are under investigation in preclinical models. 1. Introduction In January, Novavax announced that, in the British trial, the vaccine had an efﬁcacy rate of 89%. Since all the vaccines that have been administered worldwide are focused on the spike protein, which accumulates high rate of mutations during viral evolution, as evidenced in the genome sequences from the new emerging SARS-CoV-2 variants, it is imperative to evaluate the impact of those mutations on the actual efﬁcacy of COVID-19 vaccines. 2.1. B.1.1.7 (VOC 202012/01 or 20B/501Y.V1) Variant 2.1. B.1.1.7 (VOC 202012/01 or 20B/501Y.V1) Variant B.1.1.7 variant, also known as VOC 202012/01 or 20B/501Y.V1, was unveiled on 14 December 2020 by the United Kingdom (UK) authorities who declared an increase in the incidence of SARS-CoV-2 infections in eastern and south-eastern England and the London metropolitan area associated with the emergence of a new SARS-CoV-2 variant. The ﬁrst clinical sample in which it could be identiﬁed dates back to September 2020. Since then, it has largely replaced the circulating viruses, becoming the predominant variant in the UK. This variant is characterized by greater transmission, which has contributed to an increase in incidence, hospitalizations, and pressure on the health system since the second half of December 2020. Epidemiological studies and mathematical modeling suggest that it spreads 56% faster than other lineages and results in higher nasopharyngeal viral loads than the wild-type strain [8]. By January 2021, the UK reported the highest daily mortality from COVID-19 since the onset of the pandemic [9]. Retrospective observational studies estimate a 35% (12–64%) increased risk of death associated with B.1.1.7 variant, indicating that it is not only more transmissible than pre-existing linages but can also cause a more serious disease [10]. However, there was no evidence of more severe disease in children and young people [11]. y g p p Compared to ancestral viruses containing the D614G mutation, the B.1.1.7 variant has accumulated 23 mutations, and it is not phylogenetically related to the viruses circulating in the UK when it was detected. Of these mutations, 14 are non-synonymous: [[T1001I, A1708D, and I2230T] in open reading frame (ORF)1ab; [N501Y, A570D, P681H, T716I, S982A, and D1118H] in the spike (S) protein; [Q27stop, R52I, and Y73C] in ORF8; and [D3L and S235F] in the nucleocapsid (N) protein]; 6 are synonymous: [[C913T, C5986T, C14676T, C15279T, and T16176C] in ORF1ab; and [T26801C] in M (membrane) gene]; and 3 are deletions: [[SGF 3675-3677del] in ORF1ab; and [H69-V70del and Y144del] in S protein]. It is unclear how this variant achieves prominence, although the unusual genetic divergence of the B.1.1.7 lineage may have resulted, at least in part, from the evolution of the virus in an individual with chronic infection [12]. Excluding synonymous mutations, 47% of reported changes in B.1.1.7 variant occur in the gene encoding for the spike protein that contains the receptor binding domain (RBD). 2. Emerging SARS-CoV-2 Variants That Raise Global Concern Natural selection usually determines the fate of a newly arising mutation. However, other potential mechanisms such as chance events, host shifts, persistent infection in immunocompromised host, or mutations affecting the proofreading function could also drive viral evolution. Those mutations that confer a competitive advantage with respect to viral replication, transmission, or escape from immunity will increase in frequency, becoming the dominant variant. Currently, an exhaustive surveillance monitoring has being performed on three SARS-CoV-2 variants: B.1.1.7 (VOC 202012/01 or 20B/501Y.V1), B.1.351 (20H/501Y.V2), and P.1 (B.1.1.28.1). Vaccines 2021, 9, 243 3 of 13 2.1. B.1.1.7 (VOC 202012/01 or 20B/501Y.V1) Variant SARS-CoV-2 S protein is the main target of neutralizing antibodies and, hence, it has been used as vaccine antigen in most of the SARS- CoV-2 candidates under development and in the licensed vaccines that are being globally administered. The high frequency of mutations in this protein has caused global concern because these mutations, either individually or as a whole, can induce structural changes that might: (i) alter the interaction with the human angiotensin-converting enzyme-2 (hACE2) receptor, modifying the infection rate or even the interaction of the virus with the ACE2 receptor from other species supporting host shifts; (ii) modify the efﬁcacy of both neutralizing antibodies and speciﬁc T cells elicited either during natural infection or through vaccination; or (iii) alter the sensitivity to neutralization by monoclonal antibodies or sera from convalescent patients, compromising the efﬁcacy of treatments. The three mutations of B.1.1.7 with the greatest potential to affect the biological behavior of the virus are: H69-V70del, N501Y, and P681H. H69/V70 deletion is one of the recurrent mutations observed in the amino terminal domain (NTD) of S protein and emerged independently in at least six lineages of the SARS-CoV-2 virus prevalent in Europe. It is present in over 6000 sequences worldwide and often co-occurs with the RBD amino acid replacements N501Y, N439K, and Y453F [13,14]. H69/V70 deletion itself favored a two-fold increase in S protein-mediated infectivity in vitro using pseudotyped lentivirus, indicating that this effect on virus ﬁtness seems to be independent of the RBD changes [13]. Protein structure modeling shows that H69/V70 deletion could be a “permissive” mutation that modiﬁes the immunodominant epitopes located at variable loops within NTD, conferring resistance to neutralization by sera from both convalescent patients and vaccinated individuals [14]. The N501Y mutation is of major concern because it involves one of the six key amino acid residues determining a tight interaction of the SARS-CoV-2 RBD with ACE2 receptor. Modeling analysis showed that the N501Y mutation would allow a potential aromatic ring— Vaccines 2021, 9, 243 4 of 13 ring interaction and an additional hydrogen bond between RBD and ACE2 [15] and, hence, an increase in the binding afﬁnity of SARS-CoV-2 S protein for hACE2 receptor [16]. A retrospective study has recently reported a three-fold higher inferred viral loads in a group of UK individuals infected with the viral variant carrying the N501Y mutation, evidencing the high efﬁciency of infection and transmission associated with B.1.1.7 variant [17]. 2.1. B.1.1.7 (VOC 202012/01 or 20B/501Y.V1) Variant The N501Y mutation has also been associated with increased infectivity and virulence in mouse and ferret models [18]. The function of the P681H mutation is not yet clear, but it is located immediately adjacent to the amino acids 682–685, the furin cleavage site (FCS) identiﬁed at the S1/S2 in the spike protein. SARS-CoV-2 FCS is not found in closely related coronavirus and has been shown to promote the entry of the virus into respiratory epithelial cells [19]. Similarly, it has been shown that the insertion generated by FCS into SARS-CoV-2 S protein enhances transmembrane serine protease (TMPRSS)-induced cleavage ability and viral infectivity [20]. Both N501Y and P681H mutations have been reported independently, but thus far they have not appeared in combination. The mutation Q27stop in ORF8 observed in the B.1.1.7 variant truncates the ORF8 protein or makes it inactive, allowing the accumulation of additional mutations in other regions. At the beginning of the pandemic, multiple viruses with deletions that led to the loss of ORF8 expression were isolated worldwide, including a large group in Singapore. These deletions were associated with a milder clinical infection and lower post-infection inﬂammation; however, this group disappeared at the end of March after Singapore successfully implemented control measures. In subsequent reports, it was found that the deletion of ORF8 has only a very modest effect on virus replication in human primary respiratory cells compared to viruses without the deletion [21]. The rapid transmission of B.1.1.7 variant in and out of the UK suggests that this variant could become the dominant lineage responsible for the upcoming infections in Europe. As of 16 February 2021, 71.413 sequences of B.1.1.7 lineage have been detected in 64 countries, while 82 have reported cases related with this variant [22]. This emphasizes the importance of a global approach to surveillance, tracking, and vaccine deployment. How does B.1.1.7 dissemination impact the efﬁcacy of vaccines and treatments that are being administered globally? To date, low or no signiﬁcant impact of the vaccines efﬁcacy against B.1.1.7 variant has been reported. Xiu et al. show small effects of some of the mutations present in the B.1.1.7 variant on neutralization by sera from vaccinated individuals elicited after two BNT162b2 doses (Pﬁzer). Nevertheless, the modiﬁed virus used in the study lacked the full set of spike mutations described in B.1.1.7 variant [23]. Similarly, Collier et al. 2.2. B.1.351 (20H/501Y.V2) Variant 2.2. B.1.351 (20H/501Y.V2) Variant The second variant that has raised global concern is the B.1.351 variant, also known as 20H/501Y.V2, identiﬁed on 18 December 2020 by the authorities from Republic of South Africa. The ﬁrst clinical sample in which this variant could be detected dates back to 8 October 2020, and one month later, it replaced the circulating viruses in the region, becoming the dominant variant. This behavior suggests higher transmission rates, although no evidence of greater virulence or disease severity has been reported to date. As of 16 February 2021, 1383 sequences of B.1.351 lineage have been detected in 35 countries, while 40 have reported cases related with this variant [22]. p Compared to the Wuhan reference strain, the B.1.351 variant has 12 non-synonymous mutations and one deletion. Approximately 77% of these mutations are located in the spike protein [L18F, D80A, D215G, LAL 242–244 del, R246I, K417N, E484K, N501Y, D614G, and A701V] while the remaining ones are located in ORF1a [K1655N], envelope (E) [P71L], and N [T205I] viral proteins. Similar to B.1.1.7 variant, it could have emerged through intra-host evolution in one or more individuals with prolonged viral replication. However, the high number of mutations accumulated within two of the most immunodominant regions of S protein, such as the NTD and the RBD domains, suggests that it could also be originated as an escape variant to neutralization [28]. Therefore, the concern about the effect that the B.1.351 mutations might have on vaccine efﬁcacy and treatments is even greater than with the B.1.1.7 variant. B.1.351 and B.1.1.7 variants share the N501Y mutation, located in the RBD domain of the spike protein. As described above, this mutation confers an increased binding afﬁnity of S RBD for the ACE2 receptor, raising the viral transmission rate. However, in addition to the N501Y mutation, this variant accumulates two additional mutations in the same RBD domain (K417N and E484K) that could play a pivotal role in both the interaction with the receptor and the immune evasion. The E484K mutation is present in <0.02% of sequences outside South Africa. There is evidence that this mutation can modestly improve the binding afﬁnity of the virus to the receptor. 2.1. B.1.1.7 (VOC 202012/01 or 20B/501Y.V1) Variant did not observe a signiﬁcant reduction in the ability of sera from vaccinated individuals (Pﬁzer) to inhibit parental or mutant pseudoviruses including only three (H69/V70del, N501Y, and A570D) S mutations; however, a reduction in the neutraliza- tion titers was evident using pseudoviruses with the complete set of mutations described for B.1.1.7 variant [24]. Slightly reduced but overall largely preserved neutralizing titers against the B.1.1.7 lineage pseudovirus were also detected in sera from people receiving the Pﬁzer vaccine [25]. Correspondingly, modest reductions in the neutralizing activity of both plasma from convalescent patients (2.7–3.8-fold) and sera from individuals that received Moderna or Pﬁzer vaccines (1.8–2-fold) were described by Wang et al. [26]. This group also evaluated a panel of 30 monoclonal antibodies (mAbs) directed against the NTD and the RBD domains of S protein and observed that the B.1.1.7 variant is refractory to neutralization by most of the mAbs that recognized NTD and is relatively resistant to several RBD-directed mAbs, including two that have been approved for emergency treatment [26]. Finally, Wu et al. did not detect a signiﬁcant impact in the neutralizing capacity of sera from human subjects or non-human primates (NHPs) who received the mRNA-1273 (Moderna) vaccine against the B.1.1.7 variant [27]. Overall, although these in vitro studies have their limitations either by methodology, by sample size, or by only considering the humoral arm of the immune response, what they indicate is that the efﬁcacy of the vaccines that have being administered is similar or moderately lower against the B.1.1.7 variant. Vaccines 2021, 9, 243 5 of 13 2.2. B.1.351 (20H/501Y.V2) Variant show that B.1.351 variant is notably more resistant to neutralization by polyclonal antibodies derived from individuals vaccinated with Pﬁzer (6.5-fold) or Moderna (8.6-fold) vaccines [26]. Furthermore, Wu et al. reported a signiﬁcant decrease in the neutralization of B.1.351 variant by sera from either humans or NHPs who were vaccinated with mRNA-1273 (Moderna); however, the authors highlight that, despite the observed decreases, the geometric mean of the neutralization titers in sera from human vaccinated individuals against B.1.351 variant remained at ~1/300, a value that they still consider acceptable for protection [27]. Finally, a new study assaying the antibody responses and memory B cells in volunteers who received Moderna (mRNA-1273) or Pﬁzer (BNT162b2) vaccines reported a reduced activity against the SARS-CoV-2 variants that contain the E484K or the N501Y mutations or the K417N:E484K:N501Y combination [31]. Overall, these data reﬂect a more pronounced decrease in the efﬁcacy of antibody-based vaccines and therapies against this variant. p g The most worrying statistics are derived from the interim efﬁcacy results from two randomized placebo-controlled clinical trials reported in press release by Novavax and Janssen companies in South Africa, the region where B.1.351 variant prevails. Biotechnology company Novavax reports that its COVID-19 vaccine NVX-CoV2373, which includes the S protein from the SARS-CoV-2 Wuhan reference strain, has shown 85.6% efﬁcacy against the B.1.1.7 variant (95.6% against the original strain) in the phase 3 clinical trial involving 15,000 participants between 18 and 84 years old in the UK. However, in the phase 2b study developed in South Africa, involving more than 4400 individuals, the efﬁcacy in the overall population was 49.4% against B.1.351 variant. This value increases up to 60% efﬁcacy in the prevention of mild, moderate, and severe COVID-19 disease if the human immunodeﬁciency virus (HIV) positive group is eliminated from the overall count [34]. These interim data evidence a signiﬁcant decrease in the efﬁcacy of a vaccine inﬂuenced by the dominance of a viral variant such as B.1.351. Additionally, Janssen’s coronavirus vaccine has shown 72% efﬁcacy in a single dose in the ENSEMBLE trial in the United States to prevent moderate to severe COVID-19 at 28 days after vaccination. However, these values were reduced to 66% in Latin America and 57% in South Africa. Despite the reduced efﬁcacy, the rAd26 vaccine was 85% effective overall in preventing severe COVID-19, and protection was similar in all regions [35]. 2.2. B.1.351 (20H/501Y.V2) Variant Similarly, although K417 residue is a unique SARS-CoV-2 S RBD residue that interacts with ACE2 contributing to an enhanced afﬁnity of the virus for the receptor, a mutational scanning study suggests that the amino acid change of K by N minimally affects this binding [16]. Moreover, a molecular dynamics simulation study reveals that both E484K and N501Y mutations increase afﬁnity of S RBD for hACE2 and E484K in particular switches the charge on the ﬂexible loop region of S RBD, which leads to the formation of novel favorable contacts. Moreover, the combination of E484K, K417N, and N501Y mutations results in the highest degree of conformational alterations of S RBD when bound to hACE2, compared to either E484K or N501Y alone, allowing the virus a more effective escape to neutralization [29]. It should be noted that the rest of the mutations that this variant holds in the spike protein are located in the NTD region (L18F, D80A, D215G, LAL 242-244 del, and R246I), especially within or near ﬂexible variable loops that accept sequence changes without modifying the structure of the functional domains of S protein. NTD is also a preferential target of antibodies isolated from convalescent patients or vaccinated individuals. Preliminary studies highlight that a combination of RBD and NTD mutations in the B.1.351 spike protein signiﬁcantly affects the neutralization of this variant by both mAbs targeting these regions and immune sera derived from convalescent or vaccinated patients [26,27,30–33]. Studies evaluating panels of potent mAbs that have been identiﬁed against the spike protein indicate that B.1.351 variant is refractory to be neutralized by antibodies that recognize S NTD while the S RBD mutations reduce or abolish the neutralization capacity of nearly 80% of the mAbs that recognize this region [26,30,31]. In line with these ﬁndings, a signiﬁcant reduction in the neutralization capacity of polyclonal antibodies derived from convalescent patients against B.1.351 variant has been reported, being the sera from hospitalized patients with a more severe disease those who displayed higher neutralization efﬁcacy [26,31–33]. These data point to a possible decrease in the efﬁcacy of the treatments based on monoclonal or Vaccines 2021, 9, 243 6 of 13 polyclonal antibodies as well as an increase in the rate of reinfection in regions where this variant spreads dominantly. p y In terms of vaccine efﬁcacy against B.1.351 variant, the results are more worrying than those shown for B.1.1.7. Wang et al. 2.2. B.1.351 (20H/501Y.V2) Variant Finally, a clinical trial evaluating two-dose regimen of AZD1222 (AstraZeneca/Oxford vaccine) in South Africa did not show protection against mild to moderate COVID-19 due to B.1.351 variant [36]. Altogether, these results conﬁrm that it is imperative to minimize the circulation of the virus, prevent infections, and reduce the opportunities for the SARS-CoV-2 to evolve, resulting in mutations that could reduce the efﬁcacy of existing vaccines. 2.3. P.1 (B.1.1.28.1) Variant The third variant of SARS-CoV-2 that raises concerns is P.1 variant, also known as B.1.1.28.1. It was detected by Japan’s National Institute of Infectious Diseases on 6 January 2021 and was isolated from four travelers who arrived in Tokyo from Amazonas, Brazil, on 2 January 2021 at airport control. P.1 variant was later identiﬁed in Brazil, where it has become the dominant circulating virus [37]. The rapid increase in the number of hospital admissions by COVID-19 in January 2021 (six-fold higher than the number reported in December) [38] is unexpected and worrying considering that this city reached 76% seroprevalence during the summer wave [39]. P.1 variant belongs to the B.1.1.28 lineage and contains 17 non-synonymous muta- tions: [L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and V1176F] in S protein, [S1188L, K1795Q, and E5665D] in ORF1ab, [E92K] in ORF8, and [P80K] in N protein; 1 deletion: [SGF 3675-3677del] in ORF1ab; and 4 synonymous mutations. P.1 is the SARS-CoV-2 variant that accumulates the highest number of mutations Vaccines 2021, 9, 243 7 of 13 in the spike protein (12 mutations). The mutation N501Y is present in the three variants, while L18F, K417T, E484K, and D614G mutations are shared with the B1.351 variant. As described above, this set of spike mutations has important implications for transmission, reinfection rates, and evasion of antibody-mediated immunity. In fact, one clinical case of reinfection has been reported in this region [40]. As of 16 February 2021, 150 sequences of P.1 lineage have been detected in 14 countries, while 18 have reported cases related with this variant [22]. [ ] One of the most worrying mutations in terms of immune evasion is the E484K, which is shared by the P.1 and the B.1.351 variants. Recently, the effect of this mutation has been evaluated in the neutralization ability of sera from convalescent or vaccinated patients considering their SARS-CoV-2 spike immunoglobulin G (IgG) antibody titer. The efﬁcacy of serum neutralization against the virus carrying the E484K mutation was reduced in both vaccination samples and convalescent sera. However, sera with high anti-S IgG titers were still able to neutralize the virus with the mutation, indicating that it is important to induce the highest possible levels of speciﬁc antibodies through vaccination to improve protection against emerging variants of SARS-CoV-2 [41]. 2.3. P.1 (B.1.1.28.1) Variant Although this study does not use a virus containing the entire set of mutations that are present in combination with E484K in the spike protein of the different variants in order to provide a more realistic estimate of this effect, it makes clear that it is necessary to optimize the vaccination schedules to increase the possibility of counteracting the expansion of the new variants. Findings by Planas et al. using authentic clinical viral isolates to assess inherent viral ﬁtness and potential impact of additional mutations outside of the spike on sensitivity to neutralizing antibod- ies also demonstrated that low global antibody levels or declining antibody responses are associated with a loss of cross-reactivity against novel emerging variants [42]. These ﬁndings indicate that it is necessary to follow rigorously the vaccination regimen estab- lished and approved by the regulatory authorities for the different licensed vaccines. In addition, it is important to study in the clinical settings how the introduction of combined or “prime/boost” heterologous vaccination protocols could optimize the strength of both humoral and cellular immune responses [43]. There is still a long way to go into the study of this and other variants circulating in Brazil; however, considering the high number of mutations that P.1 accumulates in the spike protein, it is probable that it behaves as resistant or even more resistant than B.1.351 variant to neutralization by monoclonal antibodies and vaccinee sera. Scientists in Brazil reported on 14 January 2021 that the coronavirus vaccine of the Chinese pharmaceutical company Sinovac (CoronaVac) based on inactivated virus was 50.38% effective when tested in 12,508 volunteers, all of them health professionals in direct contact with the coronavirus [44]. It remains to be determined whether the efﬁcacy of this vaccine (which is close to the approval threshold for emergence use) is maintained against the new P.1 variant that is expanding dramatically in the country. A summary of the key features of the emergent SARS-CoV-2 variants is depicted in Table 1. aracteristics of the emergent severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants. Data uary 16 2021 (https://outbreak.info (accessed on 9 March 2021)). Table 1. Main characteristics of the emergent severe acute respiratory syndrome coronavirus-2 (SARS-CoV- updated on February 16 2021 (https://outbreak.info (accessed on 9 March 2021)). Table 1. Main characteristics of the emergent severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants. Data updated on February 16 2021 (https://outbreak.info (accessed on 9 March 2021)). 3. Other Variants of Interest The United States is the country with the highest incidence rates of COVID-19, and different states have reported the prevalence of all the emergent variants of concern. How- ever, the expansion of a novel variant descended from cluster 20C and designated CAL.20C (20C/S:452R or B.1.429) has been reported in Southern California [45]. CAL.20C variant was ﬁrst observed in July 2020 in one of 1247 samples from Los Angeles County and was not detected in Southern California again until October. Since then, the prevalence of this variant has increased, and, in January 2021, it accounted for 35% and 44% (37 of 85) of all samples collected in California state and Southern California, respectively. However, relatively few samples have been sequenced, and sequencing is not performed uniformly throughout the state, making it difﬁcult to establish a more accurate estimate of the expan- sion of this variant [46]. CAL.20C variant is deﬁned by ﬁve mutations (ORF1a: I4205V; ORF1b: D1183Y; S: S13I, W152C, and L452R). In particular, the L452R mutation in the spike protein has been found to be resistant to certain therapeutic monoclonal antibodies [47]. As clinical outcomes have yet to be established, the functional effect of CAL.20C variant regarding infectivity and disease severity remains uncertain. Another new coronavirus variant, named A.23.1, has been detected in Uganda and has quickly become the most common coronavirus in Uganda’s capital city, Kampala. The set of the spike mutations in A.23.1 includes R102I, F157L, V367F, Q613H, and P681R. Additional substitutions in non-spike regions include non-structural protein (nsp) 3: E95K; nsp6: M86I and L98F; ORF8: L84S and E92K, and N: S202N and Q418H. As of 16 February 2021, 274 sequences of A.23.1 lineage have been detected in 17 countries [48]. In addition, an emerging lineage (now designated as B.1.526) of viral isolates in the New York region that shares mutations with previously reported variants has been recently detected by West et al. using a tool to query the spike mutational landscape. The most common sets of spike mutations in B.1.526 are L5F, T95I, D253G, and E484K or S477N, D614G, and A701V. This lineage appeared in late November 2020, and it accounts for ~5% of coronavirus genomes sequenced and was deposited in Global Initiative on Sharing Avian Inﬂuenza Data (GISAID) during late January 2021 [49]. 2.3. P.1 (B.1.1.28.1) Variant Variants B.1.1.7 B.1.351 P.1 1st detection September 2020 8 October 2020 2 January 2021 Detection site United Kingdom South Africa Japan/Brazil Mutations in S protein 7 mutations: N501Y, A570D, D614G, P681H, T716I, S982A, D1118H 2 deletions: H69-V70del, Y144del 9 mutations: L18F, D80A, D215G, R246I, K417N, E484K, N501Y, D614G, A701V 1 deletion: LAL 242-244 del 12 mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F Countries reported cases 82 40 19 Countries with sequences 64 35 14 Vaccines 2021, 9, 243 8 of 13 Variants B.1.1.7 B.1.351 P.1 Potential risk - Higher transmission - Higher disease severity - Modest reduction in the neutralization efﬁcacy of sera from convalescent patients or vaccinees - Higher transmission - Higher reinfection rates - Signiﬁcant reduction in the neutralization efﬁcacy of sera from convalescent patients or vaccinees - Higher transmission - Higher reinfection rates - Signiﬁcant reduction in the neutralization efﬁcacy of sera from convalescent patients or vaccinees 3. Other Variants of Interest Although the clinical impact of the A.23.1 and the B.1.526 variants is not yet clear, it is essential to perform a careful monitoring of these variants as well as a rapid assessment of the consequences of the spike protein changes for vaccine efﬁcacy. The UK has strengthened genomic surveillance to evaluate the molecular evolution of the prevalent B.1.1.7 variant. New variants with different substitutions have emerged as a consequence of both the high replication rates of the virus and the increasing selection pressure resulting from the growth of the seroprevalent fraction of the population of England. The ones that worry the most are L18F and E484K. The introduction of the L18F mutation confers a replicative advantage to the virus [50], whereas E484K mutation could confer resistance to immunity. Moreover, other non-B.1.1.7 lineages with the E484K mutation have been identiﬁed in some UK regions such as the VUI 202102/01 (A.23.1 with E484K) or the B.1.525 (VUI 2021 02/03) with 4 mutations within the spike protein (Q52R, E484K, Q677H, and F888L). Further work is needed to establish the impact of these mutations on protective vaccines efﬁcacy in the context of the evolving variants that have acquired E484K mutation [51]. Vaccines 2021, 9, 243 9 of 13 9 of 13 There is substantial variability in the course of COVID-19, ranging from asymptomatic infection to death. One of the main topics of ongoing research is how the emergence of the new SARS-CoV-2 variants impacts patient’s outcome. However, there are no consistent data published yet, in part due to the fact that most of the genome sequences shared are not linked to clinical outcomes. One study performed by researchers from the University of Washington comparing two dominant clades of virus in circulation showed no signiﬁcant difference in outcomes of hospitalization or death between clades [52]. Similarly, clinicians and scientists working in the frontline in South Africa have not observed any differences in symptoms in people infected with the new variant P.1.351, compared with people infected with other variants [53]. Nevertheless, further analysis is necessary to screen differences in COVID-19 symptom type, severity, or duration of the disease caused by the new VOC. 4. Population Monitoring of Variant by Genomic Sequencing Worldwide expansion of genomic sequencing and data exchange is essential to detect the emergence of new variants or their introduction in a given country or region. To date, more than 528,000 sequences have been submitted to the GISAID that promotes the rapid sharing of data from the coronavirus causing COVID-19; however, most of them come from only a few countries. It is necessary that all the countries share sequence information to understand the spread of SARS-CoV-2. Improving the geographical coverage of sequencing is essential for the world to adequately capture the viral changes and establish alternative measures, as previously reported in Netherlands [54]. Increased sequencing capacity is a priority research area for the WHO. In order to achieve this objective, a number of recommendations have been established highlighting the need to have or implement a network of laboratories with sequencing experience integrated within the epidemiological surveillance system to generate useful information for the decision-making of public health measures and to ensure that resources are available to manage increasing numbers of COVID-19 detection and characterization of sample requests. 5. Deﬁning SARS-CoV-2 Variants More Resistant to Vaccine Action There are several lines of action to establish the role played by the mutations intro- duced into the SARS-CoV-2 genome on resistance to the action of the immune responses induced by current vaccines mostly targeting the spike protein. To this end, the scientiﬁc community, WHO, and companies have warned of the need to experimentally establish assays that determine to what extent the various variants already identiﬁed and those upcoming, that are spreading in the population, are due to mutational changes that confer an enhanced degree of resistance to the antibodies generated in infected or vaccinated people. Experimental approaches to be followed are: (1) demonstration in experimental animal models (humanized mice susceptible to virus, hamsters, ferrets, and macaques) that emerging variants are more virulent (higher transmission, replication, organ damage, mor- bidity, and mortality) than Wuhan’s reference strain; (2) demonstration that these variants withstand the neutralizing action of immune sera induced in current vaccination cam- paigns, such as mRNA-based vaccines (Pﬁzer, Moderna), non-replicating adenovirus-based vaccines (AstraZeneca, Janssen, Sputnik), protein subunit (Novavax), inactivated virus (Sinovac), and others; and (3) demonstration of the degree of variant control in vaccinated personnel in comparison with infection by the parental Wuhan strain In those cases in which increased resistance to antibodies was demonstrated, as indi- cated by the above experimental data, it should also be conﬁrmed that T cell responses are also affected, or if, on the contrary, the controlling effect of the infection by T lymphocytes is maintained. These experimental data in animals and humans are necessary to establish the highest sensitivity and resistance of the different variants to the immune action. g y The reduction in the efﬁcacy of different vaccine candidates in regions where the variants of concern have become prevalent has accelerated the decision of many of the companies that produce current COVID-19 vaccines to consider modifying their designs to cover circulating viral variants. The immediate consequence is that there should be on the Vaccines 2021, 9, 243 10 of 13 10 of 13 market new vaccines from the same production companies, which could be given either to those people already vaccinated as a recall dose or to those who have not yet received the vaccine dose. These new vaccines modiﬁed in their speciﬁc platforms would entail additional costs and could in turn lead to more resistant variants with additional mutations due to selective pressure from the immune system. 5. Deﬁning SARS-CoV-2 Variants More Resistant to Vaccine Action We hope this does not occur, but we must remain vigilant about the evolution. g Anything that can be done to suppress spread of SARS-CoV-2 will help to limit the emergence of new variants. However, other strategies that include multivalent designs focused on conserved regions of different viral proteins could be of great prophylactic relevance to counteract escape variants that are emerging. In this regard, a sustained effort to develop a pan-SARS-CoV-2 vaccine is warranted. Similarly, to improve the efﬁcacy of the current treatments, it will be of relevance to use a combination of antibodies directed against other viral regions in addition to the spike protein or to implement the “lethal mutagenesis” strategy as alternatives that slow the viral diversiﬁcation [55]. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Denison, M.R.; Graham, R.L.; Donaldson, E.F.; Eckerle, L.D.; Baric, R.S. 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Vaccines 2019, 7, 37. [CrossRef] [PubMed] 15. Liu, M.A. A Comparison of Plasmid DNA and MRNA as Vaccine Technologies. Vaccines 2019, 7, 37. [CrossRef] [PubMed] 16. Starr, T.N.; Greaney, A.J.; Hilton, S.K.; Ellis, D.; Crawford, K.H.D.; Dingens, A.S.; Navarro, M.J.; Bowen, J.E.; Tortorici, M.A.; 15. Liu, M.A. A Comparison of Plasmid DNA and M 16. Starr, T.N.; Greaney, A.J.; Hilton, S.K.; Ellis, D.; Crawford, K.H.D.; Dingens, A.S.; Navarro, M.J.; Bowen, J.E.; Tortorici, M.A.; Walls, A.C.; et al. Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding. Cell 2020, 182, 1295–1310.e20. [CrossRef] [PubMed] 17. 6. Concluding Remarks The appearance of variants of SARS-CoV-2 is not an unexpected virological ﬁnding but rather a result of natural selection, giving rise to mutations within the long-RNA (30,000 nucleotides) sequence of coronaviruses. In spite of a viral proof-reading exonuclease, multiple variants emerge in the population, but only those with an advantage on virus replication and dissemination prevail. This is further enhanced by the large incidence of infection rates within humans, accentuated by the presence within the viral genome of genes encoding antagonists of host defense mechanisms, such as blocking interferon action and other immune stimulatory molecules. The virus tries to counteract the host response, giving rise to mutations. Within a year since the virus ﬁrst appeared in China and its rapid spread, we are confronted with the emergence of variants of concern in different parts of the world. The main characteristics are higher binding afﬁnity for the cellular ACE-2 receptor than the parental Wuhan virus, the enhanced resistance to neutralizing antibodies, and increased virulence. To promote actions for the control of the emerging variants, a major effort is being put forward by different nations and institutions, such as WHO, CEPI (Coalition for Epidemic Preparedness Innovations), Gates Foundation, GAVI (Global Alliance for Vaccines and Immunizations), and others, with the purpose to make universal access to vaccines and to assure control of virus infection. Indeed, we have proven that current and incoming vaccines will cope with the control of variants and the potential eradication of the virus. In this regard, the results coming out from Israel on the high efﬁcacy of the Pﬁzer vaccine against SARS-CoV-2 infections are encouraging, in a country where the UK variant is prevalent. It will be only through the detailed understanding of the virus structure, biology, and vaccine developments that we can ﬁnally achieve the control of SARS-CoV-2 infections. Author Contributions: C.E.G. conceptualized and wrote the manuscript. B.P. helped in the draft writing and editing. M.E. supervised the overall conceptualization and draft writing. All authors have read and agreed to the published version of the manuscript. Funding: This investigation was supported by grants from Spanish CSIC and Obra Social La Caixa (CaixaImpulse CF01-0008). Institutional Review Board Statement: Not applicable. nstitutional Review Board Statement: Not applicable Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. 11 of 13 Vaccines 2021, 9, 243 References Impact of SARS-CoV-2 B.1.1.7 Spike Variant on Neutralisation Potency of Sera from Individuals Vaccinated with Pﬁzer Vaccine BNT162b2. medRxiv 2021. [CrossRef] 25. 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Oude Munnink, B.B.; Nieuwenhuijse, D.F.; Stein, M.; O’Toole, Á.; Haverkate, M.; Mollers, M.; Kamga, S.K.; Schapendonk, C.; Pronk, M.; Lexmond, P.; et al. Rapid SARS-CoV-2 Whole-Genome Sequencing and Analysis for Informed Public Health Decision-Making in the Netherlands. Nat. Med. 2020, 26, 1405–1410. [CrossRef] g [ ] 55. Shannon, A.; Selisko, B.; Le, N.-T.-T.; Huchting, J.; Touret, F.; Piorkowski, G.; Fattorini, V.; Ferron, F.; Decroly, E.; Meier, C.; et al. Rapid Incorporation of Favipiravir by the Fast and Permissive Viral RNA Polymerase Complex Results in SARS-CoV-2 Lethal Mutagenesis. Nat. Commun. 2020, 11, 4682. [CrossRef]
https://openalex.org/W1966219511	https://iris.unimore.it/bitstream/11380/608282/1/Clinic0l%20Cardiology%20-%202000-%20Mattioli%20-%20Relationship%20between%20mean%20right%20atrial%20pressure%20and%20doppler%20parameters%20in%20patients.pdf	English	null	Relationship between mean right atrial pressure and doppler parameters in patients with right ventricular infarction	Clinical cardiology	2,000	cc-by	4,572	Summary Background: The incidence of an inferior left ventricular in- farction involving the right ventricle is very high, ranging from 14 to 84%. Isolated right ventricular infarction accounts for < 3% of all cases of infarction. Introduction The incidence of an inferior left ventricular infarction in- volving the right ventricle is very high, ranging from 14 to 84%.'3 * Isolated right ventricular infarction accounts for < 3% of all cases of infarction3 In the presence of a right ventricular infarction, it is necessary to increase atrial contractility to over- come the increased myocardial stiffness3 Any reduction in atrial function is likely to have significant adverse effects on hemodynamics in patients with a large right ventricular infarc- t i ~ n . ~ Right atrial pressure (RAP) is highly sensitive and spe- cific for identifying right ventricular infarction and is a reliable guide for the rap^.^^^ A comprehensive evaluation of the rela- tionship between echocardiographic and Doppler parameters of right ventricular function, right atrial (RA) function, and in- ferior vena cava, and mean RAP (-) in patients with right ventricular infarction has not been performed previously. This study was undertaken to assess the relationship between Dop- pler parameters of hepatic vein and tricuspid inflow and right ventricular and W. Hypothesis: The aim of the present study was to assess the relationship between Doppler parameters of hepatic vein and tricuspid inflow, as well as mean right atrial (RA) pressure in patients with right ventricular infarction. Methods: In all, 59 consecutive patients with inferior left ventricular infarction involving the right ventricle were select- ed for the study. All patients underwent Doppler echocardio- graphic evaluation of tricuspid and hepatic vein parameters and catheterization of the right side of the heart. Patients were divided into two groups according to the presence or absence of severe tricuspid regurgitation. Results: In patients with severe tricuspid regurgitation, a significant correlation (r = 0.64; p < 0.001) between RA max- imal volume and mean right atrial pressure (RAP) was found, and the sensitivity of RA maximal volume in identifying mean RAP > 7 mmHg was 64% with a specificity of 78%. In patients without severe tricuspid regurgitation, the most sig- nificant relationship was observed between mean RAP and in- ferior vena cava collapse index. Significant correlations be- tween maximal and minimal diameters of the inferior vena cava were also observed. Relationship between Mean Right Atrial Pressure and Doppler Parameters in Patients with Right Ventricular Infarction ANNA V m m MA~OLI, M.D., ANNADELE CASTELLI, M.D., GIORGIO MATTIOLI, M.D. Department of Cardiology, University of Modena and Reggio Emilia, Modena, Italy Department of Cardiology, University of Modena and Reggio Emilia, Modena, Italy Department of Cardiology, University of Modena and Reggio Emilia, Modena, Italy Key words: right ventricular infarction, mean right atrial pres- sure, Doppler echocardiography Address for reprints: Anna Vittoria Mattioli, M.D. Department of Cardiology University of Modena and Reggio Emilia Via del pozzo, 7 1 41 100 Modena, Italy Received: April 13, I999 Accepted with revision: December 15, 1999 Clin. Cardiol. 23, 771-775 (2000) Clin. Cardiol. 23, 771-775 (2000) 19328737, 2000, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/clc.4960231015 by CochraneItalia, Wiley Online Library on [22/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA a Clin. Cardiol. Vol. 23, October 2000 772 TABLE I Hemodynamic and two-dimensional echocardiographic parameters on right-side cardiac structure and function in patients with and without severe tricuspid regurgitation Hemod ynamics With severe TR Without severe TR Mean RAP (mmHg) 17.2 ~t 7.4 (8-28) Heart rate @eats/min) 69 f 10 (56-74) Diastolic blood pressure (mmHg) 84 f 8 (78-88) 31+13(13-35) 16.5 f 6.8 (6-28) 66 f 9 (52-78) 88 f 6 (74-84) Systolic blood pressure (mmHg) 104* lO(78-130) 106* 14(72-128) Pulmonary wery systolic pressure (mmHg) Pulmonary artery diastolic pressure (mmHg) 2812(17-36) 14 + 6 (7-21) 7 f 6 (6-15) 12 -t 7 (5-19) 8 & 6 (7-1 2) Pulmonary artery wedge pressure (mmHg) Cardiac output (Vmin) 2.3 f 1.3 (1.6-4.3) 3,2& lS(1.8-4.7) Echocardiographic parameters R.4 vol max cm3 49.7 f 7.5 (38-60) 45.8 & 6.7 (36-57) R.4 vol min cm3 44.5 rt 6.5 (32-54) 41.6-~6.2(32-52) IVCmaxcm 1.71 0.8(1.5-1.85) 1.61 fOS(1.5-1.7) IVCmincm 1.69 f 0.8 (1.4-1.84) l.lO+O.4(1-1.2) IVC collapse (%) 31 f 18 (9-98) 45223(1&98) RVEF (%) 38.4 f 11 (16-58) 32.5 -t 12(17-58) Abbreviations: TR = tricuspid regurgitation, mean RAP = mean right atrial pressure, FL4 = right atrium, IVC = inferior vena cava, RVEF = right ventricular ejection fraction. Doppler echocardiogmphy was used to screen for the presence of valvular regurgitation. A pulsed Doppler recording of tri- cuspid inflow was obtained from the low parastemal and api- cal windows, with the sample volume positioned at the tips of the tricuspid valve. The flow velocity of the hepatic vein was obtained from the subcostal view. Ten cardiac cycles from each window were recorded and measured. Right ventricular function was quantified from the four-chamber view using modified Simpson’s rule.’ Study htocol Right ventricular infarction was diagnosed if patients fit at least three of the electrocardiographic, hemodynamic, angio- graphic, or echocardiographic In all patients, a standard 12-lead electrocardiogram (ECG) and right precordial leads (v3-v6R) were recorded within 10 h from the onset of symptoms. ST-segment deviations were as- sessed 0.04 s after the J point in all 16 leads. A 1 mm ST-seg- ment elevation andor Q waves in V4-V6R leads were required to diagnose the presence of right ventricular infarction.,’ All patients had ECG signs of right ventricular involvement. Maximal right atrial volume preceding the opening of the tri- cuspid valve and minimal RA volume following atrial contrac- tion were measured from the apical four-chamber view. Right atrial volumes were calculated with modified Simpson’s rule.’ p g g Radiographic and echocardiographic features of right ven- tricular infarction were evaluated as previously described in detail.7.9. 10 The inspiratory change was measured from the two-dimen- sional subcostal view, from which a percent collapse index was obtained. The inspiratory change, commonly referred to as a sniff test, was used frequently to estimate RAP. l2 The hemodynamic alterations that were considered highly indicative of a right ventricular infarction included signifi- cantly elevated RAP that exceeded the pulmonary-capillary wedge Coronary angiography was considered indicative of right ventricular infarction if there was an occlu- sion of the right coronary artery proximal to the acute margin- al branches . q y Tricuspid inflow measurements were performed from the window providing the highest overall velocities, allowing the best angulation with flow. The following parameters were measured: peak early in- flow velocity, peak late velocity, and the ratio of deceleration: acceleration time. On the basis of this protocol, 59 patients were enrolled in this study. Thrombolytic therapy was used in all eligible pa- tients; 54 patients were eligible while 4 patients had major contraindications. From the hepatic vein flow velocity, the parameters for peak velocity and time velocity integral of the systolic, diastolic, and atrial reversal waves were measured. The duration of the atrial reversal wave was measured from the beginning to the end of the atrial reversal wave. All measurements represented the average of five consecutive cycles. This approach allowed parameters to be almost identical to those obtained during end- espiratory apnea. ’ The study protocol was approved by the Ethical Committee of the University, and all patients signed an informed consent form. Echocardiographic and Doppler Evaluation Echocardiographic studies were performed with patients in the supine position. Measurements were taken at end expira- tion. Standard echocardiographic imaging was obtained from the parastemal, apical, and subcostal windows for the evalua- tion of right and left ventricular function and for assessment of the size of the right atrium and inferior vena ~ a v a . ~ Color flow Patient Population Conclusions: Echocardiographic and Doppler parameters may be useful for evaluating mean RAP in patients with right ventricular infarction. In patients with severe tricuspid regur- gitation, the more important parameters are maximal and minimal RA volumes. In patients without severe tricuspid re- gurgitation together with right atrial volume, the important parameters are acceleration and deceleration time of the tri- cuspid inflow peak E velocity and hepatic systolic and dias- tolic venous flow. In all, 74 consecutive patients with inferior left ventricular infarction involving the right ventricle were screened for the study. Fifteen patients were excluded from the study because of an inadequate recording of Doppler tracings andor inade- quate recording of pressures. The remaining 59 patients (43 men and 16 women) had a mean age of 65 f 8 years. All pa- tients had indwelling central venous catheters or underwent catheterization of the right side of the heart, and were in the Intensive Care Unit of our University Hospital. Simultaneous recordings of transthoracic echocardiogra- phy, mRAP, and standard upright posteroanterior chest x-ray film were obtained in all patients. The transthoracic echocardiographic studies were inde- pendently examined by an observer blinded to the diagnosis of the patients. Exclusion criteria included inadequate record- ings of pressures or Doppler tracings, or a chest x-ray inade- quate for a quantitative evaluation of the width of the azygos vein. Patients with bundle-branch block, previous cardiac failure, or those admitted > 10 h after the onset of symptoms were excluded from the study. Address for reprints: Anna Vittoria Mattioli, M.D. Department of Cardiology University of Modena and Reggio Emilia Via del pozzo, 7 1 41 100 Modena, Italy Received: April 13, I999 Accepted with revision: December 15, 1999 Address for reprints: Anna Vittoria Mattioli, M.D. Department of Cardiology University of Modena and Reggio Emilia Via del pozzo, 7 1 41 100 Modena, Italy Received: April 13, I999 Accepted with revision: December 15, 1999 The clinical data of the patients are presented in Table I. Results Patients were divided into two groups according to the pres- ence or absence of severe tricuspid regurgitation. Group 1 in- cluded 32 patients (23 men, 9 women, mean age 66 k 8 years) with severe tricuspid regurgitation. Group 2 included 27 pa- tients (20 men, 7 women, mean age 64 * 9 years ) without se- vere tricuspid regurgitation. Right ventricular ejection fraction averaged 32.5 ~t 12% (range 17-58%). Right atrial maximal volume averaged 45.8 k 6.7 (range 36-57), and minimal volume averaged41.6 f 6.2 (range 32-52). The mean percent collapse of the inferior vena cava was 45 k 20% (range 1&82%). Doppler and Echocardiographic Features of Patients with Severe nicuspid Regurgitation Table I shows the hemodynamic and echocardiographic pa- rameters of patients in Group l. Right ventricular ejection hction averaged 34 k 1 1 % (range 16-58%). Right atrial max- imal volume averaged 49 f 7 cm3 (range 38-60 cm3), and minimal volume averaged 44 f 6 cm3 (range 32-54 cm3). The maximal diameter of the inferior vena cava averaged 1.7 1 f 0.1 cm (range 1.5-1.8 cm) with a minimal diameter after in- spiratory effort of 1.69 * 0.8 cm (range 1.4-1.8 cm) and a per- cent collapse of 3 1 f 18% (range 9-98%). Table IV shows the correlation between echocardiographic parameters and mRAp. Among echocardiographic parame- ters, a significant relationship was observed between mRAP TABLE IIl Correlation between Doppler-derived parameter and mean right atrial pressure in patients with severe tricuspid regurgitation Tricusuid flow r I, Value Peak E velocity Peak A velocity WA ratio AFF A-wave duration Deceleration time Acceleration time Hepatic vein flow Diastolic velocity Systolic velocity Abbreviations as in Table II. 0.11 0.28 0.14 0.29 0.06 0.47 0.42 0.012 0.25 0.5 0.1 0.4 0.1 0.7 0.006 0.01 0.09 0.16 TABLE IIl Correlation between Doppler-derived parameter and mean right atrial pressure in patients with severe tricuspid regurgitation Doppler-derived parameters in patients with severe tricus- pid regurgitation are listed in Table II. Statktical Analysis p y A strong inverse relationship was observed between mean RAP and right ventricular ejection fraction. Correlations be- tween maximal and minimal diameters and the collapse in- dex of the inferior vena cava were weak and not significant. The strongest relationship between Doppler parameters and mRAP was seen with the deceleration and acceleration time of the tricuspid inflow. Data are expressed as mean f 1 standard deviation. An un- paired t-test was used to compare variables between groups of patients. To compare qualitative ventricular function assess- ment and mean RAP, the chi-square test was used. Correla- tions were performed with linear regression analysis. Step- wise multiple linear regression was subsequently performed. The difference was considered statistically significant when p < 0.05. A.V. Mattioli et al.: Mean right atrial pressure in RV infarction 773 TABLE II Mean value of Doppler-derived parameters in patients with and without severe tricuspid regurgitation Tricuspid flow With severe TR Without severe TR Peak E velocity (cds) 58.8 f 16.9 (30-82) Peak A velocity ( c d s ) 76.30k9.2 (54-91) 57.1 f 14.1 (34-80) AFF 0.27 -I- 0.05 (0.26-0.36) 0.25 f0.03 (0.2-0.31) % Deceleration time (ms) 218.6f 139(64465) 163.9 f 46 (40-220) Hepatic vein flow 68.28 k 10.9 (55-96) WA ratio 0.89 k0. 12 (0.66-1.29) 1.08-~0.39(0.5-2.17) Diastolic velocity (cds) 39.1 f 16 ( I 8-70) 34.0k 12(18-55) Systolic velocity (cds) - 14.2 f 10 [ -7(-24)] 14.4 f 5 (8-28) Abbreviations: AFF = atrial filling fraction, TR = hicuspid regurgitation. LE II Mean value of Doppler-derived parameters in patients with and without severe tricuspid regurgitation mean RAP (r = 0.64; p <0.001) (Fig. 2), and the sensitivity of RA maximal volume in identifying mean RAP >7 mmHg was 64% with a specificity of 78%. were determined at end expiration and an average of three to five cycles was obtained. Doppler and Echocardiographic Features of Patients with- out Severe nicuspid Regurgitation (Group 2) Hemodynamics and Doppler echocardiographic parame- ters in patients without severe tricuspid regurgitation are pre- sented in Tables 11 and m. 19328737, 2000, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/clc.4960231015 by CochraneItalia, Wiley Online Library on [22/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online A.V. Mattioli et al.: Mean right atrial pressure in RV infarction A.V. Mattioli et al.: Mean right atrial pressure in RV infarction Hernodynamic Recordings All readings were referenced to the midaxillaty line with the patients in the supine position. The position of the catheter was identified by chest radiograms. Pressure measurements Relation of Echocardiographic Measurements to Mean Right Atrial Pressure Table III shows the correlation between echocardiograph- ic parameters and mean RAP. A significant correlation was present for deceleration time (r = 0.47; p <0.006) (Fig. 1). A relationship was observed between RA maximal volume and 19328737, 2000, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/clc.4960231015 by CochraneItalia, Wiley Online Library on 19328737, 2000, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/clc.4960231015 by CochraneItalia, Wiley Online Library on [22/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Li Clin. Cardiol. Vol. 23, October 2000 774 23, October 2000 30.00 - 25.00 - h I" 20.00 - E 15.00 4 10.00 - E 5.00 - q< - : I Maximal right atrial volumes (cm3) FIG. 2 Scatterplot showing the relation of maximal right atrial vol- ume with mean right atrial pressure (mRAP) in patients with severe tricuspid regurgitation. + r = 0.64, p < 0.001. 30.00 - 25.00 - h I" 20.00 - E v E 15.00 - 3 10.00 - 5.00 - 0.00 - * * / -* **- - 1 30.00 - 25.00 - h I" 20.00 - E 15.00 4 10.00 - E 5.00 - q< - :* I Deceleration time FIG. 1 Scatterplot showing the relation of tricuspid deceleration time with mean right atrial pressure (mRAP) in patients with severe tricuspid regurgitation. + r = 0.47, p < 0.01. Maximal right atrial volumes (cm3) FIG. 2 Scatterplot showing the relation of maximal right atrial vol- ume with mean right atrial pressure (mRAP) in patients with severe tricuspid regurgitation. + r = 0.64, p < 0.001. 30.00 - 25.00 - h I" 20.00 - E v E 15.00 - 3 10.00 - 5.00 - 0.00 - * * / -* **- - 1 Deceleration time FIG. 1 Scatterplot showing the relation of tricuspid deceleration time with mean right atrial pressure (mRAP) in patients with severe tricuspid regurgitation. + r = 0.47, p < 0.01. 30.00 - 25.00 - h I" 20.00 - E 15.00 4 10.00 - E 5.00 - q< - :* I Maximal right atrial volumes (cm3) 30.00 - 25.00 - h I" 20.00 - E v E 15.00 - 3 10.00 - 5.00 - 0.00 - * * / -* ***- - 1 Deceleration time Maximal right atrial volumes (cm3) FIG. 1 Scatterplot showing the relation of tricuspid deceleration time with mean right atrial pressure (mRAP) in patients with severe tricuspid regurgitation. + r = 0.47, p < 0.01. FIG. Relation of Echocardiographic Measurements to Mean Right Atrial Pressure 2 Scatterplot showing the relation of maximal right atrial vol- ume with mean right atrial pressure (mRAP) in patients with severe tricuspid regurgitation. + r = 0.64, p < 0.001. and maximal and minimal right atrial volumes. Larger atrial volumes at end systole and end diastole were found to be asso- ciated with higher mRAP. The sensitivity of this parameter in identifying mRAP > 7 mmHg was 74% and specificity was 87% (Table V). astolic inflow reflects the effect of filling pressure, right ven- tricular relaxation, and net atrioventricular compliance. l3 The dependence of tricuspid filling dynamics on loading condition has been previously described. l4 Right ventricular infarction is characterized by elevated mRAl', resulting in a higher early velocity of tricuspid In patients with right ventricular infarction and severe tricuspid regurgitation, there was a sig- nificant correlation between RA maximal volume and mRAp, this parameter had high sensitivity and specificity in identify- ing patients with mRAP > 7 mmHg. A larger atrial volume at the end of ventricular systole is probably due to several factors. The severity of the hemodynamic derangements associated with right ventricular infarction is related not only to the extent of right ventricular ischemia, and consequently right ventricu- lar dysfunction, but also to the restraining effect of the peri- cardium and the resulting interaction between the ventricles. l5 The restraining effect of the pericardium induces a reduction in right ventricular systolic pressure in a patient with severe tri- cuspid regurgitation. ( ) The most significant relationship was observed between mRAP and the inferior vena cava collapse index. Significant correlations were observed between maximal and minimal diameters of the inferior vena cava. Table V shows the sensi- tivity and specificity of the best Doppler and two-dimension- al variables for separating normal from elevated mRAP. Discussion The present study was undertaken to assess the relationship between Doppler echocardiographic parameters and mRAP in patients with right ventricular infarction. A previous paper by Nagueh et al. has demonstrated that Doppler parameters of hepatic vein flow, particularly those including the systolic fill- ing wave, had the strongest relation with mRAP.I3 This feature is confirmed by the present paper in a patient population with acute right ventricular infarction. It was postulated that, as right ventricular systolic function diminishes, the atrial contraction becomes the driving force for pulmonary perfusion.'43 16 The inverse relationship between mRAP and right ventricular ejection time is probably due to the active right ventricular systolic pressure wave caused by left ventricular contraction, which bulges in piston-like fashion into the right ventricle. This generates a systolic force suffi- cient for pulmonary perfusion and a simultaneous increase in tricuspid regurgitation.17 g The strongest relation was observed between systolic and diastolic hepatic vein flow velocities and mRAP. Tricuspid di- TABLE IV Correlation between Doppler-derived parameter and mean right atrial pressure in patients without severe tricuspid regur- gitation Tricuspid flow r p Value Peak E velocity 0.13 0.5 Peak A velocity 0.41 0.03 WA ratio 0.19 0.32 AFF 0.17 0.39 A-wave duration 0.52 0.005 Deceleration time 0.64 0.0003 Acceleration time 0.65 0.0002 Hepatic vein flow Diastolic velocity 0.47 0.01 Systolic velocity 0.73 0.001 Abbreviations as in Table II. p y p tricuspid regurgitation.17 TABLE V Sensitivity and specificity of echocardiographic and Dop- pler parameters for mean right atrial pressure > 7mmHg in patients without severe tricuspid regurgitation Sensitivity Specificity RA volume max > 35 cm 74 87 Acceleration time < 65 65 80 Deceleration time < 200 76 79 Systolic velocity hepatic vein > 30 81 92 Diastolic velocity hepatic vein > 12 84 94 Abbreviation: RA = right atrial. TABLE IV Correlation between Doppler-derived parameter and mean right atrial pressure in patients without severe tricuspid regur- gitation Tricuspid flow r p Value Peak E velocity 0.13 0.5 Peak A velocity 0.41 0.03 WA ratio 0.19 0.32 AFF 0.17 0.39 A-wave duration 0.52 0.005 Deceleration time 0.64 0.0003 Acceleration time 0.65 0.0002 Hepatic vein flow Diastolic velocity 0.47 0.01 Systolic velocity 0.73 0.001 Abbreviations as in Table II. References The behavior of acceleration time is probably due to right ventricular pressure tracing, which is often bifid in right ven- tricular infarction and characterized by a “dip-plateau” pat- tern in the diastolic pressure curve with a high early gradient between the right atrium and ventricle.I8 The correlation of mRAP with deceleration time is similar to that observed in patients with constrictive peri~arditis.’~ I . Isner JM, Roberts WC: Right ventricular infarction complicating left ventricular infarction secondary to coronary artery disease. Am J Cardiol1978;42:885-894 2. Andersen HR, Nielsen D, Falk E: Right ventricular infarction: Diagnostic value of ST elevation in lead 111 exceeding that of lead II during inferior/posterior infarction. Am Heart J 1989; 1 17:82-85 In patients without severe tricuspid regurgitation, larger vol- umes were found to be associated with a hgher mRAP There was also a strong sensitivity and specificity of the acceleration and deceleration time of the tricuspid E wave. Furthermore, these parameters correlated well with mRAP. In this group, there was also a “dip-plateau’’ in the right ventricular diastolic pressure curve that reflects a decrease in compliance as well as in pericardial constraining l9 Our findings in patients without severe tricuspid regurgitation were similar to those previously ~p3rted.I~ 3. Cohn JN: Right ventricular infarction revisited. Am J Cardiol 1979;43:666-668 4. Dell’Italia LJ, Starling MR, Crawford MH, Boros BL, Chanduri TK, O’Rourke RA: Right ventricular infarction: Identification by hernodynamic measurements before and after volume loading and correlation with noninvasive techniques. J Am Coll Cardiol 1984 4:931-939 5. Kazemi H, Parson EF, Valenca LM, Strieder DJ: Distribution of pulmonary blood flow after myocardial ischemia and infarction. Circulation 1970;41: 1025-1030 6. Milne EN, Pistolesi M, Miniati M, Giuntini C: The vascular pedicle of the heart and vena azygos. Radiology 1984; 152:%17 p y p The contribution of RA contraction to pulmonary perfusion explains the correlation between mRAP and hepatic venous flow. Determinants of the systolic forward flow include atrial relaxation, the descent of the tricuspid annular plane toward the ventricle apex, and, more important, the RAP. The higher the RAP, the lower the pressure gradient between the hepatic veins and the right atrium, and thus the lower the forward sys- tolic l3 In the present study, systolic forward flow pa- rameters correlated well with RAP, and the diastolic flow also showed a strong relationship with RAP. Goldstein et al. References ob- served the right atrial wave form and demonstrated that the predominant atrial descent in diastole is blunted, reflecting an increase in resistance to diastolic filling which is proportional to RAP.^^.^^ 7. Bellamy GR, Rasmussen HH, Nasser FN, Wiseman JC, Cooper RA: Value of two-dimensional echocardiography, electrocardiog- raphy and clinical signs in detecting right ventricular infarction. Am Heart J 1986; 1 12:304-309 8. Berger PB, Ruocco NA, Timm CT, Zaret BL, Wackers FJ, and the TIM1 Investigators: The impact of thrombolytic therapy in right ventricular infarction complicating inferior myocardial infarction: Results fromTIMI II. Circularion 1989;80:313-314 Results fromTIMI II. Circularion 1989;80:313-314 9. Mattioli AV, Bastia E, Mattioli G: Doppler echocardiographic find- ings in patients with right ventricular infarction. J Ultrasound Med 1998; 17;297-301 10. Mattioli AV, Mattioli G Radiographic finding of patients with right ventricular infarction: Prognostic evaluation. Radiography 2000; 6~19-26 I I. Panidis IF‘, Ren J, Kotler MN, Mintz G, Iskandrian AM, Ross J, Kane S: Two dimensional echocardiographic estimation of right ventricular ejection fraction in patients with coronary artery dis- ease. JAm Coll Cardiol1983;2:911-918 Clinical Implication 12. Kircher BJ, Himelman RB, Schiller NB: Noninvasive estimation of right atrial pressure from the inspiratory collapse of the inferior vena cava. Am J Cardiol1990;66:493-496 A recent paper reported the reference values for right ven- tricular filling of normal persons?’ suggesting the role of right- side Doppler parameters in the comprehensive evaluation of many diseases. The role of Doppler echocardiography in the Coronary Care Unit has been increasing over the years, and we need to get more information, not only about the extension of the ischemic disease but also to assess hemodynamics and function. The Doppler echocardiographic parameters we test- ed can be useful in identifying patients with right ventricular infarction and increased mRAP when invasive techniques are not immediately available. 13. Nagueh SF, Kopelen HA, Zoghbi WA: Relation of mean right atrial pressure to echocardiographic and Doppler parameters of right atri- al and right ventricular function. Circulation 1996;93: 1 160-1 169 g 14. Sadler DB, Brown J, Mursett H, Roberts J: Impact of hemodialysis on left and right ventricular Doppler diastolic filling indices. Am J Med Sci 1992;304:83-90 15. Calvin J E Optimal right ventricular filling pressures and the role of pericardial constraint in right ventricular infarction in dogs. Circulation 199 1;84852-861 16. Kuo LC, Quinones MA, Rokey R, Sartori M, Abinader EG, Zoghbi WA: Quantification of atrial contribution to ventricular filling by pulsed Doppler echocardiography and the effect of age in normal and diseased heart. Am J Curdiol 1987;59: 1 174-1 178 17. Goldstein JA, Barzilai B, Rosamond TL, Eisenberg PR, Jaffe AS: Determinants of hernodynamic compromise with severe right ven- tricular infarction. Circulation I990;82:359-368 Discussion TABLE IV Correlation between Doppler-derived parameter and mean right atrial pressure in patients without severe tricuspid regur- gitation TABLE V Sensitivity and specificity of echocardiographic and Dop- pler parameters for mean right atrial pressure > 7mmHg in patients without severe tricuspid regurgitation Sensitivity Specificity RA volume max > 35 cm 74 87 Acceleration time < 65 65 80 Deceleration time < 200 76 79 Systolic velocity hepatic vein > 30 81 92 Diastolic velocity hepatic vein > 12 84 94 Abbreviation: RA = right atrial. TABLE V Sensitivity and specificity of echocardiographic and Dop- pler parameters for mean right atrial pressure > 7mmHg in patients without severe tricuspid regurgitation A.V. Mattioli et al.: Mean right atrial pressure in RV infarction 775 Conclusions 18. Kinch JW, Ryan TJ: Right ventricular infarction. N Engl J Med 1994;330;1211-1217 The echocardiographic and Doppler parameters may be useful for evaluating mRAP in patients with right ventricular infarction. In patients with severe tricuspid regurgitation, the more important parameters are maximal and minimal RA vol- umes. The increase of RA volumes suggested an increased mRAP > 7 d g . In patients without severe tricuspid regur- gitation together with right atrial volume, the important pa- rameters are the acceleration and deceleration times of the tri- cuspid E wave and hepatic, systolic, and diastolic venous flow. 19. Oh JK, Hatle LK, Seward JB, Danielson GK, Schaff HV, Reeder GS, Tajik AJ: Diagnostic role of Doppler echocardiography in con- strictive pericarditis. JAm Coll Cardiol1994;22 1935-1943 p 20. Goldstein JA, ’heddell JS, Barzilai B, Yagi Y, Jaffe AS, Cox JL: Importance of left ventricular function and systolic ventricular in- teraction to right ventricular performance during acute right heart ischemia. JAm Coll Cardiol1992;19:704-711 21. Klein AL, h u n g DY, Murray RD, Urban LH, Bailey KR, Tajik AJ: Effects of age and physiologic variables on right ventricular filling dynamics in normal subjects. Am J Curdiol1999;84:440-448
https://openalex.org/W4381094598	https://link.springer.com/content/pdf/10.1007/s00338-023-02397-1.pdf	English	null	Quantifying capture and ingestion of live feeds across three coral species	Coral reefs	2,023	cc-by	10,831	Coral Reefs (2023) 42:931–943 https://doi.org/10.1007/s00338-023-02397-1 REPORT REPORT Quantifying capture and ingestion of live feeds across three coral species Julia Saper1,2,3 · Lone Høj2 · Craig Humphrey2 · David G. Bourne1,2 Received: 12 March 2023 / Accepted: 29 May 2023 / Published online: 17 June 2023 © Crown 2023, corrected publication 2023 Abstract Nutrient acquisition through heterotrophy is critical for the health of reef-building corals. The optimi- zation of exogenous nutrition protocols to support a diver- sity of aquaculture corals requires improved techniques to assess feeding rates. Here, we compared the feeding rates of three coral species (Acropora millepora, Pocillopora acuta and Galaxea fascicularis) fed Artemia salina through cap- ture rate (indirect) and dissection (direct) approaches, with direct detection and enumeration within dissected polyps facilitated by fluorescent microbeads ingested by the Arte- mia. When A. millepora was provided Artemia at 3 indi- viduals ml−1 for one hour, the calculated capture rates (0.7 ind. polyp−1 h−1) overestimated prey ingested compared to prey detected directly within polyps (0.2 ind. polyp−1 h−1), and ingestion varied significantly between genotypes. In contrast, for P. acuta, capture rate calculations (1 ind. polyp−1 h−1) underestimated prey detected within polyps (3.5 ind. polyp−1 h−1) and ingestion did not vary between genotypes. For G. fascicularis, the feeding rates were similar as calculated by both capture rates (59 ind. polyp−1 h−1) and by polyp dissections (75 ind. polyp−1 h−1). Results from this study provide valuable insights into coral feeding rates of different coral species that can improve prey enrichment and feeding strategies for nutritional supplementation of corals in captivity. Keywords Coral feeding · Coral aquaculture · Fluorescence microscopy · Artemia · Feeding rates · Dissections * David G. Bourne david.bourne@jcu.edu.au 3 AIMS@JCU, James Cook University, Townsville, QLD 4811, Australia 1 College of Science and Engineering, James Cook University, Townsville, QLD 4811 , Australia 2 Australian Institute of Marine Science, PMB 3 Townsville MC, Townsville, QLD 4810, Australia Introduction 2016; Smith et al. 2016; Axworthy & Padilla-Gamino 2019). However, dissections require time, refined microscopic approaches and are complicated by small coral polyps and cryptic prey (Trager et al. 1994; Houlbrèque et al. 2004b). Importantly, though polyp dissections are an important tool to understand the amount and types of prey consumed, it is not always feasible to differentiate degraded prey from coral tissue. One approach to overcome these limitations is visual aids (e.g., dyes, fluorescent markers) to improve prey detection. The addition of fluorescent markers could extend our understanding of feeding by facilitating docu- mentation of uptake and increasing contrast between prey and coral tissue. Visualization methods using fluorescent polystyrene microbeads first emerged as a biomedical diagnostic tool (Popielarski et al. 2005; Madden et al. 2013; Bott 2014) and have since been applied to investi- gate prey preference in ornamental marine fish (Lee et al. 2018). Many zooplankton species, including copepods, rotifers and mysid shrimp, graze on particulate matter and inadvertently ingest microplastics. Direct detection of microplastics ingestion in experimentally infected zoo- plankton has been demonstrated by the application of fluo- rescent polystyrene microbeads (Setälä et al. 2014; Lee et al. 2018; Miller et al. 2020). These studies suggest that many zooplankton species can be readily incubated with fluorescent microbeads (Setälä et al. 2014; Lee et al. 2018; Miller et al. 2020). Among a range of diets employed by aquarium facili- ties, Artemia spp. nauplii is a cost-effective, commercially available option. Artemia is offered to an estimated 85% of all marine organisms in aquaculture (Trager et al. 1994; Sebens et al. 1998; Kumar & Babu 2015) and has been dem- onstrated to stimulate growth and improve survivability of a range of captive corals (Osinga et al. 2012a; Tagliafico et al. 2018a). The growth and survival of juvenile and adult aquarium reared Pocillopora acuta (Toh et al. 2013; Huang et al. 2020), Pocillopora damicornis (Conlan et al. 2018), Acropora tenuis, Favia fragum (Petersen et al. 2008) and Duncanopsammia axifuga (Tagliafico et al. 2018b) improved significantly when fed Artemia nauplii when compared to unfed corals. A range of indirect and direct approaches have assessed coral feeding abilities, each with strengths and limitations. The capture rate approach is an indirect method of assess- ment commonly used to select optimal feeding densities for aquarium reared corals (Osinga et al. 2008, 2012b; Kuanui et al. 2016). Introduction Coral aquaculture is expanding rapidly to supply a grow- ing ornamental trade and to replenish reefs that have been degraded due to anthropogenic impacts (Osinga et al. 2011; Leal et al. 2016). Across the ornamental and hobbyist indus- try, there are over 100 coral species collected from the wild and propagated in relatively small-scale aquarium systems (Borneman 2009; Tagliafico et al. 2018a). However, as the demand for coral increases, the scale at which corals are cultured in captivity must also expand to reduce impacts of wild harvesting (Barton et al. 2015). Exogenous feeds are needed to fully meet the nutritional needs of captive corals. Our limited understanding of the nutritional requirements of corals and their species-specific feeding abilities represent significant hurdles for large-scale production. Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/ s00338-023-02397-1. i Heterotrophic feeding is essential to the health of all symbiotic, reef-building corals (Brafield & Llewellyn 1982; Anthony & Fabricius 2000; Houlbrèque et al. 2004a). Sym- biotic corals are mixotrophs, obtaining energy through auto- trophic assimilation of photosynthates derived from their algal symbiotic partners (Symbiodiniaceae) and through heterotrophic feeding (Houlbrèque & Ferrier-Pagès, 2009; Ferrier-Pagès et al. 2011). Although some corals can acquire significant amounts of their energy needs from photosyn- thates, all corals require heterotrophic inputs to survive (0121 932 Coral Reefs (2023) 42:931–943 (Palardy et al. 2008; Hughes et al. 2010; Ezzat et al. 2016). Morphological characteristics can provide useful insights into a coral species’ feeding ability. For example, a fast- growing branching or tabulate coral colony, such as an Acropora sp., has small polyps (< 1 mm diameter) and the high surface to volume ratios of colonies maximizes light- acquisition which can impede capture of live prey. In con- trast, a slow-growing massive coral colony, such as a Favites sp., has larger polyps (> 5 mm diameter), which may have evolved to maximize plankton-capture (Houlbrèque et al. 2009; Conti-Jerpe et al. 2020). accurately assess coral feeding and to date have employed video (Wijgerde et al. 2011; Osinga et al. 2012b) and coral polyp dissections (Hall et al. 2015; Kuanui et al. 2016; Smith et al. 2016). For example, dissections of P. dami- cornis, A. millepora and A. nobilis polyps have been uti- lized to investigate the breakdown of Artemia nauplii in fed corals as well as the composition of ingested plankton in G. fascicularis across reef habitats (Kuanui et al. 1 3 Coral collection and maintenance Three adult colonies (genotypes) of each species (A. mille- pora, P. acuta and G. fascicularis) were collected from Davies Reef (lat.: 18°49′31″ S, long.: 147°38′50″ E) (AIMS General Permit G12/35236.1). Following collection, corals were transported to the National Sea Simulator (SeaSim) located at the Australian Institute of Marine Science (AIMS, Cape Cleveland, Australia) and acclimated to aquarium con- ditions in outdoor tanks for two weeks, with the tempera- ture set to emulate reef conditions (24 °C) at the time of collection. Adult colonies were cut into small fragments (2–5 cm length) using a Gryphon Coral Saw then adhered to either aragonite (capture rate experiment) or ceramic plugs with a wax coating to prevent biofouling (ingestion experiment). Plugs were placed on elevated PVC trays, which were moved to 250 l indoor holding tanks where fragments further accli- mated for four weeks. Holding tanks were supplied with filtered (1 µm) flow-through seawater and a circulation pump (Tunze® Turnbelle® nanostream®) for water movement. The temperature of the holding tanks was maintained at 27 ± 0.02 °C, and a light regime was programmed to match the natural day and night cycle and oscillations in intensity (maximum 200–250 µmol photons m−2 s−1). Lights were ramped up to maximum intensity over four hours, held for four hours then ramped down over four hours. Coral frag- ments in holding tanks were fed low concentrations of Arte- mia instar I nauplii and Brachionus plicatilis daily to sup- port their nutritional health prior to experimental trials. To control algal growth, tanks were manually cleaned weekly as well as via herbivorous snails (Turbo spp. and Thalo- tia strigata) and orange-shoulder surgeonfish (Acanthurus olivaceus). For the ingestion experiment’s feeding trials, prior to use, small volumes of the microbeads stock solution (3.8 × 1010 particles ml−1, in a 2% solids solution) were dispensed and diluted with ultra-filtered seawater (0.22 µm) at 1:1 ratio. The diluted solution was sonicated briefly to prevent aggre- gation, and then, 10 µl was added to 25 ml suspension cul- ture flasks with vented caps (Sarstedt, Germany) with 7 ml of Artemia nauplii (~ 600 ind. ml−1). Flasks were placed on an orbital shaker (50 rpm) for 1 h before rinsing thoroughly with seawater using a 250 µm mesh net for 5 min. Methods ingested fluorescent microbeads and their average size at this time. For the ingestion experiment, Artemia were harvested at this time point to ensure that they could ingest microbeads and hence could be visualized in fed corals. At eight fixed time points after harvest, Artemia nauplii were incubated with fluorescent microbeads, as described below. After incu- bations, nauplii were placed onto glass cavity slides with the addition of a viscous solution (1% methocel) to slow down prey movement for visualization using an inverted fluores- cent microscope (LEICA DMI6000B, LAS-X software) with fluorescent filters (ET-GFP, 450/490 nm) and images were taken (AxioCam MRc Rev. 3). Lengths of nauplii (n = 30) were measured from the eye to the end of the tail (Ekono- mou et al. 2019) using Leica Application Suite X (LAS-X) microscope software. Larval developmental stage was deter- mined by detection of fluorescence beads in the nauplii guts since microbeads could only be taken up by feeding nauplii, which occurs after their first molt into instar II nauplii, with developed mouthparts and guts. Coral collection and maintenance To assess if the washing step removed microbeads from nauplii sur- faces and the surrounding solution, aliquots of Artemia were fixed in 4% neutral buffered formaldehyde (NBF) in sea- water solution before and after and subsequently inspected by fluorescence microscopy. Similarly, an Artemia aliquot was fixed after each feeding trial to confirm gut retention of microbeads for downstream detection. Introduction Determination of mean capture rates involves counting prey in a fixed volume of water before and after a feeding period and then, normalizing to a unit of coral (e.g., surface area or polyp number). The capture of Artemia by a range of corals, including small polyp Acropora spe- cies (Hoogenboom et al. 2015; Kuanui et al. 2016; Taglia- fico et al. 2018a), P. damicornis (Hoogenboom et al. 2015; Kuanui et al. 2016) and relatively large polyp Galaxea fas- cicularis (Hii et al. 2009; Osinga et al. 2012b; Hoogenboom et al. 2015) and D. axifuga (Tagliafico et al. 2018a), has been measured through this approach. However, such indirect methods rely on problematic assumptions that prey captured equals prey consumed and do not account for the dynam- ics of prey capture, ingestion and release, or prey passively caught by mucus secretions but not ingested (Osinga et al. 2012b). Additionally, the capture rate approach calculates average capture rates per polyp based on whole fragment measurements, although there is likely a non-uniform dis- tribution of ingested prey across polyps within a fragment and across the larger colony. To aid in the direct quantification of feeding rates, this study compares the capture and ingestion abilities of three morphologically distinct scleractinian coral species (Acro- pora millepora, Pocillopora acuta and Galaxea fascicula- ris) fed Artemia nauplii. Furthermore, this study compares and develops methods that can accurately inform feeding regimes for corals in aquaculture. The ability of these cor- als to capture and ingest Artemia was investigated through two experiments. The first, referred to as “the capture rate experiment,” compared the ability of corals to capture Artemia salina instar I nauplii supplied at different initial densities, as measured by the number of prey items cleared from a fixed volume of water. The second experiment, referred to as “the ingestion experiment,” directly quanti- fied the number of prey items ingested by coral polyps via fluorescence microscopy detection of Artemia instar II nauplii, incubated with fluorescent microbeads. To quantify and assess both capture and ingestion of delivered feeds by individual coral polyps, direct approaches must be utilized. Direct approaches can more 1 3 1 3 933 Coral Reefs (2023) 42:931–943 Capture rate experiment Capture rate experiment A drop of suspended Artemia was added to each chamber to elicit a feeding response prior to delivering feeds. For P. acuta and A. millepora trials, instar I nauplii were delivered to nine chambers per species, each housing one coral fragment, per prey density treatment (3 replicate chambers per treatment: Low (1 ind. ml−1), Medium (2 ind. ml−1) and High (4 ind. ml−1). For G. fascicularis trials, an additional prey density treatment (8 ind. ml−1) was added due to the high feeding rates previously documented for G. fascicularis (Osinga et al. 2008; 2012b). For each experimental chamber with coral, there was a corresponding control chamber without coral. Trials com- menced at 10:00 h, during peak light intensity, on three con- secutive mornings. This controlled for potential differences in feeding based on light intensity fluctuations and repeat cap- ture rate measurements over consecutive days tested satiation effects (e.g., decreases in mean daily capture rates over time). Initial (C0) and final (Ct) prey concentrations were determined by counting 20 ml seawater aliquots collected five min after feed delivery and again one hour later. After feeding trials were completed, coral fragments were fixed for 24 h in 4% NBF at 4 °C and then rinsed in ultra- filtered seawater (0.22 µm) prior to placement in 4% formic acid solution for decalcification for 24 h–48 h. Decalcified fragments were rinsed in ultra-filtered seawater (0.22 µm) and placed in 2 × PBS in 50% ethanol (0.44 µm syringe fil- tered) for storage at −20 °C until dissected.i Decalcified corals were pinned to wax dissecting plates using headless stainless-steel pins (5 mm, Cat. no. E185, Australian Entomological SuppliesPTY LTD) and dissected under a dissecting stereoscope (LEICA MZ109) with an in- built GFP long-pass filter (LEICA ET-GFP LP FLUO filter; excitation filter: 480/40 nm, barrier filter: 254/511 nm) using forceps, dissecting scissors, scalpels, and fine tip dissect- ing probes (0.25 mm, product 10,140, Fine Science Tools, Inc.). Decalcified corals were cut into tissue sections and imaged one section at a time (Software: LAS V4, Camera: Leica DFC450 C). After imaging, ingested Artemia were counted within all the polyps present within each tissue sec- tion. Lastly, tissue sections were discarded to avoid double counting. Upon conclusion of the third day of trials, polyp counts were obtained for each fragment. A. millepora and P. Ingestion experiment Ingestion experiment Capture rate experiment acuta polyp counts were derived by multiplying their respective average number of polyps per square centimeter with their surface area measured using a wax dipping protocol (Veal et al. 2010). The average number of polyps per square centim- eter for A. millepora and P. acuta fragments was estimated by counting the number of polyps within a measured area using ToupView software version 4.10 (ToupTek, Zhejiang, China) from a minimum of three different sections (e.g., base, trunk, branch) of 20 representative fragments per species. Due to their larger size, G. fascicularis polyps were counted manu- ally. Mean capture of Artemia per coral polyp was calculated by the following Eq. (1): Acropora millepora fragments (L: ~ 2 cm, W: ~ 0.5 cm) were incised longitudinally with the axial corallites bisected. P. acuta fragments (L: ~ 2 cm, W: ~ 2 cm) were ‘unfolded’ and flattened. Each fragment was cut into 6 ± 2 sections. G. fascicularis fragments (L: ~ 2 cm W: ~ 2 cm) each had four to five polyps, which were dissected separately. The mouth of each G. fascicularis polyp was probed to separate clumps of Artemia from the tentacles, which auto-fluoresced. Each polyp was then longitudinally incised, flattened and pinned to the wax dish. The flattened sections were further divided to avoid double counting. A series of images were taken for all dissected samples and imported into Image J (https:// imagej.nih.gov/ij/) for analysis and annotation. In each sec- tion, polyps were outlined and numbered with prey enumer- ated using the multi-point tool. Correct detection of ingested Artemia was ensured by comparison with control images generated in pilot studies where corals were delivered (i) Artemia without beads; (ii) Artemia incubated with beads or (iii) beads only. (1) Capture rate ∶(Co −Ct )xVwater∕N (1) where Co is the initial, and Ct is the final number of nauplii in a sample volume of water at the end of an allocated feed- ing time (1 h), Vwater is the volume of water in the feeding chamber, and N is the number of coral polyps (Osinga et al. 2012b). Artemia preparation Prior to feeding trials, coral fragments were moved into 2.5 l cylindrical acrylic feeding chambers to acclimate for 24 h with flow-through filtered seawater set to one turnover per hour (2.5 l h−1). Feeding chambers were placed in freshwater baths to maintain constant temperature of 24 °C. Water pow- ered, magnetic stirrers and a magnetic stir bar were added to the bottom of each feeding chamber to homogenize delivered prey and provide water movement.l Artemia salina cysts (Sep-Art magnetic GSL Artemia cysts, INVE, Belgium) were hatched overnight and harvested daily (2.5 g cysts in 1 l seawater; pH:8, salinity 25–35 ppt). A magnetized collector tube (Sep-Art™, INVE, Belgium) sep- arated cysts from hatched nauplii. Newly harvested instar I nauplii were diluted with filtered seawater (1 µm) and gently aerated (~ 1:4 Artemia to seawater ratio; ~ 600 ind. ml−1). Repeat counts of Artemia nauplii in the diluted solution (n = 5 or until the standard error was < 10% of the mean) were conducted in a Bogrov counting chamber to determine the delivered prey densities. The diluted Artemia solution was kept at 27 ± 0.02 °C until the feeding trials. Water flow to the feeding chambers was stopped and the water volume in each chamber carefully adjusted by siphoning to 2 l (capture rate experiment) or 1 l (ingestion experiment). Feeds were delivered to the feeding chambers via syringe. Magnetic stir bars (80 revolutions min−1) ensured distribu- tion of prey without stratification. Observations on feeding An initial study was conducted to determine an aver- age time point (hours post-harvest) when all Artemia had 1 3 3 Coral Reefs (2023) 42:931–943 934 species. Each chamber housed three fragments of differ- ent genotypes (n = 12 fragments per species; four of each genotype). To calculate capture rates for the corals in the same chambers, initial and final prey counts were taken from 20 ml seawater aliquots collected immediately after feed delivery and again 70 min later, allowing 10 min for corals to elicit a feeding response to added prey. response indicators (e.g., polyp extension, tentacle move- ment, formation of mucus-prey agglomerations) were taken at 10 min post-feed delivery as well as throughout each trial. Experiment I: capture rate For A. millepora, the average surface area of fragments was 84 ± 4.5 cm2, with an average number of 46 ± 2 polyps cm−2 (3844 ± 219 mean polyps fragment−1) (Table 1). There was no significant difference in initial Artemia prey counts between control and experimental chambers (t =− 0.75, df = 52, P = 0.5) and no significant difference between ini- tial and final Artemia counts in control chambers without corals (t =− 0.04, df = 52, P = 0.96). In contrast, a signifi- cant difference was found between initial and final Artemia counts for chambers with A. millepora, with final counts being lower (t = 6.39, df = 52, P < 0.0005). The capture rates of Artemia delivered at initial concentrations of 1, 2 and 4 ind. ml−1 were 0.31 ± 0.08, 0.57 ± 0.08 and 1.22 ± 0.13 ind. polyp−1 h−1, respectively. There was a strong linear rela- tionship between capture rate and prey density as the ini- tial prey concentration increased (capture rate ~ initial ind. polyp−1, r2 = 0.88) (Fig. 1a). Capture rates, normalized to polyps across chambers, on consecutive days and delivered prey levels did not differ significantly (Analysis of Deviance, x2 = 1.33, P = 0.25 and x2 = 3.17, P = 0.20, respectively). However, coral mucus secretions were observed trapping delivered Artemia, particularly in chambers delivered 2 or 4 ind. ml−1 (Online Resource 2a). There were no significant interactions between fixed factors (Analysis of Deviance, x2 = 3.25, P = 0.19). For the ingestion experiment, count regression models assessed the effect of genotype and feeding chamber on the number of Artemia counted within polyps of each dissected section of a particular species. Dissected sections within samples were included as random effects, when this was found to improve the model. Overdispersion in the nauplii counts was handled by using the negative binomial as the family for the count regression. Analyses of deviance (car R-package) determined whether the two main effects (geno- type, chamber) influenced the number of Artemia consumed by each species. Paired t tests assessed differences in feeding rates as calculated by capture rate and ingestion rate values for a given chamber. Lastly, pairwise two-sample t tests, allowing for unequal mean variances, were used to analyze the mean feeding rates from both experiments where initial prey densities were comparable to 3 ind. Data analysis Artemia instar II nauplii, incubated with fluorescent micro- beads as described above, were delivered at a fixed den- sity (3 ind. ml−1) to four replicate feeding chambers per Statistical analyses were carried out in R-studio (version 1.4.1106, R Core Development Team, 2009) using a sig- nificance level of P < 0.05. Models that best explained the 1 3 3 935 Coral Reefs (2023) 42:931–943 trends in the feeding rate data, outlined below, were selected using Akaike Information Criterion (AIC). For the capture rate experiment, maximum-likelihood models were used to evaluate the effect of day and initial prey densities (ind. ml−1) on capture rates (ind. polyp−1 h−1) (car, lme4 R-pack- ages). Two sets of models were fitted. One examined the effects of coral species, day (i.e., satiation) and initial prey density on the prey capture rate and all interactions. This first model was then reduced to the simplest model, using AIC to compare alternative models. To clarify the signifi- cant interactions identified in the final model, each species was then analyzed separately. Additionally, the difference in initial and final Artemia nauplii concentrations between control chambers (Artemia only) and experimental chambers (Artemia + corals) was assessed by paired t tests. trends in the feeding rate data, outlined below, were selected using Akaike Information Criterion (AIC). For the capture rate experiment, maximum-likelihood models were used to evaluate the effect of day and initial prey densities (ind. ml−1) on capture rates (ind. polyp−1 h−1) (car, lme4 R-pack- ages). Two sets of models were fitted. One examined the effects of coral species, day (i.e., satiation) and initial prey density on the prey capture rate and all interactions. This first model was then reduced to the simplest model, using AIC to compare alternative models. To clarify the signifi- cant interactions identified in the final model, each species was then analyzed separately. Additionally, the difference in initial and final Artemia nauplii concentrations between control chambers (Artemia only) and experimental chambers (Artemia + corals) was assessed by paired t tests. Experiment I: capture rate ml−1 ± 1.5 to com- pare results derived from three calculations. For P. acuta, the average surface area of fragments was 153 ± 10 cm2, with an average of 70 ± 2 polyps cm−2 (10,762 ± 748 polyps fragment−1) (Table 1). Mean P. acuta capture rates ranged from 0 to 0.29 ind. polyp−1 h−1 (Fig. 1b). Initial and final Artemia counts in the control chambers were not significantly different (t =− 0.06, df = 34, P = 0.949) though initial counts made five min after feed delivery were higher in control chambers than in cham- bers with corals (t = 2.73, df = 30, P < 0.005). In chambers with P. acuta fragments, fewer Artemia were counted at the final compared to the initial time point (t = 3.69, df = 22, P < 0.005). Mean capture rates when delivered Artemia at 1, 2 and 4 ind. ml−1 were 0.06 ± 0.02, 0.06 ± 0.01 and 0.14 ± 0.09 ind. polyp−1 h−1, respectively. Mean capture rates across chambers on consecutive days and delivered prey levels did not differ significantly (Analysis of Deviance, x2 = 1.73, P = 0.22 and x2 = 1.33, P = 0.30, respectively). Artemia developmental stages For the capture rate experiment, the average length of deliv- ered Artemia nauplii was 508 ± 45 µm (instar I, 3 h post-har- vest). For the ingestion experiment, Artemia were delivered after 22 h post-harvest, as initial trials showed that all nauplii had undergone a first molt into instar II nauplii at this time with an average length of 735 ± 94 µm (Online Resource 1). Observations via fluorescence microscopy found that Artemia instar II nauplii ingested fluorescent microbeads and retained their gut contents 2 h post-rinsing, confirming that the fluorescent markers were not lost through excretion during the feeding trials. Galaxea fascicularis fragments had an average of 103 ± 5 polyps (Table 1). Mean G. fascicularis capture rates ranged from 1 to 110 ind. polyp−1 h−1 in experiments performed on consecutive days (Fig. 1c). The initial and final Arte- mia counts in control chambers as well as initial Artemia counts in control and experimental chambers did not dif- fer significantly (t =− 0.75, df = 52, P = 0.5 and t =− 0.04, df = 52, P = 0.96, respectively). Significantly fewer Artemia 1 3 Coral Reefs (2023) 42:931–943 936 ( ) hamber and genotype did not improve the model, however dding in section as a random effect did (Car package R) Table 2) (Fig. 2b). This is consistent with the observation Fig. 1 Relationship between capture rate and initial prey per polyp in corals fed Artemia Instar I nauplii. The x- axis shows number of prey items normalized to polyps per coral fragment (ind. polyp−1 h−1), and the y-axis is the number of prey caught per polyp over an hour from one coral within a chamber on any given day (ind. polyp−1). In G. fascicularis trials (c) there was an interaction between day and level on capture rates. The () shows an outlier where the day one capture rate was 110, and day three capture rate was 13.5, from the same coral fragment ▸ were counted for the final compared to the initial time point for chambers containing G. fascicularis (t = 2.95, df = 67, P < 0.005). Delivered prey density had a significant effect on capture rates (Analysis of Deviance, x2 = 25.04, P = < 0.001) and were found to be 16.15 ± 1.69, 26.16 ± 2.20, 43.67 ± 5.01 and 47.87 ± 9.56 ind. Experiment II: ingestion Dissection of coral polyps fed instar II Artemia nauplii with fluorescent microbeads identified variation in prey ingestion between coral species. For A. millepora, the total number of polyps assessed over the 12 fragments was 477 with an aver- age of 40 ± 2 polyps per fragment. Eight of 165 polyps (5% of total polyps) from genotype A fragments had ingested Artemia, with between 1 and 3 Artemia detected per polyp. Two of 150 polyps (1.3% of total polyps) from genotype B fragments were detected to have ingested Artemia, with 2 Artemia detected per polyp. In contrast, 45 out of 162 polyps from genotype C fragments ingested Artemia, with the number of Artemia ranging from 1 to 4 per polyp. The fragments from genotype C were observed to have full polyp extension at 10 min post-feed delivery and were noticeably paler than the fragments sourced from other adult colonies. A negative binominal count regression model was found to be the best fit for the A. millepora dataset. Genotype signifi- cantly influenced the Artemia counts in dissected A. mille- pora polyps (Analysis of Deviance, x2 = 15.6, P < 0.0005), and there was also a significant interaction between genotype and chamber (Analysis of Deviance, x2 = 29.8, P < 0.0005) (Table 2) (Fig. 2a). Dissection of coral polyps fed instar II Artemia nauplii with fluorescent microbeads identified variation in prey ingestion between coral species. For A. millepora, the total number of polyps assessed over the 12 fragments was 477 with an aver- age of 40 ± 2 polyps per fragment. Eight of 165 polyps (5% of total polyps) from genotype A fragments had ingested Artemia, with between 1 and 3 Artemia detected per polyp. A negative binominal count regression model was found to be the best fit for the A. millepora dataset. Genotype signifi- cantly influenced the Artemia counts in dissected A. mille- pora polyps (Analysis of Deviance, x2 = 15.6, P < 0.0005), and there was also a significant interaction between genotype and chamber (Analysis of Deviance, x2 = 29.8, P < 0.0005) (Table 2) (Fig. 2a). For P. acuta, data were collected from 1443 polyps within 72 sections of 12 fragments, with an average of 3.3 ± 0.1 Artemia per polyp. A negative binomial mixed-effects model with genotype and chamber as fixed effects was found to be the best fit for the P. acuta dataset. Artemia developmental stages polyp−1 h−1 for initial Artemia densi- ties of 1, 2, 4 and 8 ind. ml−1, respectively. Capture rates were not influenced by day (Analysis of Deviance, x2 = 1.58, P = 0.21), but there was a significant interaction between day and delivered prey level indicating effects of an outlier (Analysis of Deviance, x2 = 20.46, P = < 0.001). Capture rates from a single G. fascicularis fragment in one chamber equated to 110 on day one and 13.5 prey captured per polyp on day three. 1 3 Discussion Most aquarium-based feeding studies have calculated coral feeding rates from indirect capture rate methods (Petersen et al. 2008; Osinga et al. 2012b; Tagliafico et al. 2018a). However, direct approaches, such as calculating ingestion rates from prey enumerated in polyp dissections, produce more accurate feeding rates and provide insights into how prey is ingested across the entire coral (Sebens et al. 1998; Kuanui et al. 2016). Ingested prey is often difficult to dif- ferentiate from coral tissue, and visual markers are one way of mitigating this constraint. Here, visualization of ingested prey was achieved by incubating them with fluorescent microbeads prior to feeding the corals. For G. fascicularis, strong auto-fluorescence in replicate fragments taken from two out of the three adult colonies (i.e., two out of three genotypes) prevented enumeration of ingested prey because Artemia was unable to be distin- guished from the coral tissue. However, in four replicate fragments taken from a third adult colony (i.e., one geno- type), Artemia was able to be enumerated without auto-flu- orescence interfering. The average prey counts within these replicate fragments were 75 ± 13 ind. polyp−1 h−1 (n = 20 polyps). Experiment II: ingestion Effects of chamber and genotype did not improve the model, however adding in section as a random effect did (Car package R) (Table 2) (Fig. 2b). This is consistent with the observation 1 3 1 3 Coral Reefs (2023) 42:931–943 937 Table 1 Summary of mean surface area, polyp number and capture rates of corals fed Artemia instar I nauplii a Individual corals were placed in triplicate chambers per prey concentration level (ind. ml−1) and fed over three successive days b Capture rates are expressed as Mean ± S.E Speciesa Surface area (cm2) No. Polyps Conc. of nauplii (ind. ml −1) Capture rateb (ind. polyp−1 h−1) cm −2 fragment −1 Acropora millepora 84 ± 4.5 46 ± 2.4 3844 ± 219 1 0.31 ± 0.08 2 0.57 ± 0.08 4 1.22 ± 0.13 Pocillopora acuta 153 ± 10 70 ± 2 10,762 ± 748 1 0.06 ± 0.02 2 0.06 ± 0.01 4 0.14 ± 0.09 Galaxea fascicularis n/a n/a 103 ± 5 1 16.15 ± 1.70 2 26.16 ± 2.20 4 43.67 ± 5.01 8 47.87 ± 9.56 Table 1 Summary of mean surface area, polyp number and capture rates of corals fed Artemia instar I nauplii a Individual corals were placed in triplicate chambers per prey concentration level (ind. ml−1) and fed over three successive days b a Individual corals were placed in triplicate chambers per prey concentration level (ind. ml−1) and fed over three successive days b Capture rates are expressed as Mean ± S.E that Artemia were clumped within polyps and that polyps closer together consumed similar numbers of Artemia prey. Artemia clusters were typically present in the polyps furthest away from the fragment’s point of attachment to the ceramic plug (Online Resource 2b). Comparison of capture and ingestion rates Results from the capture rate experiment suggest that differ- ent coral species have varying abilities to capture delivered Artemia salina instar I nauplii. The capture rates increased with prey density level for A. millepora and P. acuta; how- ever, for these coral species the trend was only significant when capture rates were normalized to polyps per fragment. In contrast, for G. fascicularis, the delivered prey density treatment was found to be a significant factor for Artemia capture rate, without normalization based on polyp number. The fixed density levels were relatively similar (1–4 ind. ml−1), which partly explains why normalization was required for the resulting capture rates to display significant differ- ences. Our results confirm previous studies where capture rates of Artemia by corals increased with higher initial prey densities, normalized to polyps per fragment (Petersen et al. 2008; Osinga et al. 2012b; Tagliafico et al. 2018a). Further- more, given the large number of polyps per A. millepora Both capture rates and ingestion rates were determined in experiment II to enable direct comparison between these two measures of coral feeding. When normalized to the total number of polyps in the chamber (i.e., across three fragments), the measured capture and ingestion rates dif- fered significantly for A. millepora and P. acuta but were similar for G. fascicularis (Fig. 3). For A. millepora, cap- ture rates were higher than ingestion rates (tone-tail = 2.99, df = 4, P < 0.05), while the opposite was seen for P. acuta (tone-tail = − 14.84, df = 4, P < 0.0005). For G. fascicularis, capture and ingestion rates were not significantly different (tone-tail =− 0.83, df = 6, P = 0.21). For the latter coral spe- cies, the measured rates were based on counts from only one fragment per chamber due to tissue auto-fluorescence in the fragments from the other two genotypes. 1 3 938 Coral Reefs (2023) 42:931–943 (x = 3844) and P. acuta (x = 10,762) fragments, satiation was not reached at these low-density levels. Galaxea fascicularis has larger polyps and lower surface area to volume ratios than the other coral species in this study. G. fascicularis fragments in the capture rate experi- ment had on average of 103 polyps per fragment, and the delivered prey density was found to be a significant fac- tor of Artemia capture rate. G. fascicularis is a fast-feeding Fig. Fig. 2 Mean Artemia instar II nauplii per (a) Acropora mille- pora (n = 477) and (b) Pocillo- pora acuta (n = 1443) polyps in 12 fragments from four feeding chambers and three genotypes (A, B, C). Significant effects of genotype and chambers (“One,” “Two,” “Three,” “Four”) on the response variable, prey nauplii counts were assessed per species Comparison of capture and ingestion rates ml−1 are included from the first experiment (Capture Rate Experiment). Mean capture rates and mean ingestion rates from chambers delivered instar II nauplii and 3 ind. ml−1 are included from the second experiment’s chambers (Inges- tion Experiment). Significant values adjusted from pairwise Welch two-sample t tests to account for unequal variance of mean feeding rates per chamber. For Pocillopora acuta and Gal- axea fascicularis, an additional four chambers from experiment 2 pilot studies () were included for this analysis Fig. 3 Cross-study and cross- species comparison of mean feeding rates in corals derived from feeding chambers. Mean capture rates from chambers with initial instar I nauplii prey counts of 3 ± 1.5 ind. ml−1 are included from the first experiment (Capture Rate Experiment). Mean capture rates and mean ingestion rates from chambers delivered instar II nauplii and 3 ind. ml−1 are included from the second experiment’s chambers (Inges- tion Experiment). Significant values adjusted from pairwise Welch two-sample t tests to account for unequal variance of mean feeding rates per chamber. For Pocillopora acuta and Gal- axea fascicularis, an additional four chambers from experiment 2 pilot studies () were included for this analysis higher mean polyp counts per P. acuta fragments than A. millepora, calculated capture rates may not be a suitable measure for the comparison. At a delivered Artemia den- sity of 1 ind. ml−1 or less, A. millepora and P. acuta corals in this study captured 0.31 ind. polyp−1 h−1 and 0.06 ind. polyp−1 h−1, respectively, similar to 0.13 ind. polyp−1 h−1 and 0.05 ind. polyp−1 h−1 corals reported by Kuanui et al. (2016). A study by Hoogenboom et al. (2015) reported similar capture rates of Artemia nauplii delivered to Acropora sp., as reported in this study, though in contrast recorded higher capture rates for P.acuta at 188 nauplii cm−2 h−1 (approx. 18 ind. polyp−1 h−1 based on average polyp density per cm2 calculated here). The difference in the P.acuta capture rates reported between studies could be attributable to differences in experimental factors, such as flow rates, recently shown to significantly affect prey capture rates in Caribbean species (Geertsma et al. 2022). However, variable Artemia capture rates have previously been documented for Pocillopora sp. with capture rates from fragments within the same experiment varying signif- icantly based on the collection location; thus, differences could also be attributed to feeding variability between Pocillopora spp. or genotypes. (Kuanui et al. 2016). Comparison of capture and ingestion rates higher mean polyp counts per P. acuta fragments than A. millepora, calculated capture rates may not be a suitable measure for the comparison. At a delivered Artemia den- sity of 1 ind. ml−1 or less, A. millepora and P. acuta corals in this study captured 0.31 ind. polyp−1 h−1 and 0.06 ind. polyp−1 h−1, respectively, similar to 0.13 ind. polyp−1 h−1 and 0.05 ind. polyp−1 h−1 corals reported by Kuanui et al. (2016). A study by Hoogenboom et al. (2015) reported similar capture rates of Artemia nauplii delivered to Acropora sp., as reported in this study, though in contrast recorded higher capture rates for P.acuta at 188 nauplii cm−2 h−1 (approx. 18 ind. polyp−1 h−1 based on average polyp density per cm2 calculated here). The difference in the P.acuta capture rates reported between studies could be attributable to differences in experimental factors, such as flow rates, recently shown to significantly affect prey capture rates in Caribbean species (Geertsma et al. 2022). However, variable Artemia capture rates have previously been documented for Pocillopora sp. with capture rates from fragments within the same experiment varying signif- icantly based on the collection location; thus, differences could also be attributed to feeding variability between Pocillopora spp. or genotypes. (Kuanui et al. 2016). capture rates across our three experimental species are con- sistent with other aquarium-based studies. For example, fast-feeding corals, such as G. fascicularis and Duncanop- sammia axifuga, have been observed to consume over one hundred Artemia nauplii per polyp compared to autotrophic dominant, smaller polyp corals, such as A. millepora, or H. rigida, which may consume one nauplii per every ten polyps at the same prey densities (Kuanui et al. 2016; Tagliafico et al. 2018a). In the capture rate experiment, the lower mean cap- ture rates per delivered prey treatments (1, 2 and 4 ind. ml−1), observed in P. acuta trials as compared to A. mille- pora trials, was unexpected. Pocillopora spp. have been documented to consume significantly more Artemia instar I nauplii than Acropora spp. (Latyshev et al. 1991; Toh et al. 2013; Hoogenboom et al. 2015; Conlan et al. 2018; Geertsma et al. 2022). However, true P. acuta capture rates in this experiment were likely higher than calculated due to prey capture occurring between the time of delivery, and before initial counts were made five minutes later. Comparison of capture and ingestion rates 2 Mean Artemia instar II nauplii per (a) Acropora mille- pora (n = 477) and (b) Pocillo- pora acuta (n = 1443) polyps in 12 fragments from four feeding chambers and three genotypes (A, B, C). Significant effects of genotype and chambers (“One,” “Two,” “Three,” “Four”) on the response variable, prey nauplii counts were assessed per species Table 2 Analysis of Deviance table for the best fit fixed and mixed effect models of the number of Artemia salina instar II nauplii ingested by Acropora millepora and Pocillopora acuta × 2 df p valuea Acropora millepora Genotype 15.6 2 < 0.0005 Chamber 2.05 3 0.56 G h b 29 8 6 0 0005* (x = 3844) and P. acuta (x = 10,762) fragments, satiation was not reached at these low-density levels. Table 2 Analysis of Deviance table for the best fit fixed and mixed effect models of the number of Artemia salina instar II nauplii ingested by Acropora millepora and Pocillopora acuta Galaxea fascicularis has larger polyps and lower surface area to volume ratios than the other coral species in this study. G. fascicularis fragments in the capture rate experi- ment had on average of 103 polyps per fragment, and the delivered prey density was found to be a significant fac- tor of Artemia capture rate. G. fascicularis is a fast-feeding coral which requires high numbers of Artemia for satiation (Osinga et al. 2012b; Tagliafico et al. 2018a), and, except for one fragment that consumed a mean 110 ind. polyp−1 h−1 on day one and 13.5 ind. polyp−1 h−1 on day three, satiation was not observed here (see Fig. 1c). The differences in Artemia a Signif. codes: 0 ‘*’ 0.001 ‘’ 0.01 ‘’ 0.05 ‘.’ 0.1 ‘’ 1 × 2 df p valuea Acropora millepora Genotype 15.6 2 < 0.0005 Chamber 2.05 3 0.56 Genotype x chamber 29.8 6 < 0.0005* Pocillopora acuta Genotype 4.54 2 0.1 Chamber 2.83 3 0.4 Genotype x chamber 7.29 6 0.3 1 939 Coral Reefs (2023) 42:931–943 ( ) capture rates across our three experimental species are con- sistent with other aquarium-based studies. For example, fast-feeding corals, such as G. fascicularis and Duncanop- sammia axifuga, have been observed to consume over one hundred Artemia nauplii per polyp compared to autotrophic dominant, smaller polyp corals, such as A. millepora, or H. Comparison of capture and ingestion rates rigida, which may consume one nauplii per every ten polyps at the same prey densities (Kuanui et al. 2016; Tagliafico et al. 2018a). In the capture rate experiment, the lower mean cap- ture rates per delivered prey treatments (1, 2 and 4 ind. ml−1), observed in P. acuta trials as compared to A. mille- pora trials, was unexpected. Pocillopora spp. have been documented to consume significantly more Artemia instar I nauplii than Acropora spp. (Latyshev et al. 1991; Toh et al. 2013; Hoogenboom et al. 2015; Conlan et al. 2018; Geertsma et al. 2022). However, true P. acuta capture rates in this experiment were likely higher than calculated due to prey capture occurring between the time of delivery, and before initial counts were made five minutes later. This is suggested by significantly fewer Artemia counted in initial prey counts from experimental chambers than in control chambers. Additionally, given the significantly higher mean polyp counts per P. acuta fragments than A. millepora, calculated capture rates may not be a suitable measure for the comparison. At a delivered Artemia den- sity of 1 ind. ml−1 or less, A. millepora and P. acuta corals in this study captured 0.31 ind. polyp−1 h−1 and 0.06 ind. polyp−1 h−1, respectively, similar to 0.13 ind. polyp−1 h−1 and 0.05 ind. polyp−1 h−1 corals reported by Kuanui et al. (2016). A study by Hoogenboom et al. (2015) reported similar capture rates of Artemia nauplii delivered to Acropora sp., as reported in this study, though in contrast recorded higher capture rates for P.acuta at 188 nauplii cm−2 h−1 (approx. 18 ind. polyp−1 h−1 based on average polyp density per cm2 calculated here). The difference in the P.acuta capture rates reported between studies could be attributable to differences in experimental factors, such as flow rates, recently shown to significantly affect prey capture rates in Caribbean species (Geertsma et al. 2022). However, variable Artemia capture rates have previously been documented for Pocillopora sp. with capture rates from fragments within the same experiment varying signif- icantly based on the collection location; thus, differences could also be attributed to feeding variability between Pocillopora spp. or genotypes. (Kuanui et al. 2016). Fig. 3 Cross-study and cross- species comparison of mean feeding rates in corals derived from feeding chambers. Mean capture rates from chambers with initial instar I nauplii prey counts of 3 ± 1.5 ind. Comparison of capture and ingestion rates This is suggested by significantly fewer Artemia counted in initial prey counts from experimental chambers than in control chambers. Additionally, given the significantly 1 3 940 Coral Reefs (2023) 42:931–943 from the adult colony, would similarly need to consume more prey. Alternatively, polyps within the tips may benefit from higher surface area exposed to prey, increasing chances of an encounter. This clumped distribution pattern of prey ingestion cannot be observed by capture rate methods, which assume an even consumption of prey by polyps across a fragment, yet this observation has important implications. For example, it may be important to maximize the number of branch tips that have unrestricted exposure to the water column and maximize the vertical height of each fragment when arranging and attaching P. acuta fragments to a sub- strate in aquaculture. Regardless, studies repeatedly show that Pocillopora spp. readily feed and derive physiological benefits from Arte- mia as compared to unfed counterparts (Raymundo & Maypa 2004; Toh et al. 2013; Huang et al. 2020). In general, capture rates estimated using the initial prey density per milliliter of seawater result in inconsistent measures across studies. For example, capture rate calcula- tions of G. fasicularis delivered Artemia instar I nauplii at 2 ind. ml−1 produced rates of 9 ind. polyp−1 h−1 (Osinga et al. 2008), 26 ind. polyp−1 h−1 (this study) and 51 ind. polyp−1 h−1 (Ferrier-Pages et al. 2010). Similarly, when prey was delivered at 4 ind. ml−1, capture rates recorded in this study were 44 ind. polyp−1 h−1 compared to 93 ind. polyp−1 h−1 in a study by Wijgerde et al. (2011). However, normalizing the initial prey densities to the individual unit of the polyp results in more consistent capture rates across studies. For example, the capture rate calculated for G. fasicularis delivered Artemia instar I nauplii at a density of between 40 and 100 ind. polyp−1, fell between 10 and 70 ind. polyp−1 h−1 in this study, which is consistent with capture rates of 15 and 75 ind. polyp−1 h−1 by G. fascicularis in a different study, using the same initial density range (Osinga et al. 2012b). Delivered prey density does significantly affect capture rate calculations, though normalizing prey densities to individuals per polyp are likely to produce more compara- ble results across studies looking at multiple species (Online Resource 3). Comparison of capture and ingestion rates This study further highlights that factors such as geno- type and environmental conditions can influence ingestion rates. Here, this was particularly apparent for A. millepora, with two of the three A. millepora genotypes not displaying feeding responses such as extended tentacles. In these two genotypes, few dissected polyps (10 out of 315) contained ingested prey. In contrast, 45 out of 162 polyps from the third genotype (genotype ’C’ in Fig. 2a) contained ingested prey although the proportion of polyps with and without prey varied between fragments, likely explaining the signifi- cant interaction between genotype and chamber on Artemia counts. Notably, this third genotype contained fragments that were visually paler, indicating a possible stress response. Additionally, these A.millepora fragments were the only ones observed to have fully extended tentacles at 10 min post-delivery. Stressed corals displaying bleaching signs may compensate for lowered autotrophic energetic acquisi- tion through increasing heterotrophic feeding (Hughes & Grottoli 2013). Increased assimilation of heterotrophic car- bon has been observed in bleached Montipora capitata and Porites lobata compared to unbleached controls (Hughes & Grottoli 2013), and a similar mechanism may be at play here. Low feeding rates of Acropora spp. on Artemia instar I nau- plii have been postulated to be due to their higher depend- ence on autotrophic carbon and small polyp size (Tagliafico et al. 2018a, b). By this reasoning, A. millepora corals would be less capable of ingesting larger instar II nauplii. Our results indicate that, although possible for A. millepora to consume the larger Artemia instar II nauplii, ingestion rates are variable and depend on complex genotype and holobiont health factors, such as the need to compensate for lost nutri- tion during times of stress. Use of visual tools to assess prey ingestion Determining the ingestion rate of Artemia through gut dis- sections facilitated by fluorescent markers provides a more accurate assessment of feeding. This study confirmed that different coral species have different feeding rates on the same prey (A. millepora, 0.2; P. acuta 3.5; G. fascicularis, 75 ind. polyp−1 h−1 at 3 ind. ml−1) and showed species-spe- cific patterns in how prey is ingested across the fragment, such as the non-homogenous distribution of ingested prey between polyps observed in P. acuta. Pocillopora acuta fragments had clusters of ingested prey in polyps located in the branch tips, and fewer clusters were found in polyps near the point of adhesion to the coral plug, although the mean number of prey ingested per polyp did not differ across replicate fragments. Previous studies have demonstrated that polyps in different colony branch posi- tions exhibit significantly different nutritional profiles with high-energy nutrients catabolized more readily at the edges of colonies, such as branch tips (Conlan et al. 2018). A simi- lar mechanism could be at play in the P. acuta fragments, where the polyps at the tips need to consume more prey to meet the higher nutritional demands of proliferating cells. However, if this were the case, actively growing polyps at the base of the fragment, adjacent to the site of excision References Anthony KRN, Fabricius KE (2000) Shifting roles of heterotrophy and autotrophy in coral energetics under varying turbidity. J Exp Mar Biol Ecol 2:221–253 Understanding species-specific feeding behavior and the amount of prey corals ingest is critically important for the development of improved feeding regimes in coral aquaculture. We recommend that future coral feeding stud- ies utilize visual markers to assess feeding rates in corals fed live prey items. The potential health benefit derived from enriching live feeds depends on the ability of a coral to capture, ingest, digest and furthermore, assimilate nutri- ents from the feed. Therefore, this approach is particularly suitable for live feeds that are also suitable for nutritional enrichment. Here, we were able to confirm and compare the capture and ingestion rates of Artemia salina nauplii using fluorescent microbeads allowing for the direct detec- tion of prey within individual polyps which allowed stand- ardization across studies and comparison between coral species. Corals possess diverse feeding strategies, physi- ologies and varying degrees of reliance on autotrophic and heterotrophic nutrient acquisition pathways as seen in here and other studies. Future studies could expand the proto- cols established herein to assess feeding abilities and prey preferences to cater to the nutritional needs of different corals and develop appropriate nutritional supplementation regimes, required for the expansion of coral aquaculture. Barton JL, Willis B, Hutson K (2015) Coral propagation: A review of techniques for ornamental trade and reef restoration. Rev Aquac 22:1–19 Borneman E (2009) Aquarium corals: selection, husbandry, and natural history. TFH Publications, New Jersey, USA Bott R (2014) Brock biology of microorganisms. Pearson Education Limited, Londoni Brafield AE, Llewellyn MJ (1982) Animal energetics. Springer, New York Conlan JA, Bay LK, Severati A, Humphrey C, Francis DS (2018) Com- paring the capacity of five different dietary treatments to optimise growth and nutritional composition in two scleractinian corals. PLoS ONE. https://doi.org/10.1371/journal.pone.0207956 Conti-Jerpe IE, Thompson PD, Wong CWM, Oliveira NL, Duprey NN, Moynihan MA, Baker DM (2020) Trophic strategy and bleach- ing resistance in reef-building corals. Sci Adv. https://doi.org/10. 1126/sciadv.aaz5443i Ekonomou G, Lolas A, Castritsi-Catharios J, Neofitou C, Zouganelis G, Tsiropoulos N, Exadactylos A (2019) Mortality and Effect on growth of artemia franciscana exposed to two common organic pollutants. Water. https://doi.org/10.3390/w11081614 Ezzat L, Towle E, Irisson JO, Langdon C, Ferrier-Pagès C (2016) The relationship between heterotrophic feeding and inorganic nutrient availability in the scleractinian coral T. reniformis under a short- term temperature increase. Declarations Conflict of interest On behalf of all authors, the corresponding au- thor states that there is no conflict of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adap- tation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Development of feeding assessment methods Because the auto-fluorescence overlapped with the signal emitted from the microbeads, it obscured differentiation between ingested prey and coral tissue and therefore pre- vented accurate counts of ingested prey. These observa- tions highlight the need to select markers that maximize contrast with auto-fluorescent pigments present in the coral species and genotypes under study. Funding Open Access funding enabled and organized by CAUL and its Member Institutions. Development of feeding assessment methods Whereas the capture rate approach is a quick and effi- cient metric, a coral’s feeding behavior can influence the efficacy of this assessment method. In this study, capture and ingestion rates were calculated from the same feed- ing event enabling side-by-side comparison, with the mean values calculated by the two approaches differing 3 3 941 Coral Reefs (2023) 42:931–943 significantly for A. millepora and P. acuta. For A. mille- pora, secretion of high volumes of mucus, which capture and cause agglomeration of Artemia (Huettel et al. 2006; Naumann et al. 2009), could explain why the calculated capture rates were significantly higher than the ingestion rates. In contrast, P. acuta capture rates were lower than ingestion calculations, though the reason for this pattern is unclear. It may be associated with uneven distribution of feeding polyps across the colony, resulting in a bias dependent on which polyps were chosen to be dissected. For G. fascicularis, there was no significant difference between the capture rate and ingestion rate calculations. This could be related to the low sample size for the inges- tion rate calculations resulting from the high auto-fluores- cent tissues of two of the three G. fascicularis genotypes. Because the auto-fluorescence overlapped with the signal emitted from the microbeads, it obscured differentiation between ingested prey and coral tissue and therefore pre- vented accurate counts of ingested prey. These observa- tions highlight the need to select markers that maximize contrast with auto-fluorescent pigments present in the coral species and genotypes under study.i significantly for A. millepora and P. acuta. For A. mille- pora, secretion of high volumes of mucus, which capture and cause agglomeration of Artemia (Huettel et al. 2006; Naumann et al. 2009), could explain why the calculated capture rates were significantly higher than the ingestion rates. In contrast, P. acuta capture rates were lower than ingestion calculations, though the reason for this pattern is unclear. It may be associated with uneven distribution of feeding polyps across the colony, resulting in a bias dependent on which polyps were chosen to be dissected. For G. fascicularis, there was no significant difference between the capture rate and ingestion rate calculations. This could be related to the low sample size for the inges- tion rate calculations resulting from the high auto-fluores- cent tissues of two of the three G. fascicularis genotypes. References Limnol Oceanogr. https://doi.org/10. 1002/lno.10200 Ferrier-Pages C, Rottier C, Beraud E, Levy O (2010) Experimental assessment of the feeding effort of three scleractinian coral species during a thermal stress: Effect on the rates of photosynthesis. J Exp Mar Biol Ecol 390:118–124 Acknowledgements This study was made possible by grants pro- vided by AIMS@JCU, the College of Science and Engineering at James Cook University and in-kind support from the National Sea Simulator at the Australian Institute of Marine Science. Furthermore, we would like to thank the National Sea Simulator personnel, Tom Barker, Loni Koukoumaftsis and Andrea Severati and volunteer, Nata- lia Robledo, for technical assistance and support. Lastly, Rhondda Jones is thanked for her assistance with statistical analyses. Geertsma RC, Wijgerde T, Latijnhouwers KRW, Chamberland VF (2022) Onset of zooplanktivory and optimal water flow rates for prey capture in newly settled polyps of ten Caribbean coral spe- cies. Coral Reefs 41:1651–1664 Goulden EF, Hall MR, Bourne DG, Pereg LL, Høj L (2012) Patho- genicity and infection cycle of Vibrio owensii in larviculture of 1 3 942 Coral Reefs (2023) 42:931–943 the ornate spiny lobster (Panulirus ornatus). Appl Environ Micro- biol 78:2841–2849 pelagic–benthic coupling in the reef ecosystem. Mar Ecol Prog Ser 385:65–76fifi Osinga R, Schutter M, Griffioen B, Wijffels RH, Verreth JAJ, Shafir S, Henard S, Taruffi M, Gili C, Lavorano S (2011) The biology and economics of coral growth. Mar Biotechnol 13:658–671i Hall NM, Berry KLE, Rintoul L, Hoogenboom MO (2015) Micro- plastic ingestion by scleractinian corals. Mar Biol 162:725–732 Hii YS, Soo CL, Liew HC (2009) Feeding of scleractinian coral, Gal- axea fascicularis, on Artemia salina nauplii in captivity. Aquac Int 17:363–376 Osinga R, Schutter M, Wijgerde T, Rinkevich B, Shafir S, Shpigel M, Marco Luna G, Danovaro R, Bongiorni L, Deutsch A, Kücken M, Hiddinga B, Janse M, McLeod A, Gili C, Lavorano S, Henard S, Barthelemy D, Westhoff G, Laterveer M (2012a) The CORAL- ZOO project: A synopsis of four years of public aquarium science. Mar Biol Assoc UK 92:753–768 Hoogenboom M, Rodolfo-Metalpa R, Ferrier-Pagès C (2010) Co-var- iation between autotrophy and heterotrophy in the Mediterranean coral Cladocora caespitosa. J Exp Biol 14:2399–2409 Hoogenboom M, Rottier C, Sikorski S, Ferrier-Pagès C (2015) Among-species variation in the energy budgets of reef-building corals: scaling from coral polyps to communities. 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https://openalex.org/W2053052032	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0073633&type=printable	English	null	Restoration of the Normal Splicing Pattern of the PLP1 Gene by Means of an Antisense Oligonucleotide Directed against an Exonic Mutation	PloS one	2,013	cc-by	5,639	Introduction Pelizaeus–Merzbacher disease (PMD, MIM #312080) and X- linked paraplegia type 2 (SPG2, MIM #312920) are allelic hypomyelinating leukodystrophies caused by mutations in the PLP1 gene (MIM #300401). The resulting clinical phenotypes vary quite widely, ranging from the most severe connatal phenotype, presenting at birth with nystagmus, severe spasticity and hypotonia, to relatively mild cases of paraplegia without mental retardation [1]. Despite their abundance in CNS myelin, myelination occurs in knockout mice lacking the Plp1 gene, even though the myelin produced exhibits reduced physical stability [5]. In similar vein, male PMD patients harbouring a complete PLP1 gene deletion are relatively mildly affected [1]. However, male PMD patients with a duplication of the PLP1 gene exhibit a more severe phenotype, with severity increasing in the rarer PMD patients with three or five PLP1 gene copies [6]. Point mutations can give rise to a wide range of PMD/SPG2 phenotypes, the most severe being those that impair PLP and DM20 folding and transport [7], whilst mutations with a limited impact on PLP/DM20 protein structure are predicted to lead to milder phenotypes. Among these mild mutations are those that are located in exon 3B and which therefore involve PLP but not DM20 [3]. Duplication of a region of chromosome Xq22.2 containing the PLP1 gene is the most frequent gene defect reported in PMD (60-70% of cases). The complete deletion of the gene has been reported only rarely as a cause of PMD/SPG2, while point mutations have been identified in 20% of cases [2]. The PLP1 gene, which is mainly expressed in oligodendrocytes, encodes a 4-pass transmembrane protein, termed PLP, which is the most abundant protein in the myelin sheaths of the central nervous system (CNS) [3]. A shorter protein isoform, DM20, which lacks 35 amino acids from an intracellular domain, is however generated by alternative splicing of the same primary PLP1 gene transcript. Two competing 5’ donor splice sites, residing within the 3’ portion of exon 3 (commonly termed exon 3B) are responsible for the alternative splicing [4]. The We previously reported a mildly affected PMD patient with a c.436C>G mutation located in exon 3B, and demonstrated that this mutation, which would be expected to substitute leucine at residue 146 with a valine (p.L146V), resulted instead in the loss of the PLP isoform [8]. Stefano Regis1, Fabio Corsolini1, Serena Grossi1, Barbara Tappino1, David N. Cooper2, Mirella Filocamo1* Stefano Regis1, Fabio Corsolini1, Serena Grossi1, Barbara Tappino1, David N. Cooper2, Mirella Filocamo1* g 1 Centro di Diagnostica Genetica e Biochimica delle Malattie Metaboliche, Istituto G. Gaslini, Genova, Italy, 2 Institute of Medical Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom 1 Centro di Diagnostica Genetica e Biochimica delle Malattie Metaboliche, Istituto G. Gaslini, Genova, Italy, 2 Institute of Medical Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom Abstract An exonic missense mutation, c.436C>G, in the PLP1 gene of a patient affected by the hypomyelinating leukodystrophy, Pelizaeus–Merzbacher disease, has previously been found to be responsible for the alteration of the canonical alternative splicing profile of the PLP1 gene leading to the loss of the longer PLP isoform. Here we show that the presence of the c.436C>G mutation served to introduce regulatory motifs that appear to be responsible for the perturbed splicing pattern that led to loss of the major PLP transcript. With the aim of disrupting the interaction between the PLP1 splicing regulatory motifs and their cognate splicing factors, we designed an antisense oligonucleotide-based in vitro correction protocol that successfully restored PLP transcript production in oligodendrocyte precursor cells. Citation: Regis S, Corsolini F, Grossi S, Tappino B, Cooper DN, et al. (2013) Restoration of the Normal Splicing Pattern of the Antisense Oligonucleotide Directed against an Exonic Mutation. PLoS ONE 8(9): e73633. doi:10.1371/journal.pone.0073633 Editor: Ralf Krahe, University of Texas MD Anderson Cancer Center, United States of America Editor: Ralf Krahe, University of Texas MD Anderson Cancer Center, United States of America Received February 25, 2013; Accepted July 30, 2013; Published September 3, 2013 Copyright: © 2013 Regis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Italian Health Department ‘Finanziamento Cinque per mille e Ricerca Corrente’ and FP7-HEALTH, LeukoTreat no.241622. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Funding: This work was supported by grants from the Italian Health Department ‘Finanziamento Cinque per mille e Ricerca Corrente’ and FP7-HEALTH, LeukoTreat no.241622. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: mirellafilocamo@ospedale-gaslini.ge.it biological roles of the PLP/DM20 proteins remain to be clarified. Restoration of the Normal Splicing Pattern of the PLP1 Gene by Means of an Antisense Oligonucleotide Directed against an Exonic Mutation Stefano Regis1, Fabio Corsolini1, Serena Grossi1, Barbara Tappino1, David N. Cooper Stefano Regis1, Fabio Corsolini1, Serena Grossi1, Barbara Tappino1, David N. Cooper2, Mirella Filocamo1* Introduction Although none of the regulatory splice sites known to be involved in PLP/DM20 alternative splicing regulation [9–11] was directly altered by the mutation, 1 PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e73633 Oligonucleotide-Mediated Splicing Restoration CGCGGGCCCTCTAGACTCGA -3’, according to Grossi et al. [8]. RT-PCR from non-specifically primed reverse transcription reactions was performed using both minigene-specific primers 31GF: 5’- TGATTTCAGCCGCGCTGTACTGG-3’ and LACT2R [8]. computational analysis suggested that c.436C>G leads to the acquisition of exon splicing silencer (ESS) motifs [8]. In the present report, we have extended this analysis by identifying several splicing regulatory motifs created by the c.436C>G mutation. In vitro experimentation subsequently confirmed the predictions of the in silico hypotheses and suggested the therapeutic potential of a specific antisense oligonucleotide to correct the splicing defect by blocking the splicing regulatory motifs. To evaluate PLP transcript expression in morpholino-treated Oli-neu cells transfected with the mutant plasmid construct, we performed real-time PCR on cDNA reverse transcribed from LACT2R-primed RNA using a PLP transcript-specific primer- TaqMan probe set encompassing PLP1 exons 3 and 4 (P2), and a PLP/DM20 transcripts-specific primer-TaqMan probe set encompassing PLP1 exons 2 and 3 (P23B). Set P2 was used as target, whereas set P23B was used as a reference [8]. Using this experimental approach, we were able to derive the PLP/(DM20+PLP) ratio for cells treated with the morpholino antisense oligonucleotide. cDNA samples, reverse transcribed from LACT2R-primed RNAs, were run in quadruplicate, each well containing the cDNA obtained from 10 ng RNA. A plasmid containing the cDNA corresponding to PLP transcript isoform was used to generate the standard curves for the P2 and the P23B amplicons, respectively. Standards were run in duplicate. Standard wells contained 10-2, 10-3, 10-4, 10-5, 10-6 and 10-7 ng/µl plasmid DNA. GTTTGGGAAAATGGGAAGGACATCCCGACAAG-3’ and 146UPR: 5’- All patient samples were obtained from the “Cell Line and DNA Biobank from Patients Affected by Genetic Diseases” (G. Gaslini Institute, Genoa) and processed as previously described [8]. CTTGTCGGGATGTCCTTCCCATTTTCCCAAAC-3’) were used to introduce a further mutation (c.437T>A) adjacent to the c.436C>G in the mutant plasmid. Thus, three versions of the construct were used: the wild-type version (TGGCTAGG), the naturally occurring mutant version (TGGGTAGG), and the double mutant version (TGGGAAGG). Plasmid constructs, transfection and transcript analysis Plasmid constructs, derived from the pcDNA3.1/V5-His- TOPO/LacZ vector (Invitrogen, San Diego, CA), with a recombinant in-frame LacZ-PLP1-LacZ minigene containing the genomic region between exons 2 and 4 of the PLP1 gene, were previously prepared as wild-type and mutant (c.436C>G) versions [8]. In the present study, a double mutant construct was produced using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene Agilent, Santa Clara, CA, USA). In accordance with the manufacturer’s instructions, specific primers (146UPF: 5’- Ethical aspects Following ethical guidelines, all cell and nucleic acid samples stored in the Biobank were obtained for analysis and storage with the patients’ (and/or a family member’s) written informed consent. Consent was sought using a form approved by local Ethics Committee. Wild-type and mutant plasmid constructs were transfected into murine oligodendroglial Oli-neu cells [12] using the Lipofectamine 2000 Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. SRSF6-specific siRNA (Ambion, Austin, TX, USA) was cotransfected with the mutant (c.436G) minigene into Oli-neu cells using the lipofectamine 2000 Transfection Reagent (Invitrogen) according to the manufacturer’s guidelines. A morpholino antisense oligonucleotide (Gene Tools, Philomath, OR, USA) (5’-GATGTCCTACCCATTTTCCCAAACA-3’) was designed by the Gene Tools Oligo Design Support based upon the target sequence surrounding the c.436G mutation. When morpholino treatment was performed, various amounts of morpholino oligonucleotide were added with the EndoPorter peptide delivery system (Gene Tools) to the c.436G mutant minigene- transfected Oli-neu cells as detailed in the Results and Discussion section. Patient samples p ( GTTTGGGAAAATGGGAAGGACATCCCGACAAG-3’ and 146UPR: 5’- Results and Discussion In a previously studied PMD patient, we identified a PLP1 gene mutation, c.436C>G, located within exon 3B, the PLP- specific region [8]. Since no RNA sample was available from the patient, the functional effect of the c.436C>G mutation was investigated using a recombinant LacZ-PLP1-LacZ in-frame minigene containing a PLP1 gene exon 2 – exon 4 fragment cloned into the pcDNA3.1/V5-His-TOPO/LacZ vector. Comparison of the mRNAs generated through the transfection of the wild-type (c.436C) and mutated (c.436G) versions of the construct into Oli-neu cells, demonstrated that the c.436C>G, expected to result in a missense p.L146V mutation, led instead to the loss of the PLP transcript encoding the major isoform of the PLP1 gene, whereas the shorter DM20 version of the PLP1 transcript was produced normally [8]. Transfected cells were harvested 48 hrs after transfection. RNA was extracted using an RNeasy Plus Mini kit (Qiagen, Valencia, CA, USA) and reverse transcribed using the SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen). To avoid reverse transcription of endogenous Plp1 gene transcripts from the Oli-neu cells, when specific priming was required, first-strand cDNA synthesis was performed using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen), using a minigene-specific primer, LACT2R: 5’- To investigate the apparent association between the c. 436C>G mutation and the observed alteration in the mRNA splicing phenotype, we analyzed the mutated region in silico to ascertain whether the mutation was responsible for the gain or loss of splicing regulatory motifs. The ESEfinder 3.0 program PLOS ONE \| www.plosone.org 2 September 2013 \| Volume 8 \| Issue 9 \| e73633 Oligonucleotide-Mediated Splicing Restoration Figure 1. Schematic representation of the analysis of PLP1 gene structure with the ESEfinder, Human Splicing Finder and FAS-ESS programs. (a) Schematic representation of PLP1 gene structure (7 exons) and the alternative splicing of the PLP1 gene into the major PLP1 transcript and a minor transcript encoding the shorter DM20 isoform, which differs only in terms of the latter half of exon 3 (3B) which is post-transcriptionally spliced out. (b) Only part of the sequence of exon 3A (sequence in black type) and exon 3B (sequence in red type) mutant PLP1 transcript is represented. The mutated nucleotide (c.436C>G) is underlined. According to the ESEfinder 3.0 program, the c.436C>G change created an exonic splicing enhancer (EES, green trapezium), whereas Human Splicing Finder version 2.4.1 and FAS-ESS web server predicted the creation of two exonic splicing silencers (ESS, purple trapezia). Results and Discussion doi: 10 1371/journal pone 0073633 g001 Figure 1. Schematic representation of the analysis of PLP1 gene structure with the ESEfinder, Human Splicing Finder and FAS-ESS programs. (a) Schematic representation of PLP1 gene structure (7 exons) and the alternative splicing of the PLP1 gene into the major PLP1 transcript and a minor transcript encoding the shorter DM20 isoform, which differs only in terms of the latter half of exon 3 (3B) which is post-transcriptionally spliced out. (b) Only part of the sequence of exon 3A (sequence in black type) and exon 3B (sequence in red type) mutant PLP1 transcript is represented. The mutated nucleotide (c.436C>G) is underlined. According to the ESEfinder 3.0 program, the c.436C>G change created an exonic splicing enhancer (EES, green trapezium), whereas Human Splicing Finder version 2.4.1 and FAS-ESS web server predicted the creation of two exonic splicing silencers (ESS, purple trapezia). doi: 10 1371/journal pone 0073633 g001 doi: 10.1371/journal.pone.0073633.g001 [13,14] predicted that the c.436C>G mutation would create an exonic splice enhancer (ESE, sequence TGGGTA, mutation site underlined) specific for the SRSF6 splicing factor with a score (2.70848) that was above the threshold level (2.676); the corresponding score for the wild-type sequence (TGGCTA, score: 0.40451) was considerably lower (Table 1 and Figure 1). SRSF6 (also known as Srp55) is a splicing factor belonging to the SR protein family which, by binding to ESEs, helps to ensure the correct 5’ to 3’ linear order of exons in spliced mRNA and also prevents exon skipping [15]. It is therefore likely that SRSF6, by acting negatively on the splicing of exon 3B, plays a role in promoting PLP transcript production. The Human Splicing Finder program (version 2.4.1) [16] and the FAS-ESS web server [17] predicted the creation of two exonic splice silencers (ESS), more specifically two FAS-hex3 hexamers (GGGTAG and GGTAGG) [16] (Figure 1). FAS-hex3 hexamers are potential exonic splicing silencer motifs [17]. They were found to be contained (and were overrepresented) in decamers selected from a decamer random library by a cell- based splicing reporter system specifically designed to detect sequences with splicing silencer activity [17]. Fas-hex3 [13,14] predicted that the c.436C>G mutation would create an exonic splice enhancer (ESE, sequence TGGGTA, mutation site underlined) specific for the SRSF6 splicing factor with a score (2.70848) that was above the threshold level (2.676); the corresponding score for the wild-type sequence (TGGCTA, score: 0.40451) was considerably lower (Table 1 and Figure 1). Results and Discussion SRSF6 (also known as Srp55) is a splicing factor belonging to the SR protein family which, by binding to ESEs, helps to ensure the correct 5’ to 3’ linear order of exons in spliced mRNA and also prevents exon skipping [15]. It is therefore likely that SRSF6, by acting negatively on the splicing of exon 3B, plays a role in promoting PLP transcript production. The Human Splicing Finder program (version 2.4.1) [16] and the FAS-ESS web server [17] predicted the creation of two exonic splice silencers (ESS), more specifically two FAS-hex3 hexamers (GGGTAG and GGTAGG) [16] (Figure 1). FAS-hex3 hexamers are potential exonic splicing silencer motifs [17]. They were found to be contained (and were overrepresented) in decamers selected from a decamer random library by a cell- based splicing reporter system specifically designed to detect sequences with splicing silencer activity [17]. Fas-hex3 Table 1. Output of ESEfinder program when the wild-type and mutated PLP1 exon 3B sequences are compared. Sequence Splicing factor motif Score Threshold score TGGCTA (Wt) SRSF6 0.40451 2.676 TGGGTA (M) 2.70848 TGGGAA (DM) 0.10328 Legend: the mutated nucleotides are underlined; Wt = wild-type; M = mutant; DM = double mutant. Table 1. Output of ESEfinder program when the wild-type and mutated PLP1 exon 3B sequences are compared. hexamers could therefore be responsible for the loss of the PLP transcript isoform. By means of in silico analysis, we determined that the identified regulatory motifs (ESE, TGGGTA and ESS, GGGTAG and GGTAGG), could be functionally altered by introducing a second mutation (c.437T>A) immediately adjacent to c.436C>G. Indeed, the resulting double mutant (TGGGAA, GGGAAG and GGAAGG) not only abolished the FAS-hex3 hexamer ESSs but also led to a significant reduction PLOS ONE \| www.plosone.org 3 September 2013 \| Volume 8 \| Issue 9 \| e73633 Oligonucleotide-Mediated Splicing Restoration Figure 2. RT-PCR from Oli-neu cells transfected with the wild-type and mutated minigenes. Lane U: untransfected cells; lane WT: cells transfected with the wild-type minigene construct; lane SM: cells transfected with the minigene construct harbouring the c. 436C>G mutation; lane DM: cells transfected with the minigene construct harbouring the double mutation [c.436C>T plus c. 437T>A]; lane C: negative (no template) control; lane M: φX174 DNA HaeIII-restricted molecular weight marker. The minigene- specific primer 31GF/LACT2R-mediated RT-PCR products are 711 bp (PLP product) and 606 bp (DM20 product) in length, respectively. The asterisk denotes samples transfected with the pcDNA3.1/V5-His-TOPO/LacZ-PLP1-LacZ vector. doi: 10.1371/journal.pone.0073633.g002 Figure 2. Results and Discussion RT-PCR from Oli-neu cells transfected with the wild-type and mutated minigenes. Lane U: untransfected cells; lane WT: cells transfected with the wild-type minigene construct; lane SM: cells transfected with the minigene construct harbouring the c. 436C>G mutation; lane DM: cells transfected with the minigene construct harbouring the double mutation [c.436C>T plus c. 437T>A]; lane C: negative (no template) control; lane M: φX174 DNA HaeIII-restricted molecular weight marker. The minigene- specific primer 31GF/LACT2R-mediated RT-PCR products are 711 bp (PLP product) and 606 bp (DM20 product) in length, respectively. The asterisk denotes samples transfected with the pcDNA3.1/V5-His-TOPO/LacZ-PLP1-LacZ vector. doi: 10.1371/journal.pone.0073633.g002 On the basis of this working hypothesis, we next investigated the possibility of correcting in vitro the splicing defect caused by the c.436G mutation by preventing the interaction of the identified consensus sequences with their cognate splicing factors. To this end, we employed an antisense oligonucleotide designed to target the mutated region. When the antisense oligonucleotide, specifically a morpholino oligonucleotide, was introduced into Oli-neu cells transfected with the mutant plasmid, restoration of PLP-transcript isoform expression was observed. Although transcript rescue was not effectively achieved using less than 10µm morpholino oligonucleotide (not shown), satisfactory results were obtained with 10µM morpholino delivered with different amounts of Endoporter transfection reagent (from 2 to 10 µM) (Figure 3). in the ESE score (Table 1). Thus, the newly introduced c. 437T>A mutation would be predicted to abolish the effect of the c.436C>G mutation on splicing thereby potentially restoring PLP transcript production. To experimentally confirm this postulate, we prepared by site-specific mutagenesis a double- mutant version of the recombinant minigene construct and transfected the three versions of the minigene [(wild-type (TGGCTAGG), single mutant (TGGGTAGG) and double- mutant (TGGGAAGG)] into Oli-neu cells. As shown in Figure 2, the PLP transcript, which was present in cells transfected with the wild-type minigene but absent in cells transfected with the single mutant minigene, was expressed in cells transfected with the double-mutant minigene. This served to confirm the direct relationship between the presence of the c.436C>G mutation-introduced regulatory motifs and the loss of the PLP transcript isoform. in the ESE score (Table 1). Thus, the newly introduced c. 437T>A mutation would be predicted to abolish the effect of the c.436C>G mutation on splicing thereby potentially restoring PLP transcript production. Results and Discussion To experimentally confirm this postulate, we prepared by site-specific mutagenesis a double- mutant version of the recombinant minigene construct and transfected the three versions of the minigene [(wild-type (TGGCTAGG), single mutant (TGGGTAGG) and double- mutant (TGGGAAGG)] into Oli-neu cells. As shown in Figure 2, the PLP transcript, which was present in cells transfected with the wild-type minigene but absent in cells transfected with the single mutant minigene, was expressed in cells transfected with the double-mutant minigene. This served to confirm the direct relationship between the presence of the c.436C>G mutation-introduced regulatory motifs and the loss of the PLP transcript isoform. To evaluate quantitatively the morpholino-induced PLP isoform transcript production, we performed real-time PCR experiments using a primer-TaqMan probe-set specific for the PLP transcript (P2) and a primer-TaqMan probe-set specific for both the PLP and DM20 transcripts (P23B), as previously described [8]. Following this procedure, we determined the PLP/(PLP+DM20) ratio for the morpholino-treated Oli-neu cells tranfected with the mutant construct; as shown in Table 2, we obtained PLP/(PLP+DM20) ratios ranging from 0.079 to 0.122. These values compare with a PLP/(PLP+DM20) ratio of 0.212 for Oli-neu cells transfected with the wild-type construct. Thus, employing the morpholino oligonucleotide, we succeeded in increasing the PLP/(DM20+PLP) ratio to a level which was 58% of the wild–type ratio. Although the presence of the PLP and DM20 protein isoforms could not be directly confirmed experimentally, we suspect that the translation of the detected Having analyzed separately the potential effects of the ESE and the ESS motifs, we surmise that SRSF6, by binding to the ESE site, would direct the mRNA splicing phenotype towards the PLP isoform. To test this hypothesis experimentally, we cotransfected the mutant c.436C>G minigene construct and an SRSF6-specific siRNA into Oli-neu cells. As predicted, the mRNA splicing pattern, lacking the PLP transcript isoform, was unmodified by the siRNA-mediated inhibition of the SRSF6- ESE interaction (Figure S1). These results were consistent with the hypothesis that the observed impact of the c.436C>G mutation on splicing, leading to the loss of the PLP transcript isoform, was mediated by the ESS motifs. We therefore concluded that it was likely that the two newly created ESS motifs played a role in the loss of the PLP isoform. PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e73633 4 Oligonucleotide-Mediated Splicing Restoration Figure 3. Analysis of morpholino treatment: RT-PCR from minigene-transfected morpholino-treated Oli-neu cells. Results and Discussion RNA was extracted from cells transfected with the wild-type (Wt) and mutant (c.436C>G) minigene constructs (Mut, 2, 4, 6, 8, 10). Samples 2, 4, 6, 8 and 10 were treated with the morpholino oligonucleotide, whereas samples Mut and Wt were untreated. Treated cells received 10 mM morpholino oligonucleotide in 2, 4, 6, 8, 10 mM Endoporter reagent; lane C: no template control; lane M: φX174 DNA HaeIII restricted molecular weight marker. The minigene-specific primer 31GF/LACT2R-mediated RT-PCR products are 711 bp (PLP product) and 606 bp (DM20 product) in length, respectively. doi: 10.1371/journal.pone.0073633.g003 Figure 3. Analysis of morpholino treatment: RT-PCR from minigene-transfected morpholino-treated Oli-neu cells. RNA was extracted from cells transfected with the wild-type (Wt) and mutant (c.436C>G) minigene constructs (Mut, 2, 4, 6, 8, 10). Samples 2, 4, 6, 8 and 10 were treated with the morpholino oligonucleotide, whereas samples Mut and Wt were untreated. Treated cells received 10 mM morpholino oligonucleotide in 2, 4, 6, 8, 10 mM Endoporter reagent; lane C: no template control; lane M: φX174 DNA HaeIII restricted molecular weight marker. The minigene-specific primer 31GF/LACT2R-mediated RT-PCR products are 711 bp (PLP product) and 606 bp (DM20 product) in length, respectively. doi: 10.1371/journal.pone.0073633.g003 the likely clinical severity associated with the hypothetical PLP p.L146V mutation as compared with the complete absence of the PLP isoform. The p.L146V mutation would be located within the cytoplasmic loop of PLP. The analysis of the extent of evolutionary conservation of this loop, flanking the Leu 146 residue, in 7 orthologous PLP proteins showed that it has been conserved in amphibians but not in fish, suggesting that the PLP isoform is typical of higher vertebrates (Table S1) [19]. In general, mutations located within this loop, and, in particular, mutations located in amino acid residues in the vicinity of Leu146, are not associated with severe clinical phenotypes. Indeed, the closest reported missense mutations, H147Y [20] and H140Y [21], both of which are predicted to lead to the production of a mutant PLP isoform, have been reported in patients affected by a relatively mild SPG2 clinical phenotype. Table 2. Real-time RT-PCR analysis of morpholino-treated Oli-neu cells transfected with mutated (Mut) and wild-type (Wt) PLP1 minigene constructs. Table 2. Real-time RT-PCR analysis of morpholino-treated Oli-neu cells transfected with mutated (Mut) and wild-type (Wt) PLP1 minigene constructs. Results and Discussion Mut 2 4 6 8 10 Wt PLP/(PLP+DM20) ratio 0.002 0.122 0.094 0.079 0.094 0.116 0.212 REC % 0.9 58 44 37 44 55 100 Legend: Columns 2, 4, 6, 8 and 10 indicate samples treated with 10 µM morpholino oligonucleotide in 2, 4, 6, 8 and 10 µM Endoporter reagent, respectively. The PLP/(PLP+DM20) ratio, obtained using the P2 and P23B sets of primers and probes, is reported for each sample. Rec % denotes the proportion of recovered PLP1 transcript expression as compared to the wild-type. PLP and DM20 transcript might occur without blocking, owing to the mRNA-morpholino interactions, as the region involved is largely downstream of the ATG (approx. 1.9kb) translational start site in the LacZ-PLP1-LacZ minigene. It is already known that the blocking efficiency of morpholino oligonucleotides falls dramatically as the distance of the binding region from the initial ATG increases [18]. It should be noted that these results were obtained using Oli-neu cells as recipients in all the transfection experiments. The use of these mouse oligodendrocyte precursor cells was deemed appropriate with a view to mimicking the cellular environment in which PMD displays its effects, and also avoiding potential biases in splicing selection due to the action of tissue-specific factors. Th f l li l tid di t d t ti f th PLP and DM20 transcript might occur without blocking, owing to the mRNA-morpholino interactions, as the region involved is largely downstream of the ATG (approx. 1.9kb) translational start site in the LacZ-PLP1-LacZ minigene. It is already known that the blocking efficiency of morpholino oligonucleotides falls dramatically as the distance of the binding region from the initial ATG increases [18]. It should be noted that these results were obtained using Oli-neu cells as recipients in all the transfection experiments. The use of these mouse oligodendrocyte precursor cells was deemed appropriate with a view to mimicking the cellular environment in which PMD displays its effects, and also avoiding potential biases in splicing selection due to the action of tissue-specific factors. PLP and DM20 transcript might occur without blocking, owing to the mRNA-morpholino interactions, as the region involved is largely downstream of the ATG (approx. 1.9kb) translational start site in the LacZ-PLP1-LacZ minigene. It is already known that the blocking efficiency of morpholino oligonucleotides falls dramatically as the distance of the binding region from the initial ATG increases [18]. September 2013 \| Volume 8 \| Issue 9 \| e73633 References PubMed: 15689360. 18. Summerton J (1999) Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim Biophys Acta 1489: 141-158. doi:10.1016/S0167-4781(99)00150-5. PubMed: 10807004. 7. Gow A, Lazzarini RA (1996) A cellular mechanism governing the severity of Pelizaeus-Merzbacher disease. Nat Genet 13: 422-428. doi: 10.1038/ng0896-422. PubMed: 8696336. 19. Schweitzer J, Becker T, Schachner M, Nave KA, Werner H (2005) Evolution of myelin proteolipid proteins: gene duplication in teleosts and expression pattern divergence. Mol Cell Neurosci 31: 161-177. PubMed: 16289898. g 8. Grossi S, Regis S, Biancheri R, Mort M, Lualdi S et al. (2011) Molecular genetic analysis of the PLP1 gene in 38 families with PLP1-related disorders: identification and functional characterization of 11 novel PLP1 mutations. Orphanet J Rare Dis 6: 40. doi: 10.1186/1750-1172-6-40. PubMed: 21679407. 20. Sivakumar K, Sambuughin N, Selenge B, Nagle JW, Baasanjav D et al. (1999) Novel exon 3B proteolipid protein gene mutation causing late- onset spastic paraplegia type 2 with variable penetrance in female family members. Ann Neurol 45: 680-683. doi: 10.1002/1531-8249(199905)45:5. PubMed: 10319897. 9. Hobson GM, Huang Z, Sperle K, Stabley DL, Marks HG et al. (2002) A PLP splicing abnormality is associated with an unusual presentation of PMD. Ann Neurol 52: 477–488. doi:10.1002/ana.10320. PubMed: 12325077. 21. Saugier-Veber P, Munnich A, Bonneau D, Rozet JM, Le Merrer M et al. (1994) X-linked spastic paraplegia and Pelizaeus-Merzbacher disease are allelic disorders at the proteolipid protein locus. Nat Genet 6: 257-262. doi:10.1038/ng0394-257. PubMed: 8012387. 10. Wang E, Huang Z, Hobson GM, Dimova N, Sperle K et al. (2006) PLP1 alternative splicing in differentiating oligodendrocytes: characterization of an exonic splicing enhancer. J Cell Biochem 97: 999–1016. doi: 10.1002/jcb.20692. PubMed: 16288477. 11. Wang E, Dimova N, Cambi F (2007) PLP/DM20 ratio is regulated by hnRNPH and F and a novel G-rich enhancer in oligodendrocytes. Nucleic Acids Res 35: 4164–4178. doi:10.1093/nar/gkm387. PubMed: 17567613. 22. Dhaunchak AS, Colman DR, Nave KA (2011) Misalignment of PLP/ DM20 transmembrane domains determines protein misfolding in Pelizaeus-Merzbacher disease. J Neurosci 31: 14961-14971. doi: 10.1523/JNEUROSCI.2097-11.2011. PubMed: 22016529. 12. Jung M, Krämer E, Grzenkowski M, Tang K, Blakemore W et al. (1995) Lines of murine oligodendroglial precursor cells immortalized by an activated neu tyrosine kinase show distinct degrees of interaction with 23. Swanton E, High S, Woodman P (2003) Role of calnexin in the glycan- independent quality control of proteolipid protein. EMBO J 22: 2948– 2958. doi:10.1093/emboj/cdg300. PubMed: 12805210. 24. References 1. Garbern JY, Hobson GH (1999) PLP1-related disorders, updated 2010. In: RA PagonTD BirdCR Dolan. GeneReviews. Seattle (WA): University of Washington; (1993).. Available: http://www.ncbi.nlm.nih.gov/books/ NBK1182/. 1. Garbern JY, Hobson GH (1999) PLP1-related disorders, updated 2010. In: RA PagonTD BirdCR Dolan. GeneReviews. Seattle (WA): University of Washington; (1993).. Available: http://www.ncbi.nlm.nih.gov/books/ NBK1182/. axons in vitro and in vivo. Eur J Neurosci 7: 1245-1265. doi:10.1111/j. 1460-9568.1995.tb01115.x. PubMed: 7582098. axons in vitro and in vivo. Eur J Neurosci 7: 1245-1265. doi:10.1111/j. 1460-9568.1995.tb01115.x. PubMed: 7582098. 13. Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR (2003) ESEfinder: a web resource to identify exonic splicing enhancers. Nucleic Acids Res 31: 3568-3571. doi:10.1093/nar/gkg616. PubMed: 12824367. 2. Inoue K (2005) PLP1-related inherited dysmyelinating disorders: Pelizaeus–Merzbacher disease and spastic paraplegia type 2. Neurogenetics 6: 1–16. doi:10.1007/s10048-004-0207-y. PubMed: 15627202. 2. Inoue K (2005) PLP1-related inherited dysmyelinating disorders: Pelizaeus–Merzbacher disease and spastic paraplegia type 2. Neurogenetics 6: 1–16. doi:10.1007/s10048-004-0207-y. PubMed: 15627202. 14. Smith PJ, Zhang C, Wang J, Chew SL, Zhang MQ et al. (2006) An increased specificity score matrix for the prediction of SF2/ASF-specific exonic splicing enhancers. Hum Mol Genet 15: 2490-2508. doi: 10.1093/hmg/ddl171. PubMed: 16825284. 3. Garbern JY (2007) Pelizaeus–Merzbacher disease: genetic and cellular pathogenesis. Cell Mol Life Sci 64: 50–65. doi:10.1007/ s00018-006-6182-8. PubMed: 17115121. 15. Long JC, Caceres JF (2009) The SR protein family of splicing factors: master regulators of gene expression. Biochem J 417: 15-27. doi: 10.1042/BJ20081501. PubMed: 19061484. 4. Nave KA, Lai C, Bloom FE, Milner RJ (1987) Splice site selection in the proteolipid protein (PLP) gene transcript and primary structure of the DM-20 protein of central nervous system myelin. Proc Natl Acad Sci U S A 84: 5665–5669. doi:10.1073/pnas.84.16.5665. PubMed: 2441390. 16. Desmet FO, Hamroun D, Lalande M, Collod-Béroud G, Claustres M et al. (2009) Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res 37: e67. doi:10.1093/nar/ gkp215. PubMed: 19339519. 5. Klugmann M, Schwab MH, Pühlhofer A, Schneider A, Zimmermann F, et al. (1997) Assembly of CNS myelin in the absence of proteolipid protein. Neuron 18: 59–70. 17. Wang Z, Rolish ME, Yeo G, Tung V, Mawson M et al. (2004) Systematic identification and analysis of exonic splicing silencers. Cell 119: 831–845. doi:10.1016/j.cell.2004.11.010. PubMed: 15607979. 6. Wolf NI, Sistermans EA, Cundall M, Hobson GM, Davis-Williams AP et al. (2005) Three or more copies of the proteolipid protein gene PLP1 cause severe Pelizaeus–Merzbacher disease. Brain 128: 743–751. doi: 10.1093/brain/awh409. (DOCX) PLP isoform. If this turns out to be the case, then our oligonucleotide-mediated in vitro correction strategy should have a high likelihood of being successful in ameliorating the clinical phenotype of the patient as part of a sequence-targeted therapeutic approach in this case of PMD. Acknowledgements We thank the “Cell Line and DNA Biobank from Patients Affected by Genetic Diseases” (G. Gaslini Institute) - Telethon Network of Genetic biobanks (Project No. GTB12001) for providing the patient samples. Supporting Information Figure S1. siRNA-mediated inhibition of the SRSF6-ESE interaction. Author Contributions Conceived and designed the experiments: SR. Performed the experiments: SR FC SG BT. Analyzed the data: SR MF DNC. Contributed reagents/materials/analysis tools: SR FC SG BT MF. Wrote the manuscript: SR DNC MF. Table S1. Evolutionary comparison of the PLP1 protein intracellular loop flanking the L146V missense mutation with their orthologous counterparts in seven vertebrates. Results and Discussion It should be noted that these results were obtained using Oli-neu cells as recipients in all the transfection experiments. The use of these mouse oligodendrocyte precursor cells was deemed appropriate with a view to mimicking the cellular environment in which PMD displays its effects, and also avoiding potential biases in splicing selection due to the action of tissue-specific factors. The successful oligonucleotide-mediated restoration of the major PLP transcript expression is important because the ultimate cause of the disease in this patient is the absence of the PLP isoform. However, unlike the situation with the DM20 transcript, restoration of the PLP transcript would be expected to lead to the production of a PLP protein isoform harbouring the p.L146V substitution. We neither know, nor can we predict, In addition, mutations residing within the cytoplasmic loop do not appear to exert a particularly detrimental impact on the general structure of the PLP protein, in particular on the tetraspan structure, whose integrity prevents retention of the protein in the endoplasmic reticulum (ER) [22] with consequent potential activation of the unfolded protein response (UPR). The p.L146V mutation would not be predicted to alter any of the previously proposed putative ER-retention signals: namely, the fourth transmembrane domain of the PLP protein [23], and a heptapeptide within the intracellular loop [24]. Finally, the conservative replacement of the aliphatic hydrophobic side chain of Leu with the slightly smaller aliphatic hydrophobic side chain of Val is not inconsistent with a mild clinical phenotype. The successful oligonucleotide-mediated restoration of the major PLP transcript expression is important because the ultimate cause of the disease in this patient is the absence of the PLP isoform. However, unlike the situation with the DM20 transcript, restoration of the PLP transcript would be expected to lead to the production of a PLP protein isoform harbouring the p.L146V substitution. We neither know, nor can we predict, Taking the above considerations together, we would therefore predict that the p.L146V mutation should not give rise to a major structural alteration of PLP nor is it likely that it would result in the retention of the PLP protein isoform in the ER. We would therefore predict that the presence of the L146V-mutated PLP isoform would be less detrimental to the individual harbouring it than would the complete absence of the September 2013 \| Volume 8 \| Issue 9 \| e73633 PLOS ONE \| www.plosone.org 5 Oligonucleotide-Mediated Splicing Restoration References Southwood C, Olson K, Wu CY, Gow A (2007) Novel alternatively spliced endoplasmic reticulum retention signal in the cytoplasmic loop of proteolipid protein-1. J Neurosci Res 85: 471–478. doi:10.1002/jnr. 21153. PubMed: 17171701. PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e73633 6
https://openalex.org/W2804136752	https://www.nature.com/articles/s42003-018-0086-7.pdf	English	null	Genomic evidence for the degradation of terrestrial organic matter by pelagic Arctic Ocean Chloroflexi bacteria	bioRxiv (Cold Spring Harbor Laboratory)	2,018	cc-by	8,974	1 Department of Biology, Concordia University, 7141 Sherbrooke St. West, Montreal, QC H4B 1R6, Canada. 2 Department of Chemistry and School of the Environment, Trent University, 1600 West bank Drive, Peterborough, ON K9J 7B8, Canada. 3 Fisheries and Oceans Canada, Institute of Ocean Sciences, 9860 West Saanich Road, Sidney, BC V8V 4L1, Canada. 4 Département de biologie, Institut de Biologie Intégrative et des Systèmes (IBIS) and Québec- Océan, Université Laval, Laval, QC G1K 7P4, Canada. 5 Takuvik Joint International Laboratory, Centre National de la Recherche Scientiﬁque (France, CNRS UMI 3376), Université Laval, Laval, QC G1V 0A6, Canada. Correspondence and requests for materials should be addressed to D.A.W. (email: david.walsh@concordia.ca) Genomic evidence for the degradation of terrestrial organic matter by pelagic Arctic Ocean Chloroﬂexi bacteria Genomic evidence for the degradation of terrestrial organic matter by pelagic Arctic Ocean Chloroﬂexi bacteria David Colatriano1, Patricia Q. Tran1, Celine Guéguen2, William J. Williams3, Connie Lovejoy 4,5 & David A. Walsh1 The Arctic Ocean currently receives a large supply of global river discharge and terrestrial dissolved organic matter. Moreover, an increase in freshwater runoff and riverine transport of organic matter to the Arctic Ocean is a predicted consequence of thawing permafrost and increased precipitation. The fate of the terrestrial humic-rich organic material and its impact on the marine carbon cycle are largely unknown. Here, a metagenomic survey of the Canada Basin in the Western Arctic Ocean showed that pelagic Chloroﬂexi from the Arctic Ocean are replete with aromatic compound degradation genes, acquired in part by lateral transfer from terrestrial bacteria. Our results imply marine Chloroﬂexi have the capacity to use terrestrial organic matter and that their role in the carbon cycle may increase with the changing hydrological cycle. 1 COMMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio COMMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio COMMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio ARTICLE COMMUNICATIONS BIOLOGY \| DOI: 10.1038/s42003-018-0086-7 T he Arctic Ocean accounts for 1.4% of global ocean volume but receives 11% of global river discharge1. Up to 33% of the dissolved organic matter in the Arctic Ocean is of terrestrial origin and a major fraction of this terrestrial dissolved organic matter (tDOM) originates from carbon-rich soils and peatlands2,3. With thawing permafrost and increased precipita- tion occurring across the Arctic4, increases in freshwater runoff and riverine transport of organic matter to the Arctic Ocean are predicted, which will increase tDOM ﬂuxes and loadings5,6. The additional tDOM may represent new carbon and energy sources for the Arctic Ocean microbial community and contribute to increased respiration, which would result in the Arctic being a source of dissolved inorganic carbon to the ocean. Alternatively, as it moves from its source of origin to the Arctic Ocean tDOM could become more recalcitrant to bacterial metabolism and represent a long-term sequestration of the newly released carbon making the Arctic more carbon neutral7,8. However, an estimated 50% of Arctic Ocean tDOM is removed before being released to the Atlantic, at least in part by microbial processes9. As input of tDOM increases, knowledge on its microbial transformation will be critical for understanding changes in Arctic carbon cycling. locations and 4 separate bathypelagic metagenomes indicated that the Canada Basin Chloroﬂexi MAGs were not widely distributed in the oceans (Fig. 2c, Supplementary Data 1). These ﬁndings are evidence that the Chloroﬂexi MAGs represent genotypes that are rare outside Arctic marine waters. T The Chloroﬂexi MAGs contained many genes implicated in the degradation of aromatic compounds typically associated with humic-rich tDOM (Supplementary Data 2). A single MAG (SAR202-VII-2) from a previously undescribed clade (SAR202- VII) exhibited a striking enrichment in these genes (Fig. 3a). Partial pathways for the catabolism of aromatic compounds were recently reported from deep ocean SAR202 SAGs18. COMMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio To assess whether the abundance and diversity of SAR202-VII-2 genes involved in aromatic compound catabolism is unique to Arctic Ocean MAGs or is a more broad characteristic of marine Chloroﬂexi, we compared gene content between SAR202-VII-2 and two SAGs (SAR202-V-AB-629-P13 and SAR202-III- AAA240-O15) reported in Landry et al.18 Of the 117 SAR202- VII-2 orthologs implicated in aromatic compound degradation, 12 were identiﬁed in SAR202-III-AAA240-O15 and only one was identiﬁed in SAR202-V-AB-629-P13, implying distinct and less diverse pathways in deep ocean SAR202 compared to the Arctic Ocean populations (Supplementary Data 2). The marine SAR202 is a diverse and uncultivated clade of Chloroﬂexi bacteria that comprise roughly 10% of planktonic cells in the dark ocean10–14. SAR202 is also common in marine sediments and deep lakes15–17. It has long been speculated that SAR202 may have a role in the degradation of recalcitrant organic matter11,14, and the recent analysis of SAR202 single-cell-ampliﬁed genomes (SAGs) lends support to this notion18. More generally, Chloroﬂexi, including those in the SAR202 clade, are also present in the upper layers of the Arctic Ocean19, leading to the hypothesis that recalcitrant organic compounds present in high Arctic tDOM could be utilized by this group. p p ( pp y ) Proteins for the modiﬁcation and degradation of monoaryl and biaryl compounds were predicted, including a diversity of aro- matic ring-cleaving dioxygenases24–26. A total of 42 ring-cleaving dioxygenases targeting compounds related to catechol, proto- catechuate and gentisate were present in the six MAGs, with 25 dioxygenases predicted in SAR202-VII-2 alone (Fig. 3a, b). Ring demethylation, hydroxylation, and decarboxylation are important prerequisite steps to prime diverse aromatic compounds for downstream oxidative cleavage27,28. Thirty ring-demethylating monooxygenases, ten ring-hydroxylating dioxygenases, and ele- ven ring-decarboxylases were annotated in the SAR202-VII-2 MAG (Fig. 3c, Supplementary Data 2). Proteins involved in the conversion of ring-cleavage products to central intermediates of the citric acid cycle were also present in the SAR202-VII-2 MAG, including dehydrogenases (i.e., 2,3-dihydroxy-2,3-dihy- drophenylpropionate dehydrogenase), decarboxylases (i.e., oxa- loacetate B-decarboxylase), aldolases (i.e., HMG aldolase and 4-carboxymuconolactone decarboxylase), hydratases (i.e., 4- oxalmescanoate hydratase and 2-oxopent-4-enoate hydratase), isomerases (i.e., mycothiol maleulpyruvate isomerase and muconolactone isomerase) and hydrolases (i.e., 3-oxoadipate enol-lactonase and β-ketoadipate enol-lactone hydrolase) (Sup- plementary Data 2, Supplementary Figure 2). We note that we were unable to identify a single complete reference pathway for humic-like aromatic compound degradation. COMMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio Since estimated genome completeness for SAR202-VII-2 was 99%, it is unlikely the genes were missed due to an incomplete genome. Another explanation is that marine Chloroﬂexi genomes encode novel pathway variants. Indeed, numerous metal-dependent hydrolases, hydrolases of the HAD family and NAD(P)-dependent dehydrogenase were clustered in genomic regions with the ring- modifying oxygenases, decarboxylases, and demethylases descri- bed above. In addition to the array of aromatic compound degradation genes, the SAR202-VII-2 MAG also contained 34 copies of the ﬂavin mononucleotide (FM)/F420-dependent monooxygenase catalytic subunit (FMNO) proteins previously implicated in activation of recalcitrant organic compounds in the deep ocean18. These results are consistent with Chloroﬂexi in the Arctic Ocean having the metabolic potential to access carbon and energy available in aromatic compounds typically associated with tDOM. COMMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio Results h In this study, we analyzed Chloroﬂexi metagenome-assembled genomes (MAGs) generated from samples collected from the vertically stratiﬁed waters of the Canada Basin in the Western Arctic Ocean (Fig. 1a). A metagenomic co-assembly was gener- ated from samples originating from the surface layer (5–7 m), the subsurface chlorophyll maximum (25–79 m) and a layer corre- sponding to the terrestrially-derived DOM ﬂuorescence (FDOM) maximum previously described within the cold Canada Basin halocline comprised of Paciﬁc-origin waters (177–213 m)20. The Paciﬁc-origin FDOM maximum is due to sea ice formation and interactions with bottom sediments on the Beaufort and Chukchi shelves, which themselves are inﬂuenced by coastal erosion and river runoff20. Binning based on tetranucleotide frequency and coverage resulted in 360 MAGs from a diversity of marine microbes (Fig. 1b). Six near-complete Chloroﬂexi MAGs were identiﬁed. Based on 16S rRNA gene phylogeny, these MAGs represented three distinct SAR202 subclades (SAR202-II, -VI, -VII), the AncK29 clade and the TK10 clade (Fig. 2a). Estimated MAG completeness ranged from 77 to 99%, while contamination ranged from 0 to 2.3% (Table 1). All MAGs exhibited highest coverage just below the subsurface chlorophyll maximum (Fig. 2b) which is consistent with earlier ﬁndings on SAR202 distribution in the North Paciﬁc Ocean12. However, the con- centration and composition of the FDOM maximum in the Canada Basin is signiﬁcantly different compared to the North Paciﬁc Ocean21 and the North Atlantic Subtropical Gyre22. A concatenated protein phylogeny demonstrated that the SAR202 MAGs were distinct from previously published MAGs from the deep ocean18 and oxygen minimum zones23 (Supplementary Figure 1). Fragment recruitment of 21 TARA Ocean metage- nomic datasets spanning epipelagic to mesopelagic waters at 7 The diversity of SAR202-VII-2 genes implicated in aromatic compound degradation lead us to hypothesize that they may have COMMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio 2 COMMUNICATIONS BIOLOGY \| DOI: 10.1038/s42003-018-0086-7 ARTICLE originated by lateral gene transfer from terrestrial bacteria. To test this, we targeted aromatic compound degradation genes (the ring-cleaving dioxygenases, speciﬁcally) in the Chloroﬂexi MAGs for in-depth phylogenetic analyses. The genomic diversity of marine Chloroﬂexi was expanded in our analysis by including 130 Chloroﬂexi MAGs recently assembled and binned from the TARA Oceans project29. Results h A number of the SAR202-VII-2 ring- cleaving dioxygenase homologs were most closely related to Relative coverage 0 0.4 0.6 0.8 1 Completeness FDOM max SCM Surface Completeness FDOM max SCM Surface USA CANADA CB2 CB4 CB8 CB11 80°N a b 70°N 60°N 50°N 40°N 72°N 73°N 74°N 75°N 76°N 77°N 78°N 79°N 80°N Ocean data view Ocean data view Depth (m) Temperature (°C) Salinity (PSU) Chlorophyll fluorescence (mg/m3) FDOM (mg/m3) 0.75 0 –0.75 –1.5 33 30 27 0.3 0.2 0.1 4.5 3.75 3 2.25 0 300 0 300 0 300 0 300 CB2 CB4 CB8 CB11 150°W 120°W 90°W 60°W 0.2 Arc ha ea Eu kar yot es Cy ano ba c Chl oro fle xi Act ino ba cte ria Ba cte roi de te s MG A Ver ruc o P lan cto Ga mm a-p rot eob act eri a Bet a-p rot eob act eria Alp ha- pro teo bac ter ia Del ta- pro teo Aci dob act eria 72°N 73°N 74°N 75°N 76°N 77°N 78°N 79°N 80°N Ocean data view Depth (m) Temperature (°C) Salinity (PSU) Chlorophyll fluorescence (mg/m3) FDOM (mg/m3) 0.75 0 –0.75 –1.5 33 30 27 0.3 0.2 0.1 4.5 3.75 3 2.25 0 300 0 300 0 300 0 300 CB2 CB4 CB8 CB11 a USA CANADA CB2 CB4 CB8 CB11 80°N a 70°N 60°N 50°N 40°N Ocean data view 150°W 120°W 90°W 60°W Relative coverage 0 0.4 0.6 0.8 1 Completeness FDOM max SCM Surface Completeness FDOM max SCM Surface b 50°N 40°N 72°N 73°N 74°N 75°N 76°N 77°N 78°N 79°N 80°N Ocean data view Ocean data view FDOM (mg/m3) 4.5 3.75 3 2.25 300 150°W 120°W 90°W 60°W 0.2 Arc ha ea Eu kar yot es Cy ano ba c Chl oro fle xi Act ino ba cte ria Ba cte roi de te s MG A Ver ruc o P lan cto Ga mm a-p rot eob act eri a Bet a-p rot eob act eria Alp ha- pro teo bac ter ia Del ta- pro teo Aci dob act eria Fig. 1 Metagenomic survey of microbial diversity in the Canada Basin. a Sampling locations and environmental proﬁles of the Canada Basin. Sa locations and the associated environmental datasets were plotted using Ocean Data View version 4.7.742. b Concatenated protein phylogeny of 36 Ocean MAGs inferred by MetaWatt and visualized in iTOL. Results h The three inner tracks present relative coverage of MAGs averaged across samples c from surface waters, subsurface chlorophyll maximum (SCM) and the ﬂuorescent dissolved organic matter maximum (FDOM max). The outer presents estimated MAG completeness as inferred by MetaWatt. MAG completeness ranged from 25 to 94% COMMUNICATIONS BIOLOGY \| (2018)1 90 \| DOI 10 1038/ 42003 018 0086 7 \| t / bi Relative coverage 0 0.4 0.6 0.8 1 Completeness FDOM max SCM Surface Completeness FDOM max SCM Surface b 0.2 Arc ha ea Eu kar yot es Cy ano ba c Chl oro fle xi Act ino ba cte ria Ba cte roi de te s MG A Ver ruc o P lan cto Ga mm a-p rot eob act eri a Bet a-p rot eob act eria Alp ha- pro teo bac ter ia Del ta- pro teo Aci dob act eria Metagenomic survey of microbial diversity in the Canada Basin. a Sampling locations and environmental proﬁles of the Canada Basin. S ns and the associated environmental datasets were plotted using Ocean Data View version 4.7.742. b Concatenated protein phylogeny of 3 MAGs inferred by MetaWatt and visualized in iTOL. The three inner tracks present relative coverage of MAGs averaged across samples Relative coverage 0 0.4 0.6 0.8 1 Completeness FDOM max SCM Surface b 0.2 Arc ha ea Eu kar yot es Cy ano ba c Chl oro fle xi Act ino ba cte ria Ba cte roi de te s Alp ha- pro teo bac ter ia Del ta- pro teo Aci dob act eria b Fig. 1 Metagenomic survey of microbial diversity in the Canada Basin. a Sampling locations and environmental proﬁles of the Canada Basin. Sample locations and the associated environmental datasets were plotted using Ocean Data View version 4.7.742. b Concatenated protein phylogeny of 360 Arctic Ocean MAGs inferred by MetaWatt and visualized in iTOL. The three inner tracks present relative coverage of MAGs averaged across samples collected from surface waters, subsurface chlorophyll maximum (SCM) and the ﬂuorescent dissolved organic matter maximum (FDOM max). The outer track presents estimated MAG completeness as inferred by MetaWatt. MAG completeness ranged from 25 to 94% Fig. 1 Metagenomic survey of microbial diversity in the Canada Basin. a Sampling locations and environmental proﬁles of the Canada Basin. Sample locations and the associated environmental datasets were plotted using Ocean Data View version 4.7.742. Results h b Concatenated protein phylogeny of 360 Arctic Ocean MAGs inferred by MetaWatt and visualized in iTOL. The three inner tracks present relative coverage of MAGs averaged across samples collected from surface waters, subsurface chlorophyll maximum (SCM) and the ﬂuorescent dissolved organic matter maximum (FDOM max). The outer track presents estimated MAG completeness as inferred by MetaWatt. MAG completeness ranged from 25 to 94% 3 COMMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio ARTICLE COMMUNICATIONS BIOLOGY \| DOI: 10.1038/s42003-018-0086-7 a SAR202 cluster SAR202-V-AB-629-P13 SAR202-III-AAA240-O15 Surface SCM Mesopelagic Bathypelagic SAR202-II-3 SAR202-II-177A SAR202-VII-2 SAR202-VI-29A TK10-74A Anck29-46 CB2 b c CB4 CB8 CB11 CB2 CB4 CB8 CB11 CB2 CB4 CB8 CB11 Surface SCM FDOM max 0 0.5 2.5 5 0 1 2 5 10 20 50 Coverage (x) RPKG II CL500-11 cluster Deep ocean cluster Anck29 cluster TK10 cluster I VII V VI VIII IV III SAR202-II-3 SAR202-II-177A SAR202-VII-2 SAR202-VI-29A TK10-74A Anck29-46 60°N 30°N 30°S 60°S 150°W 120°W 90°W 60°W 30°W 30°E Ocean data view 0° EQ SAR202-II-3 SAR202-II-177A SAR202-VII-2 SAR202-VI-29A TK10-74A Anck29-46 CB2 b CB4 CB8 CB11 CB2 CB4 CB8 CB11 CB2 CB4 CB8 CB11 Surface SCM FDOM max 0 1 2 5 10 20 50 Coverage (x) SAR202 cluster SAR202-V-AB-629-P13 SAR202-III-AAA240-O15 Surface SCM Mesopelagic Bathypelagic SAR202-II-3 SAR202-II-177A SAR202-VII-2 SAR202-VI-29A TK10-74A Anck29-46 CB2 b c CB4 CB8 CB11 CB2 CB4 CB8 CB11 CB2 CB4 CB8 CB11 Surface SCM FDOM max 0 0.5 2.5 5 0 1 2 5 10 20 50 Coverage (x) RPKG 0.08 Station Aloha AAA240-O15 (HQ675645.1) Pacific Ocean sediment MBAE74 (AJ567614.1) Western Arctic Ocean Ga0133547_100409911 Saanich Inlet SGPZ645 (GQ346975.1) Crater Lake CL500-9 (AF316763.1) Sargasso Sea SAR317 (AY534093.1) Lake Michigan CL500-11LM (Denef et al. Results h 2016) Lake Michigan Station Aloha AAA240O05 (HQ675640.1) Sponge-associated clone AncK29 (FJ900348.1) Western Arctic Ocean Ga0133547_10017273 MAG SAR202 Anck29-46 (Ga0133547_10007268) Anck29 (Ga0133547_10000780) SAR202-I (Ga0133547_11227957) Gulf of Mexico 86 (HQ015617.1) Sargasso Sea SAR261 (AY534091.1) Pacific Ocean sediment MBMPE42 (AJ567556.1) Arctic Ocean Arctic95A18 (AF355054.1) Seawater clone D92_22 (AY534099.1) Sargaso Sea Sargasso Sea SAR202 (U20797.1) North Atlantic Ocean AB-629-P13 v2 (2640444700) Atlantic Ocean AAA007-M09 (HQ675482.1) Trichloroethene-contaminated site FTL276 (AF529110.1) Seawater clone SAR259 (AY534094.1) Sargasso Sea Sponge TK17 (AJ347036.1) Thermomicrobia Atlantic Ocean AAA001-F05 (HQ675371.1) SAR202 (Ga0133547_10004625) Sargasso Sea SAR194 (AY534098.1) Chloroflexia MAG SAR202-II-177A (Ga0133547_1000294) SAR202-II (Ga0133547_11014312) Atlantic Ocean MOC-1-361 (KX427939.1) MAG SAR202-VI-29A (Ga0133547 10002442) Sargasso Sea SAR307 (U20798.1) Anaerolineae Chloroflexi (Ga0133547_10365966) Chloroflexia MAG SAR202-II-3 (Ga0133547_10001736) Sponge XC1005 (JN596666.1) Western Arctic Ocean Ga0133547_10010958 Forrest soil C083 (AF507700.1) Crater Lak CL500-11 (AF316759.1) Crater Lake, 500 m North Aegean Sea AEGEAN_116 (AF406538.1) Southeast India sediment bac140 (JX138712.1) MAG SAR202-VII-2 (Ga0133547_10000360) SAR202-II (Ga0133547_10310572) Station Aloha AAA240-N13 (2264260034) SAR202-I (Ga0133547_11319260) Hotoke-Ike Lake MPB1-195 (AB630577.1) Western Arctic Ocean Ga0133547_10472218 Ardenticatena maritima (AB576167.1) Sargasso Sea SAR242 (AY534089.1) Sargasso Sea Sargasso Sea SAR188 (AY534087.1) Canada Basin CB0563b.27 (GQ337107.1) Station Aloha HF770_09E03 (EU361081.1) Caldilineaeceae Ktedonobacteria DOC (Ga0133547_10351421) Sponge Klon-I-93-voll (HF912758.1) Chloroflexi (Ga0133547_10005725) Station Aloha HF500_13N24 (EU361068.1) Red Sea Sponge C433/GW1046 (HG423495.1) Western Arctic Ocean Ga0133547_10002546 SAR202-II (Ga0133547_10453494) Western Arctic Ocean Ga0133547_10016871 MAG SAR202-TK10-74A (Ga0133547_10008775) Crater Lake CL500-10 (AF316764.1) SAR202-II (Ga0133547_10001767) Dehalococcoidia Western Arctic Ocean Ga0133547_10035846 Gulf of Mexico DS34_F10 (KU578722.1) Arctic Ocean Arctic96BD6 (AF355053.1) SAR202-II (Ga0133547_10125571) Chloroflexi (Ga0133547_10000969) Anck29 (Ga0133547_10183849) Saanich Inlet SGPZ590 (GQ346947.1) Thermoflexus hugenholtzii JAD2 (KC526151.1) SAR202-II (Ga0133547_10341800) Gulf of Mexico 52331 (JN018849.1) SAR202-II Ga0133547_10000170 Sargasso Sea SAR272 (AY534088.1) Anck29 (Ga0133547_10009981) 100 83 89 94 72 100 100 100 90 100 99 100 91 85 100 91 100 100 100 100 100 99 84 98 100 100 100 100 100 100 87 84 100 97 100 100 100 86 99 100 100 100 99 91 100 100 100 100 95 100 Lake Gatun 5C231021 (EU903451.1) Lake Zurich ZS4311 (FN668371.2) II CL500-11 cluster Deep ocean cluster Anck29 cluster TK10 cluster I VII V VI VIII IV III SAR202-II-3 SAR202-II-177A SAR202-VII-2 SAR202-VI-29A TK10-74A Anck29-46 60°N 30°N 30°S 60°S 150°W 120°W 90°W 60°W 30°W 30°E Ocean data view 0° EQ Fig. 2 Diversity and distribution of Arctic Ocean Chloroﬂexi MAGs. a Maximum likelihood tree of Chloroﬂexi based on partial 16S rRNA gene sequences. Blue taxa are from Canada Basin metagenomes and red taxa are from the six Chloroﬂexi MAGs. COMMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio Results h Bootstrap values of >70% are shown (100 replicates). b Distribution of MAGs in the Canada Basin based on metagenome coverage at the surface, subsurface chlorophyll maximum (SCM) and ﬂuorescent dissolved organic matter maximum (FDOM max). c Distribution of MAGs in global ocean metagenomes based on fragment recruitment. Two deep ocean SAR202 SAGs from Landry et al.18 were included for comparison. Location of TARA ocean metagenomes (red) and bathypelagic metagenomes (green) are shown on the map generated using Ocean Data View version 4.7.742 SAR202-V-AB-629-P13 SAR202-III-AAA240-O15 Surface SCM Mesopelagic Bathypelagic c 0 0.5 2.5 5 RPKG SAR202-II-3 SAR202-II-177A SAR202-VII-2 SAR202-VI-29A TK10-74A Anck29-46 60°N 30°N 30°S 60°S 150°W 120°W 90°W 60°W 30°W 30°E Ocean data view 0° EQ SAR202-V-AB-629-P13 SAR202-III-AAA240-O15 Surface SCM Mesopelagic Bathypelagic c 0 0.5 2.5 5 RPKG SAR202-II-3 SAR202-II-177A SAR202-VII-2 SAR202-VI-29A TK10-74A Anck29-46 c c 60°N 30°N 30°S 60°S 150°W 120°W 90°W 60°W 30°W 30°E Ocean data view 0° EQ 150°W 120°W 90°W 60°W 30°W 30°E 0° Fig. 2 Diversity and distribution of Arctic Ocean Chloroﬂexi MAGs. a Maximum likelihood tree of Chloroﬂexi based on partial 16S rRNA gene sequences. Blue taxa are from Canada Basin metagenomes and red taxa are from the six Chloroﬂexi MAGs. Bootstrap values of >70% are shown (100 replicates). b Distribution of MAGs in the Canada Basin based on metagenome coverage at the surface, subsurface chlorophyll maximum (SCM) and ﬂuorescent dissolved organic matter maximum (FDOM max). c Distribution of MAGs in global ocean metagenomes based on fragment recruitment. Two deep ocean SAR202 SAGs from Landry et al.18 were included for comparison. Location of TARA ocean metagenomes (red) and bathypelagic metagenomes (green) are shown on the map generated using Ocean Data View version 4.7.742 proteins from the TARA Ocean Chloroﬂexi MAGs and other marine originating genomes, particularly the catechol dioxy- genases (Supplementary Figure 3), gentisate 1,2-dioxygenases (Fig. 4) and methylgallate dioxygenases (Supplementary Figure 4) indicating that aromatic compound degradation in Chloroﬂexi is not restricted to the Arctic Ocean. However, lateral gene transfer from terrestrial bacteria was also evident. For example, an annotated gentisate dioxygenase gene was positioned within a clade of terrestrial Actinomycetes (Fig. 4). Additional genes involved in the degradation of structures related to catechol, protocatechuate and gentisate were phylogenetically associated with homologs from terrestrial Acidobacteria (Supplementary Figure 5), Actinobacteria (Supplementary Figure 6), Armatimo- nadetes (Fig. Results h of genes a ligA Decarboxylation ligB 2_0234 2_0233 4_0522 4_0523 4_0200 4_0237 4_0238 2_0146 4_0244 60_0179 103_0019 360_0073 8610_0002 8610_0007 Hydroxylation 2_0074 2_0240 2_0408 2_0449 45_0109 60_0003 60_0202 297_0090 336_0078 2_0528 Gentisate dioxygenase Catechol dioxygenase Demethylation 5,5′-dehydrodivanillate O-demethylase 4,5-dihydroxyphthalate decarboxylase 3-Phenylpropionate/ Trans-cinnamate dioxygenase Biphenyl 2,3-dioxygenase 4_0172 c b Dearomatization Terrestrial clade Marine clade Mixed clade HO HO HO HO HO HO HO HO HO HO HO OH O OH HO OH O O O O O O O OH OH OH O OH OH OH O O HO OH OH O HO HO HO HO HO HO O O O H HO HO HO HO O O O O O O O O O O O O O O O O O O O O O O O O OH OH OH HO OH OH OH OH HO OH OH OH OH OH OH OH OH OH OH OH OH OH CH3 CH3 CH3 CH3 CH3 CH3 H3C OH O O ligA ligB 2_0234 2_0233 4_0522 4_0523 4_0200 4_0237 4_0238 2_0146 4_0244 60_0179 103_0019 360_0073 8610_0002 8610_0007 2_0074 2_0240 2_0408 2_0449 45_0109 60_0003 60_0202 297_0090 336_0078 2_0528 Gentisate dioxygenase Catechol dioxygenase 4_0172 b Dearomatization Terrestrial clade Marine clade Mixed clade HO HO O O O OH OH HO HO O O O H HO HO HO HO O O O O O O O O O O O O O O O O O OH OH OH HO OH OH OH OH HO OH OH OH OH OH OH OH OH OH OH OH CH3 CH3 CH3 H3C OH O O ligA Decarboxylation ligB 2_0234 2_0233 4_0522 4_0523 4_0200 4_0237 4_0238 2_0146 4_0244 60_0179 103_0019 360_0073 8610_0002 8610_0007 Hydroxylation 2_0074 2_0240 2_0408 2_0449 45_0109 60_0003 60_0202 297_0090 336_0078 2_0528 Gentisate dioxygenase Catechol dioxygenase Demethylation 5,5′-dehydrodivanillate O-demethylase 4,5-dihydroxyphthalate decarboxylase 3-Phenylpropionate/ Trans-cinnamate dioxygenase Biphenyl 2,3-dioxygenase 4_0172 c b Dearomatization Terrestrial clade Marine clade Mixed clade HO HO HO HO HO HO HO HO HO HO HO OH O OH HO OH O O O O O O O OH OH OH O OH OH OH O O HO OH OH O HO HO HO HO HO HO O O O H HO HO HO HO O O O O O O O O O O O O O O O O O O O O O O O O OH OH OH HO OH OH OH OH HO OH OH OH OH OH OH OH OH OH OH OH OH OH CH3 CH3 CH3 CH3 CH3 CH3 H3C OH O O Fig. Results h c Examples of predicted aromatic ring-modifying enzymatic reactions identiﬁed in SAR202-VII-2 Table 1 Genomic characteristics of MAGs Size (Mb) Cov (x) GC (%) Completeness (%) Contamination (%) N50 (kb) # of Contigs SAR202-II-3 1.36 68 39 80 0 35 46 SAR202-II-177A 1.62 101 42 82 1 36 52 SAR202-VII-2 2.78 16 59 99 0 246 8 SAR202-VI-29A 1.52 16 46 97 2 59 2 TK10-74A 2.45 12 69 81 2.3 38 76 Anck29-46 1.11 11 32 77 0 75 22 COMMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio 5 117 8 13 9 3 20 SAR202-VII-2 SAR202-II-3 SAR202-II-177A SAR202-VI-29A Anck29-46 TK10-74A 0 10 20 30 40 Protocatechuate 4,5-dioxygenase Gentisate 1,2-dioxygenase Catechol 2,3-dioxygenase Ring-hydroxylating dioxygenase Ring-decarboxylases O-demethylases Flavin-dependent monooxygenases No. of genes a ligA Decarboxylation ligB 2_0234 2_0233 4_0522 4_0523 4_0200 4_0237 4,5-dihydroxyphthalate decarboxylase c b Dearomatization HO HO HO HO OH O HO HO O O O O O O O O O O O OH OH OH HO OH OH OH OH OH OH OH CH3 H3C OH O O Table 1 Genomic characteristics of MAGs Size (Mb) Cov (x) GC (%) Completeness (%) Contamination (%) N50 (kb) # of Contigs SAR202-II-3 1.36 68 39 80 0 35 46 SAR202-II-177A 1.62 101 42 82 1 36 52 SAR202-VII-2 2.78 16 59 99 0 246 8 SAR202-VI-29A 1.52 16 46 97 2 59 2 TK10-74A 2.45 12 69 81 2.3 38 76 Anck29-46 1.11 11 32 77 0 75 22 Table 1 Genomic characteristics of MAGs Size (Mb) Cov (x) GC (%) Completeness (%) Contamination (%) N50 (kb) # of Contigs SAR202-II-3 1.36 68 39 80 0 35 46 SAR202-II-177A 1.62 101 42 82 1 36 52 SAR202-VII-2 2.78 16 59 99 0 246 8 SAR202-VI-29A 1.52 16 46 97 2 59 2 TK10-74A 2.45 12 69 81 2.3 38 76 Anck29-46 1.11 11 32 77 0 75 22 Table 1 Genomic characteristics of MAGs 117 8 13 9 3 20 SAR202-VII-2 SAR202-II-3 SAR202-II-177A SAR202-VI-29A Anck29-46 TK10-74A 0 10 20 30 40 Protocatechuate 4,5-dioxygenase Gentisate 1,2-dioxygenase Catechol 2,3-dioxygenase Ring-hydroxylating dioxygenase Ring-decarboxylases O-demethylases Flavin-dependent monooxygenases No. Results h 4), Delta-proteobacteria (Supplementary Figures 3 COMMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio 4 ARTICLE COMMUNICATIONS BIOLOGY \| DOI: 10.1038/s42003-018-0086-7 117 8 13 9 3 20 SAR202-VII-2 SAR202-II-3 SAR202-II-177A SAR202-VI-29A Anck29-46 TK10-74A 0 10 20 30 40 Protocatechuate 4,5-dioxygenase Gentisate 1,2-dioxygenase Catechol 2,3-dioxygenase Ring-hydroxylating dioxygenase Ring-decarboxylases O-demethylases Flavin-dependent monooxygenases No. of genes a ligA Decarboxylation ligB 2_0234 2_0233 4_0522 4_0523 4_0200 4_0237 4_0238 2_0146 4_0244 60_0179 103_0019 360_0073 8610_0002 8610_0007 Hydroxylation 2_0074 2_0240 2_0408 2_0449 45_0109 60_0003 60_0202 297_0090 336_0078 2_0528 Gentisate dioxygenase Catechol dioxygenase Demethylation 5,5′-dehydrodivanillate O-demethylase 4,5-dihydroxyphthalate decarboxylase 3-Phenylpropionate/ Trans-cinnamate dioxygenase Biphenyl 2,3-dioxygenase 4_0172 c b Dearomatization Terrestrial clade Marine clade Mixed clade HO HO HO HO HO HO HO HO HO HO HO OH O OH HO OH O O O O O O O OH OH OH O OH OH OH O O HO OH OH O HO HO HO HO HO HO O O O H HO HO HO HO O O O O O O O O O O O O O O O O O O O O O O O O OH OH OH HO OH OH OH OH HO OH OH OH OH OH OH OH OH OH OH OH OH OH CH3 CH3 CH3 CH3 CH3 CH3 H3C OH O O Fig. 3 Aromatic compound degradation genes and pathways in Arctic Ocean Chloroﬂexi MAGs. a Abundance of aromatic compound degradation genes in Chloroﬂexi MAGs from the Canada Basin (pie chart) and a breakdown of those found speciﬁcally in SAR202-VII-2 (column plot). b Predicted aromati ring-opening enzymatic reactions identiﬁed in SAR202-VII-2 with gene loci displayed in the colored boxes. Genes in green and blue boxes were most closely related to homologs from terrestrial or marine bacteria, respectively. Genes in the blue/green boxes were in clades containing diverse environmental bacteria. COMMUNICATIONS BIOLOGY \| DOI: 10.1038/s42003-018-0086-7 0.4 Chloroflexi bacterium 5419 \| Bioreactor (A0A1Q3SS95) Pseudomonas alcaligenes \| Experimnetally validated (Q9S3U6) Deltaproteobacteria bacterium \| Terrestrial, Soil (A0A1F9K2A2) Streptomyces odonnellii \| Terrestrial, Soil (UPI0004C7D6A2) Deltaproteobacteria bacterium \| Terrestrial, Soil (A0A1F9KKT3) Kyrpidia tusciae strain DSM 2912 \| Freshwater, Pond (D5WS41) Hydrothermal vent metagenome \| Marine, Hydrothermal vent (A0A160V9W9) Armatimonadetes bacterium \| Terrestrial, Soil (A0A1Q7Q8F3) Microbacterium sp. Root53 \| Terrestrial, Arabidopsis thaliana root (A0A0Q7UW44) Leadbetterella sediminis \| Freshwater sediment (A0A0P7C872) Ca. Rokubacteria bacterium \| Terrestrial, Soil (A0A1Q7GJ09) Sulfolobales archaeon AZ1 \| Freshwater, Acidic hot spring (W7KL35) TOBG SP-2981 Chryseobacterium piperi \| Freshwater, Creek (A0A086BJU5) Streptomyces sp. AcH 505 \| Terrestrial, Soil (A0A0C2AZL0) Deltaproteobacteria bacterium \| Terrestrail, Soil (A0A1F9JXN1) Dyadobacter beijingensis \| Terrestrial, Soil (UPI00037A3AB9) Chloroflexi bacterium \| Terrestrial, Soil (A0A1Q7MBJ3) Pseudonocardia spinosispora \| Terrestrial, Soil (UPI0003FB56B0) MAG SAR202-VII-2 (Ga0133547_10000002_0146) MAG SAR202-VII-2 (Ga0133547_10000004_0172) Streptomyces sp. \| Experimentally validated (Q7X284) Deltaproteobacteria bacterium \| Terrestrial, Soil (A0A1F9KQT30 Acidobacteria bacterium \| Terrestrial, Soil (A0A1Q6XSE4) Deltaproteobacteria bacterium \| Terrestrial, Soil (A0A1F9KR35) MAG SAR202-VII-2 (Ga0133547_10000004_0224) Hydrothermal vent metagenome \| Marine, Hydrothermal vent (A0A160V8U3) Chloroflexi bacterium \| Terrestrial, Soil (A0A1Q7QL69) Betaproteobacteria bacterium \| Freshwater, Groundwater (A0A1F3ZZ05) Puniceibacterium sp. IMCC21224 \| Marine, Coastal waters (A0A0J5QC59) Pseudonocardia acaciae \| Terrestrial, Acacia auriculiformis rhizosphere (UPI00048CFA4D) Betaproteobacteria bacterium \| Freshwater, Groundwater (A0A1F3ZF54) Marinobacter algicola DG893 \| Marine (A6F042) Hydrogenophaga pseudoflava \| (UPI00082636C0) MAGSAR202-VII-2 (Ga0133547_10000002_0528) Actinoplanes globisporus \| Terrestrial, Soil (UPI00035C3EFA) MAG SAR202-VII-2 (Ga0133547_10000360_0073) Hydrothermal vent metagenome \| Marine, Hydrothermal vent (A0A160V9N6) Rhizobiales bacterium 6217 \| Bioreactor (A0A1Q4BBC1) Metallosphaera yellowstonensis MK1 \| Freshwater, Thermal spring (H2C979) Streptomyces avicenniae \| Terrestrial, Avicennia mariana rhizosphere (UPI00069C8037) Ca. Rokubacteria \| Terrestrial, Soil (A0A1Q6Z3E1) Mycobacterium sp. JS623 \| Human, Soft tissue lesion (L0ITX6) Photorhabdus temperata \| Insect symbiont (W3V0B8) Cohnella kolymensis \| Terrestrial, Permafrost soil (A0A0C2QCW1) Allosalinactinospora lopnorensis \| Terrestrial, Tamarisk tree rhizosphere (UPI000623DDAE) Conexibacter woesei \| Terrestrial, Soil (D3FAR9) Stigmatella aurantiaca \| (A0A1H8DFG8) Pusillimonas noertemannii \| Freshwater, River water (UPI0002D6E76C) TOBG EAC-693 TOBG SP-320 Deltaproteobacteria bacterium \| Terrestrial, Soil (A0A1Q7J4E1) Micromonospora rhizosphaerae \| Terrestrial, Mangrove rhizosphere (A0A1C6SGZ1) Deltaproteobacteria bacterium \| Terrestrial, Soil (A0A1F9KR04) Azohydromonas lata \| (UPI000829F41D) Solirubrobacter sp. URHD0082 \| Terrestrial, Soil (UPI000422E0D1) Paracoccus versutus \| (A0A099FP16) Actinoplanes globisporus \| Terrestrial, Soil (UPI000371AF25) Magnaporthe oryzae \| Terrestrial (Q2KEQ4) Chloroflexi bacterium \| Terrestrial, Soil (A0A1Q8AAT0) Deltaproteobacteria bacterium \| Terrestrial, Soil (A0A1Q7JAY6) TOBG NAT-93 Ca. Acidianus copahuensis \| Freshwater, Acidic hot spring (A0A031LPX2) Frankia sp. Results h 3 Aromatic compound degradation genes and pathways in Arctic Ocean Chloroﬂexi MAGs. a Abundance of aromatic compound degradation genes in Chloroﬂexi MAGs from the Canada Basin (pie chart) and a breakdown of those found speciﬁcally in SAR202-VII-2 (column plot). b Predicted aromatic ring-opening enzymatic reactions identiﬁed in SAR202-VII-2 with gene loci displayed in the colored boxes. Genes in green and blue boxes were most closely related to homologs from terrestrial or marine bacteria, respectively. Genes in the blue/green boxes were in clades containing diverse environmental bacteria. c Examples of predicted aromatic ring-modifying enzymatic reactions identiﬁed in SAR202-VII-2 5 Decarboxylation Hydroxylation Demethylation 5,5′-dehydrodivanillate O-demethylase 4,5-dihydroxyphthalate decarboxylase 3-Phenylpropionate/ Trans-cinnamate dioxygenase Biphenyl 2,3-dioxygenase c HO HO HO HO HO HO HO HO HO OH O OH HO OH O O O O OH OH OH O OH O O HO OH OH O HO HO HO HO O O O O O O O H OH OH CH3 CH3 CH3 b c Decarboxylation 4,5-dihydroxyphthalate decarboxylase Demethylation Fig. 3 Aromatic compound degradation genes and pathways in Arctic Ocean Chloroﬂexi MAGs. a Abundance of aromatic compound degradation genes in Chloroﬂexi MAGs from the Canada Basin (pie chart) and a breakdown of those found speciﬁcally in SAR202-VII-2 (column plot). b Predicted aromatic ring-opening enzymatic reactions identiﬁed in SAR202-VII-2 with gene loci displayed in the colored boxes. Genes in green and blue boxes were most closely related to homologs from terrestrial or marine bacteria, respectively. Genes in the blue/green boxes were in clades containing diverse environmental bacteria. c Examples of predicted aromatic ring-modifying enzymatic reactions identiﬁed in SAR202-VII-2 COMMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio 5 5 ARTICLE COMMUNICATIONS BIOLOGY \| DOI: 10.1038/s42003-018-0086-7 Methods S li Sampling and DNA extraction. Twelve samples for metagenomics were collected in September 2015 during the Joint Ocean Ice Study cruise to the Canada Basin. For each sample, 4–8 L of seawater was sequentially ﬁltered through a 50 μm pore mesh, followed by a 3 μm pore size polycarbonate ﬁlter and a 0.22 μm pore size Sterivex ﬁlter (Durapore; Millipore, Billerica, MA, USA). Filters were preserved in RNAlater and stored at −80 °C until processed in the laboratory. DNA was extracted from the Sterivex ﬁlter using the following method: ﬁlters were thawed on ice and RNAlater was removed. The Sterivex was then rinsed twice with a sucrose- based lysis buffer, and ﬁlled with 1.8 mL of the lysis buffer. Filters were treated with 100 μL of 125 mg mL−1 lysozyme and 20 μL of 10 μg mL−1 RNAse A and left to rotate at 37 °C for 1 h. After incubation, 100 μL of 10 mg mL−1 proteinase K and 100 μL of 20% SDS was added. Filters were left to rotate for 2 h at 55 °C. Lysate was removed from the ﬁlters. Protein was precipitated and removed with 0.583 volumes of MPC Protein Precipitation Reagent (Epicentre, Madison, WI, USA) and cen- trifugation at 10,000×g at 4 °C for 10 min. The supernatant was transferred to a clean tube. DNA was precipitated with cold isopropanol, and resuspended in low TE buffer. Metagenomic fragment recruitment. The distribution of the Canada Basin MAGs in the global ocean was determined using best-hit reciprocal blast analysis similar to Landry et al.18 Unassembled metagenomic data from 25 samples (Supplementary Data 1) was ﬁrst recruited to the six Canada Basin Chloroﬂexi MAGS as well as two SAR202 SAGs originating from the deep North Paciﬁc Ocean from the Hawaiian ocean time series (HOTS) and the deep North Atlantic Ocean18. Metagenomes from the TARA Ocean project used here were repre- sentative of the surface, chlorophyll maximum and mesopelagic waters from the North Atlantic, South Atlantic, North Paciﬁc, Coastal North Paciﬁc, South Paciﬁc, Coast of Brazil and the Antarctic peninsula. To reduce computational demand, only part 1 (1 Gbp of a random subset of reads) of each metagenomic dataset available at EBI was used (Supplementary Data 1). Additional bathypelagic metagenomes from the North Paciﬁc and South Atlantic Oceans (LineP P04, P12 and P26, and Knorr S15 2500 m) were also included. Conclusions l h In total, these results are consistent with Chloroﬂexi having a role in tDOM transformation in waters of the Arctic Ocean. This is the ﬁrst study to our knowledge to associate a speciﬁc microbial group with tDOM metabolism in the Arctic Ocean and it expands on recent studies contributing to our understanding of the metabolic diversity of the abundant yet uncultivated marine Chloroﬂexi18,23. Moreover, lateral gene transfer from terrestrial bacteria appears to have contributed to the evolution of aromatic compound degradation capabilities within marine Chloroﬂexi, particularly in regions of the Arctic Ocean impacted by terrestrial input. Single protein and concatenated protein phylogenies. A concatenated protein phylogeny was constructed using 30 Chloroﬂexi reference genomes and the 6 Canada Basin Chloroﬂexi MAGs. Orthologous genes in the 36 Chloroﬂexi genomes were identiﬁed using ProteinOrtho38. Fifty orthologs present in at least 34 of the 36 genomes were selected for concatenated phylogenetic analysis (Sup- plemetary Data 3). Each orthologous protein family was aligned using MUSCLE (implemented in MEGA6) and alignment positions were masked using the prob- abilistic masker ZORRO39, masking columns with weights <0.5. The concatenated alignment consisted of 14,815 amino acid positions. Phylogenetic reconstructions were conducted by maximum likelihood using MEGA6-v.0.6 and the following settings: JTT substitution model, gamma distribution with invariant sites model for the rate variation with four discrete gamma categories, and the nearest-neighbor interchange (NNI) heuristic search method40 with a bootstrap analysis using 100 replicates. p The majority of MAGs were restricted to the humic-rich Paciﬁc-origin halocline of the Canada Basin, however it is the surface waters that will be most immediately affected by increased freshwater input1. Hence, our initial observations suggest a need for further research on the distribution of tDOM-utilizing microbes in other Arctic water masses with an aim to establish how common and phylogenetically widespread tDOM metabo- lism is in the Arctic Ocean. These water masses could include coastal surface waters at the mouth of the Mackenzie River, as well as regions of differing DOM composition such as the East Siberian Sea20. Moreover, metagenomic studies such as this are, in essence, hypothesis-generating and future work that includes targeted cultivation, in situ gene expression analysis, and rate measurement-based approaches are required to validate and quantify microbial metabolic contributions to nutrient cycling. Conclusions l h Overall, it is likely that marine Chloroﬂexi have the capacity to degrade tDOM, and their role in the Arctic carbon cycle may increase as Arctic warming leads to greater inputs of terrestrial organic matter. p For phylogenetic analysis of ring-cleaving dioxygenase sequences identiﬁed in SAR202-VII-2, query sequences were searched against UniRef90 and 130 Chloroﬂexi MAGs constructed from the TARA Oceans dataset29. The TARA Ocean Chloroﬂexi MAGs were used as is with no manual curation. UniRef90 sequences and the top TARA Ocean MAG hits for each dioxygenase were aligned with their respective SAR202-VII-2 homologs with MUSCLE (implemented in MEGA6) and alignment positions were masked using the probabilistic masker ZORRO39, masking columns with weights less than 0.5. Phylogenetic reconstruction was conducted using the same settings as the concatenated phylogeny. Comparative genomics and metabolic reconstruction. The distribution of orthologs across Arctic Ocean genomes, as well as the identiﬁcation of orthologs shared with the deep ocean SAGs, was determined using proteinortho38. Inference of protein function and metabolic reconstruction was based on the IMG annota- tions provided by the JGI, including KEGG, Pfam, EC numbers, and Metacyc annotations. Metabolic reconstruction was also facilitated by generated pathway genome databases for each MAG using the pathologic software available through Pathway Tools41. ARTICLE The taxonomic identity of MAGs was assessed using a concatenated phylogenetic tree based on 138 single copy conserved genes as implemented in MetaWatt32. Visualization of the tree and mapping of data on to taxa was performed with iTOL33. Estimation of MAG completeness and contamination was performed using CheckM34. Six Chloroﬂexi MAGs were selected for further analysis based on the presence of a 16S rRNA gene, high completeness and low contamination. Manual curation of the six Chloroﬂexi MAGs was performed and suspected contaminating scaffolds (single copy genes most similar to non-Chloroﬂexi taxa) were removed prior to further analysis of MAGs. 16S rRNA phylogenetic analysis. Chloroﬂexi diversity in the metagenomic assembly was assessed by 16S rRNA gene analysis. All 16S rRNA genes in the co- assembly were assigned to taxonomic groups using mothur35 and the Wang method with a bootstrap value cutoff of 60%36. Chloroﬂexi 16S rRNA genes greater than 360 bp were included in a phylogenetic analysis with Chloroﬂexi reference sequences. A multiple sequence alignment was generated using MUSCLE (imple- mented in MEGA6)37. Phylogenetic reconstructions were conducted by maximum likelihood using MEGA6-v.0.6 and the following settings: general time reversible model, gamma distribution model for the rate variation with four discrete gamma categories, and the nearest-neighbor interchange (NNI) heuristic search method38 with a bootstrap analysis using 100 replicates. ARTICLE ARTICLE and 6), Beta-proteobacteria (Supplementary Figure 7) and a clade of diverse terrestrial phyla (Supplementary Figure 8). Addition- ally, 2 gentisate 1,2-dioxygenase genes and 1 protocatechuate dioxygenase ligB gene were phylogenetically associated to a clade of genes from both terrestrial Delta-proteobacteria and marine microbes (Fig. 4 and Supplementary Figure 8). These putative gene acquisitions were unlikely due to contaminating scaffolds because the genes were located on long scaffolds that were assigned to Chloroﬂexi with high conﬁdence based on tet- ranucleotide frequencies and the phylogenetic identity of house- keeping genes. Such a phylogenetic pattern supports the hypothesis that marine Chloroﬂexi acquired the capacity for aromatic compound degradation, at least in part, by lateral gene transfer from terrestrial bacteria. Paired-end sequences of 2 × 150 bp were generated for all libraries. A metagenome co-assembly of all raw reads was generated using MEGAHIT30 with kmer sizes of 23,43,63,83,103,123. Gene prediction and annotation was performed using the DOE Joint Genome Institute’s Integrated Microbial Genomes (IMG) database tool (version 4.11.0)31. Metagenomic binning was performed on scaffolds ≥10 kb in length using MetaWatt32. Relative weight of coverage binning was set to 0.75 and the optimize bins and polish bins options were set to on. The taxonomic identity of MAGs was assessed using a concatenated phylogenetic tree based on 138 single copy conserved genes as implemented in MetaWatt32. Visualization of the tree and mapping of data on to taxa was performed with iTOL33. Estimation of MAG completeness and contamination was performed using CheckM34. Six Chloroﬂexi MAGs were selected for further analysis based on the presence of a 16S rRNA gene, high completeness and low contamination. Manual curation of the six Chloroﬂexi MAGs was performed and suspected contaminating scaffolds (single copy genes most similar to non-Chloroﬂexi taxa) were removed prior to further analysis of MAGs. Paired-end sequences of 2 × 150 bp were generated for all libraries. A metagenome co-assembly of all raw reads was generated using MEGAHIT30 with kmer sizes of 23,43,63,83,103,123. Gene prediction and annotation was performed using the DOE Joint Genome Institute’s Integrated Microbial Genomes (IMG) database tool (version 4.11.0)31. Metagenomic binning was performed on scaffolds ≥10 kb in length using MetaWatt32. Relative weight of coverage binning was set to 0.75 and the optimize bins and polish bins options were set to on. COMMUNICATIONS BIOLOGY \| DOI: 10.1038/s42003-018-0086-7 Iso899 \| Terrestrial, Soil (UPI0003B34178) MAG SAR202-VII-2 (Ga0133547_10000060_0179) Burkholderiaceae arationis LMG 29324 \| Terrestrial, Soil (A0A158B6L5) Sulfitobacter geojensis \| Marine, Coastal waters (UPI00046AA492) Acidobacteria bacterium \| Terrestrial, Soil (A0A1Q7MJD6) Streptomyces sp. MnatMPM17 \| Terrestrial, Soil (A0A1C5CS47) TOBG SAT-91 Acidobacteria bacterium \| Freshwater, Groundwater (A0A1F2T6K4) Armatimonadetes bacterium CSP13 \| Freshwater sediments (A0A0T6ART8) TOBG SP-253 Marine sediment metagenome \| Marine sediments (A0A0F9CXY8) Alphaproteobacteria bacterium \| Terrestrial, Soil (A0A1Q7BII2) Marine microorganism HF4000ANIW93N21 \| Marine (B3T345) MAG SAR202-VII-2 (Ga0133547_10000103_0019) Nodosilinea nodulosa \| Marine (UPI0003025B02) Haloferax sp. \| Experimentally validated (Q330M9) Deltaproteobacteria bacterium \| Freshwater, Groundwater (A0A1F8XVR5) Chromobacterium sp. LK11 \| Terrestrial (A0A0J6NGJ2) Nocardia arthritidis \| Human (UPI0007A3EF410) Pseudonocardia sp. CNS139 \| Marine, Sediment (A0A1Q9SZW9) 87 100 96 100 78 100 98 100 92 100 100 75 83 100 93 99 100 100 100 73 100 100 100 100 98 94 65 100 74 100 100 64 100 97 100 60 95 74 100 93 Actinomycetales kelihood tree of predicted gentisate 1,2-dioxygenases. Bootstrap values of >60% are shown (100 replicates). Homologs ed in red and homologs from TARA Ocean MAGs are highlighted in blue Fig. 4 Maximum likelihood tree of predicted gentisate 1,2-dioxygenases. Bootstrap values of >60% are shown (100 replicates). Homologs from SAR202- VII-2 are highlighted in red and homologs from TARA Ocean MAGs are highlighted in blue Fig. 4 Maximum likelihood tree of predicted gentisate 1,2-dioxygenases. Bootstrap values of >60% are shown (100 replicates). Homologs from SAR202- VII-2 are highlighted in red and homologs from TARA Ocean MAGs are highlighted in blue Fig. 4 Maximum likelihood tree of predicted gentisate 1,2-dioxygenases. Bootstrap values of >60% are shown (100 replicates). Homologs from SAR202- VII-2 are highlighted in red and homologs from TARA Ocean MAGs are highlighted in blue Fig. 4 Maximum likelihood tree of predicted gentisate 1,2-dioxygenases. Bootstrap values of >60% are shown (100 replicates). Homologs from SAR202- VII-2 are highlighted in red and homologs from TARA Ocean MAGs are highlighted in blue COMMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio 6 COMMUNICATIONS BIOLOGY \| DOI: 10.1038/s42003-018-0086-7 References Biochemistry of Marine Dissolved Organic Matter 2nd ed (Academic Press, Amsterdam, 2015). 34. Parks, D. H., Imelfort, M., Skennerton, C. T., Hugenholtz, P. & Tyson, G. W. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 25, 1043–1055 (2015). 9. Kaiser, K., Benner, R. & Amon, R. M. W. The fate of terrigenous dissolved organic carbon on the Eurasian shelves and export to the North Atlantic. J. Geophy. Res. 122, 4–20 (2017). 35. Schloss, P. D. et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537–7541 (2009). 10. 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Oceanogr. 44, 2017–2022 (1999). 30. Li, D., Liu, C.-M., Luo, R., Sadakane, K. & Lam, T.-W. MEGAHIT: an ultra- fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics 31, 1674–1676 (2015). 4. Bintanja, R. & Andry, O. Towards a rain-dominated Arctic. Nat. Clim. Change 7, 263–267 (2017). 31. Huntemann, M. et al. The standard operating procedure of the DOE-JGI Metagenome Annotation Pipeline (MAP v.4). Stand. Genom. Sci. 11, 1–17 (2016). 5. Frey, K. E. & McCelland, J. W. Impacts of permafrost degradation on arctic river biogeochemistry. Hydrol. Process 23, 169–182 (2009). g y y 6. Vonk, J. E. et al. Activation of old carbon by erosion of coastal and subsea permafrost in Arctic Siberia Nature 489 137 140 (2012) 6. Vonk, J. E. et al. Activation of old carbon by erosion of coastal and subsea permafrost in Arctic Siberia. Nature 489, 137–140 (2012). 32. Strous, M., Kraft, B., Bisdorf, R. & Tegetmeyer, H. E. 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Characterization of the gallate dioxygenases are involved in syringate degradation by Sphingomonas paucimobilis SYK-6. J. Bacteriol. 187, 5067–5074 (2005). 26. Werwath, J., Arfmann, H.-A., Pieper, D. H., Timmis, K. N. & Wittich, R.-M. Biochemical and genetic characterization of a gentisate 1,2-dioxygenase from Sphingomonas sp. Strain RW5. J. Bacteriol. 180, 4171–4176 (1998). Methods S li All hits from the initial blast were then reciprocally queried against the Canada Basin Chloroﬂexi MAGs, bathypelagic SAR202 SAGs, and 130 Chloroﬂexi MAGs constructed from the Metagenomic sequencing, assembly, annotation, and binning. DNA sequen- cing of 12 samples was performed at the Department of Energy Joint Genome Institute (Walnut Creek, CA, USA) on the HiSeq 2500-1TB (Illumina) platform. 7 COMMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio MMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio COMMUNICATIONS BIOLOGY \| DOI: 10.1038/s42003-018-0086-7 COMMUNICATIONS BIOLOGY \| DOI: 10.1038/s42003-018-0086-7 TARA oceans data. The best-hit was reported. Only hits with an alignment length ≥100 bp and a percent identity of 95% or more were counted (lower % identity cut- offs did not alter the number of reads recruited in any signiﬁcant manner). To compare the results among the different datasets, the number of recruited reads was normalized to total number of reads in each sample. The ﬁnal coverage results were expressed as the number of reads per kilobase of the MAG per gigabase of metagenome (rpkg). 18. Landry, Z., Swan, B. K., Herndl, G. J., Stepanauskas, R. & Giovannoni, S. J. SAR202 genomes from the dark ocean predict pathways for the oxidation of recalcitrant dissolved organic matter. mBio 8, e00413–e00417 (2017). 19. Bano, N. & Hollibaugh, J. T. Phylogenetic composition of bacterioplankton assemblages from the Arctic ocean. Appl. Environ. Microbiol. 68, 505–528 (2002). 20. Guéguen, C. et al. The nature of colored dissolved organic matter in the southern Canada Basin and East Siberian Sea. Deep Sea Res. Part 2 81-84, 102–113 (2012). Data availability. The metagenomic data generated in this study are available in the Integrated Microbial Genomes database at the Joint Genome Institute at https://img.jgi.doe.gov, GOLD Project ID: Ga0133547. Metagenome-assembled genome projects have been deposited at DDBJ/ENA/GenBank under the Bioproject PRJNA471535 and accession numbers QGNM00000000 (for SAR202-II-3), QGNN00000000 (for SAR202-II-177A), QGNO00000000 (for SAR202-VI-29A), QEVV00000000 (for SAR202-VII-2), QGNP00000000 (for Anck29-46), and QGNQ00000000 (for TK10-74A). The versions described in this paper are versions QGNM01000000 (for SAR202-II-3), QGNN01000000 (for SAR202-II-177A), QGNO01000000 (for SAR202-VI-29A), QEVV01000000 (for SAR202-VII-2), QGNP01000000 (for Anck29-46), and QGNQ01000000 (for TK10-74A). 21. Dainard, P. G. & Guéguen, C. Distribution of PARAFAC modeled CDOM components in the North Paciﬁc Ocean, Bering, Chukchi and Beaufort Seas. Mar. Chem. 157, 216–223 (2013). 22. Dainard, P. G., Guéguen, C., McDonald, N. & Williams, W. J. Photobleaching of ﬂuorescent dissolved organic matter in Beaufort Sea and North Atlantic Subtropical Gyre. Mar. Chem. 177, 630–637 (2015). p y 23. Thrash, J. C. et al. Metabolic roles of uncultivated bacterioplankton lineages in the Northern Gulf of Mexico “dead zone”. mBio 8, e01017–e01017 (2017). 24. Barry, K. P. & Taylor, E. A. Characterizing the promiscuity of LigAB, a lignin catabolite degrading extradiol dioxygenase from Sphingomonas paucimobilis SYK-6. Biochemistry 52, 6724–6736 (2013). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Research Chair Program (D.W., C.G.) are acknowledged. D.C. was supported by FRQNT and Concordia’s Institute for Water, Energy and Sustainable Systems. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. COMMUNICATIONS BIOLOGY \| DOI: 10.1038/s42003-018-0086-7 Competing interests: The authors declare no competing interests. DE-AC02-05CH11231. Funding from the Canadian Natural Science and Engineering Research Council (NSERC) Discovery grants (D.W., C.G. and C.L.) and the Canada Research Chair Program (D.W., C.G.) are acknowledged. D.C. was supported by FRQNT and Concordia’s Institute for Water, Energy and Sustainable Systems. DE-AC02-05CH11231. Funding from the Canadian Natural Science and Engineering Research Council (NSERC) Discovery grants (D.W., C.G. and C.L.) and the Canada Acknowledgements 16. Urbach, E., Vergin, K. L., Larson, G. L. & Giovannoni, S. J. Bacterioplankton communities of Crater Lake, OR: dynamic changes with euphotic zone food web structure and stable deep water populations. Hydrobiologia 574, 161–177 (2007). The data were collected aboard the CCGS Louis S. St-Laurent in collaboration with researchers from Fisheries and Oceans Canada at the Institute of Ocean Sciences and Woods Hole Oceanographic Institution’s Beaufort Gyre Exploration Program and are available at http://www.whoi.edu/beaufortgyre. We would like to thank both the 17. Yamada, T. & Sekiguchi, Y. Cultivation of uncultured Chloroﬂexi subphyla: signiﬁcance and ecophysiology of formerly uncultured Chloroﬂexi “subphylum I” with natural and biotechnological relevance. Microbes Environ. 24, 205–216 (2009). Captain and crew of the CCGS Louis S. St-Laurent and the scientiﬁc teams aboard. The work was conducted in collaboration with the U.S. Department of Energy Joint Genome Institute, a DOE Ofﬁce of Science User Facility, and was supported under Contract No. 8 COMMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio ARTICLE COMMUNICATIONS BIOLOGY \| DOI: 10.1038/s42003-018-0086-7 Authors contributions D.C. and D.W. designed and carried out the metagenomic experiments. W.J.W., C.L. and C.G. designed the sampling strategy. W.J.W. provided oceanographic data. D.W. col- lected samples. D.C. extracted the environmental genomic DNA and analyzed sequen- cing data. P.T. contributed to the bioinformatic analyses. D.C. and D.W. with contributions from C.G. and C.L. wrote the manuscript. All authors commented on the manuscript. 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Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Additional information l f © The Author(s) 2018 Supplementary information accompanies this paper at https://doi.org/10.1038/s42003- 018-0086-7. © The Author(s) 2018 COMMUNICATIONS BIOLOGY \| (2018) 1:90 \| DOI: 10.1038/s42003-018-0086-7 \| www.nature.com/commsbio 9
https://openalex.org/W4385718095	https://www.researchsquare.com/article/rs-3215723/latest.pdf	English	null	Factors affecting public transportation in the Covid-19 period	Research Square (Research Square)	2,023	cc-by	8,966	Factors affecting public period Tünde Kovács (  kovacs.tunde.zita@econ.unid University of Debrecen László Huzsvai University of Debrecen Adrián Nagy University of Debrecen András Nábrádi University of Debrecen Szabolcs Tóth University of Debrecen Beáta Bittner University of Debrecen Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Sustainability on October 26th, 2023. See the published version at https://doi.org/10.3390/su152115310. Page 1/24 Page 1/24 Abstract In the European Union Member States, the share of public transport use from the turn of the millennium to the beginning of the pandemic period was 17–18%, while in Hungary, it was 27%. The number of public transport users has fallen due to the Covid-19 virus to 13% in the EU and 21% in Hungary. The decrease can be attributed to changes in travel habits and the impact of the measures taken in the context of the virus situation. In Debrecen, Hungary's second-largest city, the situation is similar. During a shorter period of the first wave of the virus, the public service operator realized only 30% of its usual revenue. The present study analyzes the relationship between the number of active cases of Covid-19, the impact of the measures taken and the number of paying passengers on public transport in Debrecen. Four hypotheses were put forward: 1) travel tickets, 2) general passes, 3) discount passes and 4) supplement passes were influenced by the evolution of Covid-19's active caseload. The data were collected from the Debrecen Transport Company (DKV) for 2020.01–2021.12, and the active Covid-19 case numbers were collected from the Worldometers.info database. Statistical analyses were performed using the ARMA (autoregressive and moving-average) model. We found that all four of our hypotheses had to be rejected, as Covid's active caseload did not influence sales of tickets and various passes. 1. Introduction With the intensification of urbanization processes, the evolution of urban mobility and the possibilities for its development are nowadays a focus of the transport profession (Barbosa et al., 2021). In recent decades, private transport has played an increasingly important role in passenger transport in the Member States of the European Union (EU). In contrast, the use of public transport has gradually decreased, which was also observed in Hungary (Hungarian Central Statistical Office (KSH), 2021). Increasing road congestion means it takes longer to reach our destinations (Pouliakas, 2018). These processes are becoming an increasing problem for the livability of cities, with more and more vehicles on the roads. Several factors influence the decline in the number of passengers using public transport. In addition to the dynamic increase in the number of cars, the development of cycling, scooter transport and ridesharing systems may also reduce the number of people using public transport (Anagnostopoulou et al., 2020). In recent years, in addition to the underlying decline in passenger numbers, the effects of the Covid-19 pandemic and the measures taken during this period have also reduced the number of people using public transport. According to Continental's Mobility Study 2020, the Covid-19 pandemic has increased the importance of private or individual transport (Continental AG, 2020; Furcher et al., 2021). At the same time, the number of people using public transport has fallen, for example, by half in Germany and over 50% in Japan and China. An analysis of the proportion of individual and public transport users in the EU Member States shows that from the turn of the millennium until the beginning of the pandemic, the proportion of people using public transport for passenger transport was around 17–18%. The share of rail and suburban and local transport users (Fig. 1) remained relatively stagnant during these years. Although these figures still show a more favourable picture for Hungary than the EU average, they show a significant downward trend over this period. The overall 38% in the early 2000s had fallen below Hungary's 27% modal split by the year before the pandemic. On 11 March 2020, the World Health Organization (WHO) declared the outbreak of the new coronavirus a global pandemic (WHO, 2020). 1. Introduction At the same time, the Hungarian government declared a state of emergency and a state Page 2/24 Page 2/24 Page 2/24 of public health emergency for the whole country on the same day (Ministry of Justice, 2020b). The decree was followed by a series of restrictive measures, all with one thing in common: limiting personal contacts to prevent the spread of the epidemic. These provisions had severe economic and social implications, including mobility in all its forms. Today, the proportion of people using social networking tools has fallen further due to the viral situation, to 13% in the EU and 21% in Hungary. It can be argued that this is due to changes in travel habits during the pandemic period (people's fear of the virus spreading faster in confined spaces) and the effects of the measures taken in the context of the epidemic situation, which has had a significant impact on public transport in the city of Debrecen. The seat occupancy rate and fare revenues have been significantly reduced. During the first, shorter period of the first wave of the virus, the public service operator realized about 30% of its regular revenues. Transport experts had already predicted a slower "bounce-back" at the beginning of the virus' spread, expecting the population to return to public transport after up to five years. That ridership would return to the levels seen in previous years. The two years since then confirm these predictions, with a slow recovery in ridership and revenue. This study examines the impact of the Covid-19 epidemic and the measures taken to evolve the number of paying passengers in the city of Debrecen. The following working hypotheses have been formulated: H1: Sales of travel tickets were influenced by the evolution of active caseloads. H2: Sales of general passes were influenced by the evolution of active caseloads. H3: Sales of discounted passes were influenced by the evolution of active caseloads. H3: Sales of discounted passes were influenced by the evolution of active caseloads. H4: Sales of supplement passes were influenced by the evolution of active caseloads. H4: Sales of supplement passes were influenced by the evolution of active caseloads. 2. Literature review Their analysis highlights that, instead of strict lockdowns that might seriously damage the economy, milder travel restrictions could have a similar impact on controlling the domestic transmission of Covid-19 without devastating economic damage. Tirachini & Cats (2020) stated if public transportation is perceived as unsafe and unhealthy by large segments of the population, it will not be able to fulfill the societal roles that it is set to serve, including accessibility, sustainability, and equity. Our societies need public transportation services to prosper and to address key societal challenges that are paramount and persistent. It is therefore critical to avoid contributing to stereotyping the use of public transportation as unhealthy, which may outlive the pandemic itself and hinder the long-term prospects of public transportation services. The results of the above studies, while different in approach, confirm that over-restrictive measures and closures impose more burdens on the economy than they bring benefits. In the present study, we examine this at the firm level, from the perspective of a public transport operator, by looking at the effects of Covid-19 measures. 2. Literature review In this paper, we look at the impact of COVID-19 on public transport. The study's uniqueness lies in the fact that we analyzed not only the relationship between the number of active cases of Covid-19 but also the impact of the measures taken and the number of paying passengers on public transport. Many studies have examined the impact of the pandemic on the service sector since its spread in 2020 (Bittner & Gavaldi, 2021; Bittner & Posta, 2020; Dominiak, 2022; Gunay & Kurtulmus, 2021; Lee & Eom, 2023; Mazzucato & Kattel, 2020; Prentice et al., 2021; Vida & Popovics, 2020; Xiang et al., 2021). Still, only 44 in the Web of Science (WoS) database collection have examined the effects of Covid-19 restrictions on public transport. Research by Meena & Sharma (2020) and Tóth et al. (2022) shows that since the pandemic, consumers have preferred to travel individually rather than by public transport. The study by Fumagalli et al. (2022) in Curitiba, Brazil, showed an 80% drop in public transport in March 2020, with 1,36 million daily passengers down to 200.000. In line with our present research, they also showed that this was partly due to the spread of the virus, but they also highlighted that it was partly due to strict measures such as cancelling more flights. However, they did not investigate the role of case numbers and measures in this decline. They found that 90% of passengers use the same route every day, given that a large proportion of the travelling public are commuters and students, and suggested that their travel habits should be studied to design ways and services that are convenient for them. Page 3/24 Page 3/24 Proper scaling and scheduling to connect people's daily activities can bring more comfort and safety during the Covid-19 pandemic and give users more time synchronized with their routines. Luo et al. (2022) also came to this conclusion when studying public transport in New York. Based on the Covid-19 pandemic, they stated that a well-planned subway system in New York City could sustain 88% of transit flow while reducing the risk of disease transmission by 50% relative to fully-loaded public transit systems. Wang et al. (2022) analyzed the effects of government measures by comparing data from 121 countries on morbidity and mortality. They highlighted misguided measures and their consequences, focusing on public transport. 2. Literature review They concluded that without public transportation closures, cases and deaths would have been reduced by 40% and 10%, respectively. In Japan, Murano et al. (2021) examined the impact of government measures in the context of public transport, but they looked at the effect on the spread of the virus, not the economics, as this study does. However, many of their conclusions align with the present study's findings. Their analysis highlights that, instead of strict lockdowns that might seriously damage the economy, milder travel restrictions could have a similar impact on controlling the domestic transmission of Covid-19 without devastating economic damage. Tirachini & Cats (2020) stated if public transportation is perceived as unsafe and unhealthy by large segments of the population, it will not be able to fulfill the societal roles that it is set to serve, including accessibility, sustainability, and equity. Our societies need public transportation services to prosper and to address key societal challenges that are paramount and persistent. It is therefore critical to avoid contributing to stereotyping the use of public transportation as unhealthy, which may outlive the pandemic itself and hinder the long-term prospects of public transportation services. Proper scaling and scheduling to connect people's daily activities can bring more comfort and safety during the Covid-19 pandemic and give users more time synchronized with their routines. Luo et al. (2022) also came to this conclusion when studying public transport in New York. Based on the Covid-19 pandemic, they stated that a well-planned subway system in New York City could sustain 88% of transit flow while reducing the risk of disease transmission by 50% relative to fully-loaded public transit systems. Wang et al. (2022) analyzed the effects of government measures by comparing data from 121 countries on morbidity and mortality. They highlighted misguided measures and their consequences, focusing on public transport. They concluded that without public transportation closures, cases and deaths would have been reduced by 40% and 10%, respectively. In Japan, Murano et al. (2021) examined the impact of government measures in the context of public transport, but they looked at the effect on the spread of the virus, not the economics, as this study does. However, many of their conclusions align with the present study's findings. 3. Public transportation in Debrecen The evolution of passenger numbers in the city of Debrecen also shows changes in the modal split at the European and national transport levels (Tóth et al., 2022). In the years preceding the pandemic, passenger numbers showed a slight increase due to the stability and improvements in service quality. Nearly 100 million trips are made to the city each year. Commuters mainly use the services on their daily commute to work and school. In Debrecen, tourism has become increasingly important in recent years, so the number of ad-hoc trips is also significant. These passengers mainly buy one-way travel tickets or day tickets. Thanks to its geographical location and role as an economic, educational and health centre, the city is a prime destination for daily commuters from the agglomeration and the surrounding cities. In addition to the travel tickets, these residents can buy supplement passes to accompany general passes. The city's sales figures show that around 25% of fare revenue comes from travel tickets and 75% from passes. Within passes, combined general passes represent about 51%, discounted passes 36%, and supplement passes 13% (Fig. 2). The most significant number of passengers buying passes comprises workers and students, representing over 90% of total passes. Page 4/24 Page 4/24 Page 4/24 In addition to fare revenue, a significant share of revenue is represented by social policy fare subsidies, which are the supplementary fare for discounted passes and the subsidy for free travel. In addition to fare revenue, a significant share of revenue is represented by social policy fare subsidies, which are the supplementary fare for discounted passes and the subsidy for free travel. Data from travel ticket and pass sales cannot always be linked to the actual use of the service. While the actual use time can be well determined for general passes and travel tickets, this cannot be reported for pre-purchase tickets. Tickets are sold through external partners in addition to service ticket offices, ticket vending machines and onboard points. These partners tend to stock larger quantities at a time, which may distort the time series derived from the sales data and the time series characterizing the use of fare products. Typically, they occur at the beginning and end of the year, but also continuously. Travel tickets are used in local public transport for ad-hoc journeys, including tourists arriving in the city, but the less frequent traveller population buys these fare products. 3. Public transportation in Debrecen A segment of the population also mainly uses these services in combination with private transport, for example, people who use these means of transport to access the city centre less frequently, to do business or for entertainment. Many car users also park their cars in external nonpaying zones and use public transport to reach their destination due to the high parking tariffs in the city centre. A slight increase in the consumption of travel tickets is most noticeable in the summer months, which is to a lesser extent linked to the school holidays when students no longer travel daily. However, more frequent, daily travellers will buy longer distance, unlimited-use fare products, e.g. (monthly/yearly) passes, after considering whether the network and the associated timetable service is an appropriate transport alternative to reach their destination. Seasonality can also be observed for these passes, as, for example, a significant part of annual holidays is spent in the summer and winter months, so fractional months are solved by using a travel ticket for a shorter period or ad hoc travel or even by other means of transport. Discounted passes also show a diverging trend during school and non-school periods, primarily reflecting the travel habits of students. The long-term trend in selling discounted passes for pensioners is affected by the constantly decreasing number of people entitled, as the number of retirees under 65 is falling yearly. 4.1. Used databases The sales records of the DKV for the period between 01.01.2020 and 31.12.2021 were provided by the company's sales department. The sales data were provided monthly, but in the case of the means of transport entitlement, we aggregated the data and created four main groups, as follows: The group of travel tickets include the following items: single advance tickets, single mobile tickets, a single ticket from the driver, a block of 11 tickets, small group tickets, one our mobile tickets, one-, three- and seven-day tickets, tickets for student groups and three-day family tickets. The group of travel tickets include the following items: single advance tickets, single mobile tickets, a single ticket from the driver, a block of 11 tickets, small group tickets, one our mobile tickets, one-, three- and seven-day tickets, tickets for student groups and three-day family tickets. General passes include half-monthly, monthly, yearly, and transferable passes General passes include half-monthly, monthly, yearly, and transferable passes as a group rate. The following passes have been grouped under discounted passes: student monthly combined passes, pensioner monthly combined passes and passenger with young children pass. The following passes have been grouped under discounted passes: student monthly combined passes, pensioner monthly combined passes and passenger with young children pass. Page 5/24 Page 5/24 The supplement passes include the general monthly pass supplements and the student monthly pass supplements. The supplement passes include the general monthly pass supplements and the student monthly pass supplements. The groups representing each travel entitlement are based on the DKV recommendation. Data related to the Covid-19 epidemic are from Worldometers.info (2022). Worldmeters' publisher is a small, independent, US-based digital media company, and the operating team is made up of developers, cadres and volunteers. Worldmeter's data on the outbreak is obtained from official government reports or indirectly from local media sources deemed reliable by the publisher. For our calculations of the outbreak data, we used the Active Case Rate indicator, which is calculated using the formula below: The Active Cases = (total cases) - (total deaths) – (recovered) so the active case figure represents the current number of people detected and confirmed to be infected with the virus. Data published on the Worldmeters.info website are daily. Sometimes, we must combine daily data and produce monthly statistics. 4.2. Correlation analysis and stochastic time series ARMA model Pearson's correlation coefficient (1901) was used to test the correlation between the different means of providing transport entitlements. The correlation between active caseloads and travel entitlements was performed using stochastic time series analysis. We chose this method because we had a relatively short time series with only 24 months of data. On the other hand, we believe that random effects and stresses on the process are built into the phenomenon and have a process-building role in the longer term. Time series that considers the role of chance, stochastic time series, started to become known in the 1970s. During this period, Box and Jenkins popularized the use of autoregressive integrated moving average (ARIMA) models (Bartholomew, 1971; Box & Tiao, 1975). First, we examined the stationarity of the data series. For this, we used the Augmented Dickey-Fuller (ADF) test. The null hypothesis of this test is that the time series has a unit root, i.e. it is not stationary. Based on the ADF test, we obtained significant results for all travel entitlements; the null hypothesis was rejected, i.e. the processes are stationary. The stationary time series do not contain trend effects, and the time series values fluctuate around a constant mean value with a constant standard deviation. Stationary time series is also characterized by the temporal constancy of the autocorrelation coefficients of the time series, representing the internal relationships between the data of the time series. The values of the autocorrelation coefficients depend on the distance between variables, on lags, but not on time. The autocorrelation coefficients as a function of the lag are given by the autocorrelation function, also known as the correlogram. In the theory and practice of time series analysis, autoregressive and moving average (ARMA) processes have gained significant importance since the 1970s because ARMA processes are mathematically tractable, and many of the random processes that occur in practice and follow stationary behaviour can be well approximated and described by them (Zdjelar et al., 2019). Stationary processes can be well-modelled with ARMA models. Such processes can be economical, biological, industrial, chemical and others. Thus, the use of ARMA processes is indeed wide-ranging. Page 6/24 In this model, the number of active cases can be included as a regressor. Its coefficient shows how Covid-19 affects the evolution of different travel entitlements. The indicator autoregressive (AR) indicates that the process can be described as a linear regression in its history. 4.2. Correlation analysis and stochastic time series ARMA model Furthermore, the moving average (MA) indicator expresses that the "error term" of the linear regression is the moving average of the white noise εt, i.e. the linear combination of the present and the finite past. The first-order ARMA(1, 1) model with regressor (R) was of the following form: yt = C + ϕ1yt−1 + βRt + ϵt + θ1ϵt−1 The first-order ARMA(1, 1) model with regressor (R) was of the following form: yt = C + ϕ1yt−1 + βRt + ϵt + θ1ϵt−1 he first-order ARMA(1, 1) model with regressor (R) was of the following form: yt = C + ϕ1yt−1 + βRt + ϵt + θ1ϵt−1 yt = C + ϕ1yt−1 + βRt + ϵt + θ1ϵt−1 where: yt Amount of travel entitlement for the t time; yt Amount of travel entitlement for the t time; φ1 Coefficient of the autoregressive term; : Moving average coefficient; θ1 εt−1 Error term of t-1 time. εt−1 Error term of t-1 time. 5. Public transportation in Debrecen during the Covid-19 pandemic In Hungary, and with it in Debrecen, public transport has been significantly affected by the government and municipal measures in the emergency. The measures taken in the context of the emergency declared in March 2020 significantly impacted passenger numbers and, thus, the revenue from passengers and the costs incurred to facilitate the protection. The measures that have had the most significant impact on public transport ridership are presented in Table 1. The changes in travel patterns brought about by the virus, the measures taken, and the emergence of atypical employment and distance learning have significantly impacted travel demand and the development of paying passenger numbers. The public service operator in the city of Debrecen has also suffered a significant drop in demand for travel and, thus, a significant loss of revenue. In the first months of the pandemic, with the launch of distance learning, sales of student passes fell to 6%, but sales of general passes also fell to 40%. Ad-hoc travel also dropped sharply, with ticket volumes below 13% in the first month of the pandemic. Page 7/24 Page 7/24 Table 1 Government's restrictive measures during the Covid-19 pandemic Measure Begin End Government decision number Remote learning I. 16 March 2020 15 June 2020 1102/2020 (Ministry of Justice, 2020c) Curfew I. 28 March 2020 30 April 2020 71/2020 (Ministry of Justice, 2020a) Wearing mask I. 01 May 2020 26 June 2021 168/2020 (Ministry of Justice, 2020e) Free travel entitlement 01 April 2020 30 June 2022 486/2020 (Ministry of Justice, 2020f) Free parking I. 06 April 2020 17 June 2020 87/2020 (Ministry of Justice, 2020d) Curfew II. 04 November 2020 21 May 2021 479/2020 (Ministry of Justice, 2020h) Free parking II. 04 November 2020 21 May 2021 478/2020 (Ministry of Justice, 2020i) and 512/2020 (Ministry of Justice, 2020g) Remote learning II. 10 November 2020 10 May 2021 at the individual discretion of educational institutions Wearing mask II. 21 November 2021 07 March 2022 597/2021 (Ministry of Justice, 2021) Source: Own data collection and editing Several waves of the Covid-19 epidemic can be distinguished in Hungary, the periods shown in Table 2. The Table 1 Several waves of the Covid-19 epidemic can be distinguished in Hungary, the periods shown in Table 2. The identification of each wave is based on the current active case numbers. 5. Public transportation in Debrecen during the Covid-19 pandemic Page 8/24 Page 8/24 Page 8/24 Table 2 Covid-19 pandemic waves in Hungary Begin End Wave 1 04 March 2020 17 July 2020 Wave 2 18 July 2020 16 February 2021 Wave 3 17 February 2021 02 September 2021 Wave 4 03 September 2021 04 January 2022 Wave 5 05 January 2022 22 June 2022 Wave 6 23 June 2022 currently Source: Own editing based on Worldometer's (Worldometers.info, 2022) data Table 2 Covid 19 pandemic waves in Hungary Table 2 During the pandemic wave and to adapt to the measures, several timetable changes had to be made while the service remained in operation. Driver ticket sales were suspended, and contactless purchase options were introduced, which also required service improvements in the city. During the second wave, increasing the proportion of articulated vehicles with a higher capacity was necessary to ensure adequate passenger spacing. The continuous daily disinfection of vehicles and public spaces also increased operator costs. Combining the evolution of the active case numbers and the restrictive measures the government took, we get the following very suggestive graph (Fig. 3). The first wave of the epidemic is barely noticeable on the graph, but each subsequent wave is much more pronounced. It is also clear that the measures more specific to public transport, such as distance learning, curfew, and parking fees, were introduced almost simultaneously. These measures were repealed before the first wave (17 July 2020), even though the active caseload was no less than when the measures were introduced (Table 1). The second and third waves are not distinctly different, with curfews, free parking and distance learning being reintroduced during their duration. It should be noted that distance learning does not affect all students, only those in secondary and higher education (Zeng & Zhang, 2019). The above measures will expire in May 2021, well before the end of the third wave (02 September 2021). The fourth and fifth waves will not be sharply different, but during this period, the only daily restrictions that public transport users will face will be wearing masks. However, we highlighted a crucial moment in (Fig. 3) − 26.12.2020, marking the start of vaccination against Covid-19 uniformly in the EU countries. Our graph shows that the start of vaccination in Hungary coincides with the peak of the second wave. Source: Own editing The relationship between general and discounted passes is also not correlated, primarily because students did not purchase travel entitlements during the period under study because of the multiple distance learning restrictions, while the restrictions for the working population were lower, resulting in less change in their travel habits and options. The dependency between Supplement and General passes is because many supplement pass users are commuters from rural areas. Students who bought supplement passes during this period could travel to the county centre to access health and other services, despite distance learning. 6.2. ARMA model We used the ARMA model to investigate how the evolution of different travel entitlements is influenced by the rate/shape of active case numbers during the epidemic. To conduct the study, we examined each travel entitlement separately. 6.1. Correlation matrix Correlation analysis aims to determine the extent to which entitlements for each mode of transport change simultaneously. The correlation coefficients are significant in all cases, with positive, medium and high Page 9/24 relationships between variables. We find higher correlation coefficients between the long-distance and unlimited- use fare products, with a strong relationship (0.795) for the General and Discounted passes. A solid dependent relationship for the General passes, the supplement passes, the Discount passes, and the supplement passes, with correlation coefficients above 0.9. The correlation coefficient between ticket-type vouchers and any fare product providing unlimited use over a more extended period shows a medium strength, typically between 0.48 and 0.57 (Table 3). The results of the correlation study in the transport professional context show that no strong relationship exists between the evolution of sales volumes of ad-hoc travel tickets and frequent travel passes. Table 3: Pearson's correlation Source: Own editing Source: Own editing 6.2.1. Analysis of the correlation between travel ticket sales and active cases There was a sharp drop in ad-hoc travel of almost 90% in the first period of the pandemic. In Fig. 4, we have graphically depicted the evolution of ticket types and active case numbers by month for 2020 to 2021. The graph shows the duration of each Covid-19 wave and the trends in ticket sales over this period. However, it is not suitable to establish a clear correlation between whether the trend in active caseloads is due to a decrease in ticket sales or other external influences. In our case, by external influence, we mean the government measures listed in Table 1. Applying the first-order ARMA(1,1) model to the time series of travel ticket sales and active case numbers, the parameters given in Table 4 are obtained. Page 10/24 Page 10/24 Table 4 Parameters of the ARMA(1,1) model describing the effect of Active cases on Travel ticket sales in Debrecen, Hungary Coefficient Estimate Standard error t value Pr (>\|t\|)1 Intercept 172138,927 24398,210 7,06 < 0,0001* ϕ 1 0,603 0,206 2,93 0,0083 * β1 -0,199 0,127 -1,56 0,1333 θ1 0,390 0,170 2,29 0,0328* 1 * significance level P < 0.05 (dup: 6 ?) Table 4 Source: Own calculations The most important is the value of the coefficient β1. As shown in Table 4, the coefficient is negative (β1 = -0,199), indicating an opposite shift in travel ticket sales and active caseload evolution. However, the coefficient is not significant, as Pr is greater than 5%, so the working hypothesis H1 could not be confirmed. In conclusion, active case numbers did not affect travel ticket sales during the Covid-19 outbreak. There was a significant drop in travel ticket sales in the first months of the first wave of the pandemic, which is certainly related to the fact that several government measures during this period resulted in a significant reduction in the use of community facilities. People feared a greater risk of infection in the confined spaces of public vehicles and preferred to use private transport. With the gradual lifting of restrictions, a more significant increase in travel ticket volumes was observed after the first, second, third and fourth waves, which occurred close together in time, peaked. The pandemic has led to a change in travel habits, with a return to greater use of public transport, albeit on an ad hoc basis. 6.2.1. Analysis of the correlation between travel ticket sales and active cases With the lifting of restrictions, domestic tourism also resumed, bringing occasional visitors and passengers to the city. 6.2.2. Analysis of the correlation between general pass sales and active cases The pandemic has also brought about significant changes in travel patterns in this segment. The spread of atypical forms of employment has reduced daily commuting, and the lack of comfort for passengers in public transport has led to a preference for individual transport to reach their destination. Similar fluctuations can be observed for the general pass in the first wave, but there were no increases in magnitude after the subsequent waves (Fig. 5). The general passes are mainly used by employees who are not eligible for discounted passes, usually for frequent travel, even several times a day. This group is most likely to be able to afford to maintain and even use their vehicle daily, thus requiring more time for them to return to public transport. Applying the first-order ARMA(1,1) model to the time series of general passes and active caseloads, the parameters given in Table 5 are obtained. Page 11/24 Page 11/24 Table 5 Parameters of the ARMA(1,1) model describing the effect of Active cases on the General pass sales in Debrecen, Hungary Coefficient Estimate Standard error t value Pr (>\|t\|)1 Intercept 13486,750 1761,697 7,66 < 0,0001* ϕ1 0,635 0,2691 2,36 0,0285 * β1 -0,005 0,0107 -0,49 0,6296 θ1 0,067 0,2371 0,28 0,7795 evel P < 0.05 Table 5 1 * significance level P < 0.05 Source: Own calculations Source: Own calculations The coefficient β is also negative for general rents. Although it is very low (-0.005), it is not significant in this case, so we cannot confirm working hypothesis H2. In this case, our conclusion is that the number of active cases did not influence the sales of general passes. The coefficient β is also negative for general rents. Although it is very low (-0.005), it is not significant in this case, so we cannot confirm working hypothesis H2. In this case, our conclusion is that the number of active cases did not influence the sales of general passes. Source: Own calculations Source: Own calculations Source: Own calculations The coefficient β is also negative in the case of concessionary season tickets. Although low (-0.016), it is not significant, so we cannot confirm working hypothesis H3. In this case, we conclude that the active caseload did not influence discounted pass sales. 6.2.3. Analysis of the correlation between discounted pass sales and active cases Several factors also influenced the development of discounted passes. Mandated remote learning is reflected in the declining number of student pass sales, with the biggest drop in this case also being caused by the first wave, with a significant "bounce back" in 2020 at the start of school in September (Fig. 6). The trend was quickly broken by the new measures taken in November, with volumes remaining low until the end of the school year due to remote learning. With the fourth wave, remote learning was not mandated, and sales resumed upward until winter break. The decline in December is due to the usual seasonal effect when a large part of the population travels by ticket or other means of transport because of the holidays. The discounted passes also include passengers under 65 who are already retired. In their case, the fear of viral infection may have been the main factor influencing the change in travel habits. Applying the first-order ARMA(1,1) model to the time series of discounted passes and active caseloads, the parameters in Table 6 are obtained. Page 12/24 Page 12/24 Table 6 Parameters of the ARMA(1,1) model describing the effect of Active cases on the Discounted pass sales in Debrecen, Hungary Coefficient Estimate Standard error t value Pr (>\|t\|)1 Intercept 16382,925 2604,826 6,29 < 0,0001* ϕ1 0,457 0,259 1,76 0,0932 β1 -0,016 0,018 -0,87 0,3968 θ1 0,307 0,264 1,16 0,2578 gnificance level P < 0.05 1 * significance level P < 0.05 6.2.4. Analysis of the correlation between supplement pass sales and active cases When examining the trend in the number of supplement passes, it can be seen that the trend is similar to that of discounted passes (Fig. 7). After a significant recovery following the first wave, there was a significant decline during the second and third waves. The decline was also due to the introduction of remote learning. In the fourth wave, as with the discounted pass type, a larger volume increase was observed due to the normal schooling regime. A smaller increase reduces the spike intensity in sales of supplement passes for employees. The seasonal decline at the end of the year can also be observed for this type of pass. Applying the first-order ARMA(1,1) model to the time series of supplement pass sales and active caseloads, we obtain the parameters in Table 7. Page 13/24 Table 7 Parameters of the ARMA(1,1) model describing the effect of Active cases on the Supplement pass sales in Debrecen, Hungary Coefficient Estimate Standard error t value Pr (>\|t\|)1 Intercept 5812,682 909,627 6,39 < 0,0001* ϕ1 0,484 0,266 1,82 0,0840 β1 -0,054 0,007 -0,83 0,4193 θ1 0,2083 0,253 0,82 0,4199 1 * significance level P < 0.05 Source: Own calculations Table 7 Parameters of the ARMA(1,1) model describing the effect of Active cases on the Supplement pass sales in Debrecen, Hungary Coefficient Estimate Standard error t value Pr (>\|t\|)1 Intercept 5812,682 909,627 6,39 < 0,0001* ϕ1 0,484 0,266 1,82 0,0840 β1 -0,054 0,007 -0,83 0,4193 θ1 0,2083 0,253 0,82 0,4199 Table 7 Parameters of the ARMA(1,1) model describing the effect of Active cases on the Supplement pass sales in Debrecen, Hungary 1 * significance level P < 0.05 The coefficient β for the supplement passes are also negative, very low (-0.054), but not significant, so we cannot confirm working hypothesis H4. In this case, our conclusion is that active caseloads did not influence the sales of supplement passes. The coefficient β for the supplement passes are also negative, very low (-0.054), but not significant, so we cannot confirm working hypothesis H4. In this case, our conclusion is that active caseloads did not influence the sales of supplement passes. 7. Discussion and conclusion The emergence and spread of the Covid-19 virus have changed our lives dramatically, affecting our daily lives in several distinct waves of active cases (Fig. 3). In Hungary, government measures to control and slow the spread of the virus have affected our mobility patterns in the city of Debrecen. The analysis showed that most measures affecting public transport were taken during the first two waves regarding their number and restrictive effect. These included the introduction of remote learning, curfews and free parking. The mandatory use of masks and distance control has further increased the minimization of the use of public spaces. Travel habits changed radically during this period, with the fear of contamination by the virus making private transport the preferred form of mobility for the population. However, the ARMA model results show that the evolution of active caseloads did not influence the evolution of sales volumes. All of our hypotheses, whether logical or evident, had to be rejected. Many factors may have influenced the decline in demand for public transport, but this study only examined government action as an objective factor. The Oxford Coronavirus Government Response Tracker (Hale et al., 2021) scored countries based on governmental stringency measures, with Hungary's stringency (Fig. 8) index ranking among the most stringent throughout the pandemic. Declarations Authors The authors confirm that neither the manuscript nor any part of its content is currently under consideration or published in another journal. Authors The authors confirm that neither the manuscript nor any part of its content is currently under consideration or published in another journal. Authors have no financial or non-financial interests that are directly or indirectly related to the work subm publication. Authors have no financial or non-financial interests that are directly or indirectly related to the work submitted for publication. Limitations and further research directions The present research only examines the relationship between active caseloads, travel entitlements sold and government measures, not other factors influencing travel. The conclusions drawn are valid only for the region under study. In the future, the impact of the measures and Covid-19 on public transport should be further investigated, and the results used to help decision-makers to reorient travel demand towards public transport. In the future, the impact of the measures and Covid-19 on public transport should be further investigated, and the results used to help decision-makers to reorient travel demand towards public transport. Source: Hale et al. (2021) Limitations and further research directions Source: Hale et al. (2021) Source: Hale et al. (2021) Of the two impact factors examined, the number of active cases had a minor impact on the decline in ticket and pass sales, while government measures had a more significant impact. Our analysis, although different in approach, confirms the studies by Meena & Sharma (2020), as well as Tóth et al. (2022), Fumagalli et al. (2022) and Wang et al. (2022), where these authors highlight that strict restriction that severely damages the economy are not essential to control the spread of Covid-19. Stochastic, i.e. probabilistic, systems can be modelled using random walk models, even if these phenomena are not necessarily random. Such or similar correlations exist in the field of Covid-19 evolution. The stochastic random walk process leads to the best solution in systems for which we do not know how it works (Malkiel, 2007). We assume that the economic disadvantages of direct intervention are more significant for DKV than if there had been no governmental-municipal restrictive action. In contrast to the sudden and significant negative impact on the transport sector at the onset of the virus, the reversal of travel patterns during the pandemic is likely to be a long process, lasting several years. The DKV has had to take several forced steps to ensure the sustainability of the service. To adapt to changes in travel demand, it has had to adjust its capacity, sometimes by reducing the number of timetables and increasing them. Unfortunately, these measures have led to an even more unfortunate shift away from public transport. The effects of these measures can still be felt today in the travel habits of the city of Debrecen and the proportion of people using public transport because travel habits have shifted towards individual modes of transport (Bittner Page 14/24 Page 14/24 et al., 2023; Tóth et al., 2022). To encourage people to choose public transport again, it is necessary to rationalize timetables and improve service quality. et al., 2023; Tóth et al., 2022). To encourage people to choose public transport again, it is necessary to rationalize timetables and improve service quality. A significant challenge for public transport operators in the coming period will be to attract people back from private transport to public transport. 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Exploration on the Construction of Statistical Index System of Sharing Economy Based on Problem Orientation. Paper presented at the 5th Annual International Conference on Social Science and Contemporary Humanity Development (SSCHD), Wuhan, PEOPLES R CHINA. (2019) Figures Figure 1 The percentage share of modal split transportation Source: Own editing based on the Eurostat database (European Commission, 2022) Page 18/24 Social Science and Contemporary Humanity Development (SSCHD), Wuhan, PEOPLES R CHINA. (2019) Figures Figure 1 The percentage share of modal split transportation Source: Own editing based on the Eurostat database (European Commission, 2022) Figure 1 The percentage share of modal split transportation Source: Own editing based on the Eurostat database (European Commission, 2022) Page 18/24 Page 18/24 Figure 2 Figure 2 Distribution of fare revenues Distribution of fare revenues Source: Own editing Figure 3 Trends in active caseloads and various restrictive measures Source: Own editing based on Worldometer's (2022) data and authors' data collection Figure 3 Figure 3 Trends in active caseloads and various restrictive measures Source: Own editing based on Worldometer's (2022) data and authors' data collection Source: Own editing based on Worldometer's (2022) data and authors' data collection Page 19/24 Page 19/24 Page 19/24 Figure 4 Travel ticket sales and evolution of active cases between 2020 - 2021 Source: Own editing Page 20/24 Figure 4 Travel ticket sales and evolution of active cases between 2020 - 2021 Source: Own editing Figure 4 Figure 4 Travel ticket sales and evolution of active cases between 2020 - 2021 Source: Own editing Source: Own editing Source: Own editing Page 20/24 Figure 5 General pass sales and evolution of active cases between 2020 - 2021 Source: Own editing Figure 5 General pass sales and evolution of active cases between 2020 - 2021 General pass sales and evolution of active cases between 2020 - 2021 Source: Own editing Source: Own editing Source: Own editing Page 21/24 Page 21/24 Figure 6 Discounted pass sales and evolution of active cases between 2020 – 2021 Source: Own editing Figure 6 Discounted pass sales and evolution of active cases between 2020 – 2021 Source: Own editing Page 22/24 Figure 7 Supplement pass sales and evolution of active cases between 2020 - 2021 Source: Own editing Figure 7 Figure 7 Supplement pass sales and evolution of active cases between 2020 - 2021 Supplement pass sales and evolution of active cases between 2020 - 2021 Page 23/24 Source: Own editing Figure 8 Page 23/24 Source: Own editing Figure 8 Figure 8 Page 23/24 Covid-19 stringency index in Hungary Source: Hale et al. (2021) Covid-19 stringency index in Hungary Source: Hale et al. (2021) Page 24/24
https://openalex.org/W2291685772	https://bmccancer.biomedcentral.com/track/pdf/10.1186/s12885-016-2221-5	English	null	Stroke related to androgen deprivation therapy for prostate cancer: a meta-analysis and systematic review	BMC cancer	2,016	cc-by	5,192	© 2016 Meng et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Meng et al. BMC Cancer (2016) 16:180 DOI 10.1186/s12885-016-2221-5 Meng et al. BMC Cancer (2016) 16:180 DOI 10.1186/s12885-016-2221-5 * Correspondence: yuanjieniu68@hotmail.com †Equal contributors 2Department of Urology, Second Hospital of Tianjin Medical Unversity, Tianjin Institute of Urology, 23 Pingjiang Road, Tianjin 300211, China Full list of author information is available at the end of the article Stroke related to androgen deprivation therapy for prostate cancer: a meta-analysis and systematic review Fanzheng Meng1,2†, Shimiao Zhu2†, Jinsheng Zhao1†, Larissa Vados3, Lei Wang4, Yusheng Zhao5, Dan Zhao1 and Yuanjie Niu2* Abstract Background: Whether androgen deprivation therapy (ADT) leads to stroke morbidity is still unclear because of inconsistent evidence. We performed a systematic review and meta-analysis to evaluate if ADT used in men with prostate cancer (PCa) is associated with stroke. Methods and results: Medline, Embase and Cochrane Library databases up to September 30th 2014 were systematically searched with no date or language restriction, and reports from potentially relevant journals were complementally searched. Both randomized controlled trials and observational studies were included. Two reviewers independently extracted data and assessed study quality. Six observational studies finally met inclusion criteria, with 74,538 ADT users and 85,947 non-ADT users reporting stroke as an endpoint. Although no significant association was observed in pooled estimates, the incidence of stroke in ADT users was 12 % higher than control groups, (HR = 1.12, 95 % confidence interval [CI]: 0.95 to 1.32; P = 0.16). In subgroup-analyses of different ADT types, stroke was found to be significantly associated with gonadotropin-releasing hormone (GnRH) alone (HR = 1.20, 95 % CI: 1.12 to 1.28; P < 0.001), GnRH plus oral antiandrogen (AA) (HR = 1.23, 95 % CI: 1.13 to 1.34; P < 0.001) and orchiectomy (HR = 1.37, 95 % CI: 1.33 to 1. 46; P = 0.001), but not with AA alone (HR = 1.06, 95 % CI: 0.71 to 1.57; P = 0.78). Conclusions: GnRH alone, GnRH plus AA and orchiectomy is significantly associated with stroke in patients with PCa. Keywords: Stroke, Androgen deprivation therapy, Prostate cancer, Meta-analysis Keywords: Stroke, Androgen deprivation therapy, Prostate cancer, Meta-analysis increasingly used as a treatment for PCa, this effect on prolonging life expectancy is unclear or even negative in several clinical studies [5, 6]. In our previous study [7], we found that ADT was positively associated with car- diovascular disease. Because both cardiovascular and cerebrovascular diseases share many common risk factors including atherosclerosis, dyslipidemia, visceral obesity, arterial endothelial dysfunction, and hyperten- sion [8–12], ADT may also be associated with stroke. Additionally, one population-based cohort study [13] demonstrated that, GnRH agonists could significantly in- crease the risk of stroke (adjusted rate ratio [RR], 1.18; 95 % confidence interval [CI], 1.00–1.39). However, con- flicting results were also reported. In a nation-wide population-based cohort study [14], authors found that ADT was associated with decreased stroke risk (adjusted hazard ratio [HR], 0.88; P = 0.001). Therefore, there is * Correspondence: yuanjieniu68@hotmail.com Search strategy and study selection We systematically searched Medline, Embase and Cochrane Library databases up to September 30th 2014, with all possible combinations of the keywords as follows: prostate cancer or prostate tumor or prostate carcinoma, androgen deprivation or androgen suppression or endo- crine treatment or ADT or AST; and stroke or cerebrovas- cular or transient ischemic attack or hemiplegia or TIA or cardiovascular (Additional file 1: Methods S1). No lan- guage, date, or other restrictions was used. Publications from potentially relevant journals were complementally searched. Data extraction and quality assessment Two reviewers (Meng & Zhu) independently extracted the data from eligible and potentially relevant publica- tions, with differences resolved by the third reviewer (Niu) as necessary. General characteristics of each in- cluded publication were recorded: first author’s name, year of publication, medical center, study design, sample size, population characteristics, follow-up period, inter- ventions, definition of stroke morbidity, HRs and corre- sponding 95 % CIs of estimates in each comparisons. Definition of stroke was according to what descripted in each included publication. Our meta-analysis involved different types of ADT including AA, GnRH agonists, orchiectomy, and two or more types above combined. We used Begg adjusted rank correlation test and Egger linear regression test to evaluate publication bias. All meta-analyses were conducted with Review Manage (version 5.3; The Cochrane Collaboration, Oxford) and STATA software (version 11.0; College Station, Texas). Two-tailed P < 0.05 indicated significant difference statistically. Background Prostate cancer (PCa) is the most prevalent malignancy and remains a major healthcare problem in men in the United States [1]. Because the development and growth of PCa cells depends on androgens [2, 3], Androgen deprivation therapy (ADT) undoubtedly plays an import- ant role to treat PCa, and recently, approximately 40 % of men diagnosed with PCa within 6 months have been treated with ADT in the US [4]. ADT is a palliative therapy, including different types of treatments such as gonadotropin-releasing hormone (GnRH), oral antiandrogen (AA), orchiectomy, and two or more types above combined. Although ADT is * Correspondence: yuanjieniu68@hotmail.com †Equal contributors 2Department of Urology, Second Hospital of Tianjin Medical Unversity, Tianjin Institute of Urology, 23 Pingjiang Road, Tianjin 300211, China Full list of author information is available at the end of the article Meng et al. BMC Cancer (2016) 16:180 Page 2 of 7 agreement reached. Level of evidence (LOE) of all eli- gible publications were evaluated using the classifica- tions of Phillips et al’s, [17]. still no consensus regarding that ADT is associated with stroke. Based on the controversy of this clinical issue, we per- formed a meta-analysis and systematic review to investi- gate whether ADT is associated with stroke in patients with PCa. Subgroups analyses In order to minimize the influence of concomitant treat- ments (e.g. radiotherapy and prostatectomy), subgroup analysis of ADT monotherapy vs watchful waiting or active surveillance (WW/AS) for stroke morbidity was carried out. ADT monotherapy was defined as a single therapeutic that in addition to ADT, no other previous therapy was used in intervention group. Considering the significance of existing heterogeneity in overall-analysis, additional subgroup-analyses for various types of ADT (e.g. GnRH, AA, GnRH + AA and Orchiectomy) vs non- ADT were also performed. Statistical analysis h Using the same methods as in our previous study [18], weighted HRs and 95 % CIs were estimate to compare all of these dichotomous variables. Different methods were employed to calculate the HRs on the basis of the data provided in the studies. When studies compared more than one types of ADT with the same control group severally (for example, GnRH vs Control, Orchi- ectomy vs Control), random effects meta-analyses were used to combine these results together as necessary. Studies were included if they fulfilled the following in- clusion criteria: 1) Patients diagnosed with PCa only; 2) Intervention groups must include ADT (either mono- therapy or combination therapy); 3) Treatments in con- trol groups were non-ADT (e.g. radical prostatectomy, radiotherapy, active surveillance.); 4) Studies must have the data of risk estimates with 95 % CIs; 5) Studies must report comparative data. If more than one study were identified from the same population, we extracted data from all available informations, rather than just a single publication. Statistical heterogeneity among studies was evaluated with the Cochrane’s Q statistic [19]. In addition, incon- sistency was quantified by I2 statistic (100 % × [(Q-df)/ Q]), different I2 values (25, 50, and 75 %) denote differ- ent levels (low, medium, and high levels) of heterogen- eity [20]. Using the Der-Simonian and Laird method, we chose random-effects models throughout this analysis no matter whether heterogeneity existed or not. Study characteristics and study quality Study characteristics and study quality HRs and 95 % CIs were directly given in two publications [14, 21], and four studies [13, 14, 23, 24] respectively com- pared different types of ADT with control groups. All of these observational studies were of high LOE (2a). Details of the eligible studies were summarized in Table 1. According to the assessment of NOS for observational studies, all eligible studies were high-quality with scores more than seven stars (Additional file 1: Table S2). ADT is considered to be effective when serum testos- terone is declined to the recommended levels of 50 ng/ dl, according to the 2012 NCCN (National Comprehen- sive Cancer Network) guidelines [26]. However, How- ever, as reported in our previous study [7], low level of serum testosterone is likely related to many stroke risk factors including high triglyceride and low-density lipo- protein cholesterol levels, endothelial dysfunction and proinflammatory factors [12, 27–29]. In addition, previ- ous studies [11, 30] showed that testosterone deficiency was significantly associated with hypertension, high body mass index, hypercoagulable states, and hyperfibrinogen- emia [31]. All of these adverse effects may put patients at a high risk of stroke. Results Based on the titles, abstracts, and full text screening, we finally identified five cohort studies [14, 21–24] and one nested case–control study [13] that met the inclusion criteria. All articles included were published in English. Details of reasons for exclusion of articles through full text screening are shown in Additional file 1: Table S1. Figure 1 shows the literature search and study selection process of our meta-analysis. Study qualities of the selected trials were assessed by the Jadad score [15]. Trails were considered to be of high quality if they achieved more than 4 scores. Newcastle-Ottawa quality assessment scale (NOS) [16] was used to assess the observational studies. Studies with more than 6 scores were considered high-quality. Two authors (Zhu & Meng) respectively addressed the assessments and discussed the discrepancies until Meng et al. BMC Cancer (2016) 16:180 Page 3 of 7 Fig. 1 Flow Diagram of Search Strategy and Study Selection subgroup-analyses of orchiectomy vs non-ADT. Figure 3 showed the subgroup analyses for the effect of different types of ADT vs control on stroke events. Stroke was sig- nificantly associated with GnRH alone (HR = 1.20; 95 % CI 1.12–1.28; P < 0.001), GnRH plus AA (HR = 1.23; 95 % CI 1.13-1.34; P < 0.001), and orchiectomy (HR = 1.37; 95 % CI 1.33–1.64; P = 0.001), but not with AA alone (HR = 1.06; 95 % CI 0.71–1.57; P = 0.78). Details of meta-analyses for each type of ADT were shown in Additional file 1: Figure S1. Additionally, two studies [23, 24] with 81,402 pa- tients were included for subgroup analysis of ADT monotherapy vs WW/AS. 6150 stroke events were re- corded, containing 3317 events from ADT users (8.2 %) and 2349 from WW/AS groups (5.5 %). Pooled result re- vealed that ADT monotherapy could significantly increase the risk of stroke, with a higher incidence of 16 % than WW/AS (HR = 1.16, 95%CI: 1.03–1.31, P = 0.01; Fig. 2b). Discussion Although the occurrence of stroke in men undergoing ADT with PCa has been an emerging problem over re- cent years, the relationship between ADT and stroke morbidity is still unclear. This meta-analysis including five population-based observational studies showed that ADT has a tendency to increase the risk of stroke. Evi- dence was directly proved by Azoulay et al. [13], show- ing that ADT could significantly increase the risk of stroke over a median follow-up of 3.9 years in men with newly diagnosed PCa (HR = 1.34, P = 0.0001). Another cohort study [24] involving 29,443 ADT users, and 19,527 with surveillance showed the standardized mor- tality ratios of stroke was 1.17. Meta-analysis results Six studies [13, 14, 21, 23–25] involving 160,485 partici- pants were identified for inclusion criteria. Figure 2a showed the impact of ADT vs non-ADT on the end point of fatal or non-fatal stroke morbidity. 5578 (7.4 %) stroke events occurred among 74,538 ADT users compared with 5134 events (5.7 %) within control participants. Pooled HR showed that the incidence of stroke morbidity in ADT group was 12 % higher than non-ADT users, although sta- tistically significant difference was not observed (HR = 1.12; 95 % CI, 0.95–1.32; P = 0.16). As to subgroup-analyses of different types of ADT, four studies [13, 23–25] were identi- fied: three studies [13, 23, 24] respectively compared AA alone, GnRH alone and GnRH plus AA with control groups, four studies [13, 23–25] were available for the Out of the six studies we analyzed, only one [14] did not show the positive relationship between ADT and stroke (HR = 0.88; P = 0.001). This inconsistency was likely due to the contamination bias caused by radical prostatectomy. To reduce this bias, a sensitivity analysis was performed comparing ADT monotherapy with WW/AS. When ADT users undergoing other treatments were excluded, more significantly increased risk of Table 1 Characteristics of Studies Investigating Stroke Related to ADT Table 1 Characteristics of Studies Investigating Stroke Related to ADT First author year Design, LOE Database source (Duration) Definition of Stroke (ICD codes) Types of ADT Treatments of control No. of ADT/ Control Age ya(SD) of patients Follow-up, (ya) Hazard Ratios(95%CI) Jespersen et al. [25] 2013 Cohort, 2a Danish Cancer Registry (2002–2010) Ischemic Stroke/TIA (ICD-8 codes 433, 434.09/99, 436.01/436.90, ICD-10 codes DI63.x, DI64.x) GnRH/AA non-ADT 9204 20,307 71 3.3 (1.8 to 5.2) 1.19(1.06,1.35)c 1.17 (0.94, 1.50)d Orchiectomy 2060 1.11(0.90,1.36)c Hemelrijck et al. [24] 2010 Cohort, 2a NPCR of Sweden (1997–2007) Stroke (ICD-10: 160–164, G45) GnRH agonist RP 9066 26,432 ≤65: 19,153 3.8 4.4 1.21(1.11,1.32)b 1.16 (1.01, 1.32)d AA 3391 4 0.88(0.76,1.00)b GnRH + AA WW/AS 11,646 19,527 66 to 74: 27,737 3.3 4.7 1.25(1.15,1.35)b Orchiectomy 5340 ≥75: 13,110 3.1 1.30(1.18,1.44)b Other types 1199 - - Alibhai et al. [14] 2009 Cohort, 2a ICES (1995–2005) Stroke (ICD-9-CM codes 430–438) ADT non-ADT 19,079/19,079 75 ± 6.3 6.47 0.88(0.81,0.96)c Keating et al. Abbreviations: LOE level of evidence, ADT androgen deprivation therapy, GnRH gonadotropin-releasing hormone (leuteinizing hormone releasing hormone, LHRH), AA oral antiandrogens, RP radical prostatectomy/cura- tive treatment, WW/AS watchful waiting (WW)/active surveillance (AS), SD standard deviation, NA not applicable, NPCR National Prostate Cancer Register, GPRD UK general practice research database, ICES institute for clinical evaluative sciences amean or median bcompared with WW/AS cHR was directly given in the publication dCombined estimates from all types of ADT with random effect meta-analysis Abbreviations: LOE level of evidence, ADT androgen deprivation therapy, GnRH gonadotropin-releasing hormone (leuteinizing hormone releasing hormone, LHRH), AA oral antiandrogens, RP radical prostatectomy/cura- tive treatment, WW/AS watchful waiting (WW)/active surveillance (AS), SD standard deviation, NA not applicable, NPCR National Prostate Cancer Register, GPRD UK general practice research database, ICES institute for clinical evaluative sciences Abbreviations: LOE level of evidence, ADT androgen deprivation therapy, GnRH gonadotropin-releasing hormone (leuteinizing hormone releasing hormone, LHRH), AA oral antiandrogens, RP radical prostatectomy/cura- tive treatment, WW/AS watchful waiting (WW)/active surveillance (AS), SD standard deviation, NA not applicable, NPCR National Prostate Cancer Register, GPRD UK general practice research database, ICES institute for clinical evaluative sciences amean or median bcompared with WW/AS cHR was directly given in the publication Meta-analysis results [23] 2010 Cohort, 2a Veterans Healthcare Administration (2001–2004) Ischemic Stroke/TIA (ICD-9 codes 433.XX −435.XX) GnRH agonist WW/AS 14,037 22,846 66.9 ± 8.6 2.6 1.18(1.02,1.36)c 1.18 (0.91, 1.51)d AA 1229 0.89(0.46,1.73)c GnRH + AA 1838 0.91(0.60,1.39)c Orchiectomy 308 1.81(1.15,2.84)c Huang et al. [21], 2014 Cohort, 2a Queen Mary Hospital, Hong Kong (1998–2011) Ischemic Stroke (NA) ADT non-ADT 517/228 72.2 ± 0.3 5.3 0.94 (0.35, 2.45)c Azoulay et al. [13] 2011 Nested Case– control, 2a GPRD (1988–2008) Stroke/TIA (NA) GnRH agonist non-ADT 3274 3960 72.3 ± 3.9 3.9 1.18(1.00,1.39)c 1.34 (1.15, 1.55)d AA 457 1.47(1.08,2.01)c GnRH + AA 481 1.26(0.93,1.72)c Orchiectomy 295 1.77(1.25,2.51)c Other types 142 1.42(0.84,2.39)c Abbreviations: LOE level of evidence, ADT androgen deprivation therapy, GnRH gonadotropin-releasing hormone (leuteinizing hormone releasing hormone, LHRH), AA oral antiandrogens, RP radical prostatectomy/cura- tive treatment, WW/AS watchful waiting (WW)/active surveillance (AS), SD standard deviation, NA not applicable, NPCR National Prostate Cancer Register, GPRD UK general practice research database, ICES institute for Meng et al. BMC Cancer (2016) 16:180 Page 5 of 7 Fig. 2 a. HRs of Stroke Related to ADT. b. HRs of Stroke Related to ADT Monotherapy vs WW/AS Fig. 2 a. HRs of Stroke Related to ADT. b. HRs of Stroke Related to ADT Monotherapy vs WW/AS stroke was observed in ADT monotherapy users (Fig. 2b). heterogeneity, and showed that stroke morbidity was sig- nificantly associated with GnRH alone, GnRH plus AA, and prostatectomy. The US Food and Drug Administra- tion announced a safety warning that GnRH agonists could increase the risk of stroke in men receiving these drugs for treating PCa [1]. As previously reported [32], There may be bias in the results due to different types of ADT that were used in some studies [13, 23–25]. Therefore, we carried out subgroup analyses stratified by different types of ADT in order to reduce this Fig. 3 HRs of Subgroup Analyses for Stroke Related to Different Types of ADT Fig. 3 HRs of Subgroup Analyses for Stroke Related to Different Types of ADT Meng et al. Conclusion In conclusion, there is a tendency that ADT could in- crease the risk of stroke. Significant association of ADT monotherapy with stroke was observed after removing patients with prostatectomy and radiotherapy. Addition- ally, GnRH, GnRH plus AA, and orchiectomy can sig- nificantly result in stroke. These findings may help clinicians be aware of the potential risks of ADT and en- sure clinical management when prescribing this treat- ment. Additional studies should also focus on the different definitions of stroke since they require different approaches to treatment. This meta-analysis and systematic review has several strengths. First, the included studies were all large-scale observational studies with long term of follow-up. Sec- ond, if the HRs were not available in eligible studies, all the data which could be used to calculate these were ad- justed for the durations of follow-up. Finally, funnel plots showed balance in our assessment of publication bias. Begg’s and Egger’s tests also indicated that no sig- nificant publication bias existed (Table 2). Additionally, there was no obvious publication bias as shown in Add- itional file 1: Figure S2, since points are distributed around the verticals. Therefore, the findings in this meta-analysis can be considered credible. Authors’ contributions NY had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. MF, ZS and ZJ made substantial contributions to concept, design, and acquisition of data, statistical analysis and interpretation of data. V helped to edit English expression. WL and ZD drafted the manuscript, ZY revised this manuscript critically for important intellectual content and offering a lot of revise opinions. All authors read and approved the final manuscript. Competing interests No competing interests exit in the submission of this manuscript, and manuscript is approved by all authors for publication. All authors have contributed significantly, and are in agreement with the content of the manuscript. Meta-analysis results BMC Cancer (2016) 16:180 Page 6 of 7 Table 2 Pooled Results and Publication Bias for All Comparisons Measurement na Case/control Heterogeneity Pooled rate/HR Begg’s test (P) Egger’s test (P) P I2 (%) (95 % CI) Stroke morbidity ADT vs Non-ADT 5 74538/85947 <0.001 85 1.13 (0.95–1.33) 0.806 0.261 AA vs Non-ADT 3 5078/47309 0.010 78 1.06 (0.71–1.57) 1.000 0.653 GnRH vs Non-ADT 3 49292/47309 0.930 0 1.20 (1.12–1.28) 1.000 0.125 GnRH plus AA vs Non-ADT 3 13906/47309 0.360 3 1.23 (1.13–1.34) 0.296 0.501 Orchiectomy vs Non-ADT 4 7963/67616 0.060 59 1.37 (1.33–1.64) 0.734 0.456 a Number of included studies Table 2 Pooled Results and Publication Bias for All Comparisons ischemic events as the endpoint. Finally, the certain characteristics of patients that may contribute to stroke were different in each included study, which might con- found the presented results. Therefore, adjusted data were extracted when available to minimize the bias. GnRH agonist may cause the development of metabolic syndrome, which in turn could accelerate the athero- sclerotic process and then lead to increased stroke mor- bidity. One included cohort study [23] investigating the relationship between GnRH and stroke over a median follow-up of 2.6 years, concluded that GnRH was signifi- cantly associated with stroke morbidity (adjusted HR = 1.18, P = 0.03). All of these listed above was in accord- ance with our findings. Additional file Additional file 1: Methods S1. Literature Search Strategy. Table S1. List of Excluded Full-text Articles with Reasons for Exclusions. Table S2. Newcastle-Ottawa Scale Quality Assessment of Included Studies. Figure S1. Details of Subgroup Analyses for Stroke Related to Different Types of ADT. Figure S2. Funnel plots for Meta-analyses. (DOC 180 kb) Additional file 1: Methods S1. Literature Search Strategy. Table S1. List of Excluded Full-text Articles with Reasons for Exclusions. Table S2. Newcastle-Ottawa Scale Quality Assessment of Included Studies. Figure S1. Details of Subgroup Analyses for Stroke Related to Different Types of ADT. Figure S2. Funnel plots for Meta-analyses. (DOC 180 kb) y However, we acknowledge that several limitations should be taken into consideration with the results found in this meta-analysis. First, all eligible reports were retrospective observational studies, which may introduce recall limitation, so the integrity of records may weaken the reliability of the results to some extent. Second, selection bias may have influenced our results. To minimize this bias, we carried out a predesigned search strategy with independent selection, and data was extracted by two reviewers. Third, incomplete data in some included publications [24, 25] may have influenced the overall result. As described in detail in our previous study [7], we have tried to minimize this limitation as much as possible. Furthermore, the stroke definition (is- chemic, hemorrhagic, or TIA) was not specified in some studies [13, 14, 24], introducing potential bias in stroke incidence estimate. However, most of events in these eli- gible studies were defined as ischemic events, and this bias is possibly minimized because these overall stroke rates were similar to the study [23] only including References 1. FDA drug safety communication. FDA requests label changes and single- use packaging for some over-the-counter topical antiseptic products to decrease risk of infection. Clin Infect Dis. 2014;58(3):i–ii. 25. Jespersen CG, Norgaard M, Borre M. Androgen-deprivation therapy in treatment of prostate cancer and risk of myocardial infarction and stroke: a nationwide Danish population-based cohort study. Euro Urol. 2014:65(4): 704–9. 2. Huggins C. Endocrine-induced regression of cancers. 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Marin P, Holmang S, Gustafsson C, Jonsson L, Kvist H, Elander A, et al. Androgen treatment of abdominally obese men. Obes Res. 1993;1(4):245–51. 31. Glueck CJ, Glueck HI, Stroop D, Speirs J, Hamer T, Tracy T. Endogenous testosterone, fibrinolysis, and coronary heart disease risk in hyperlipidemic men. J Lab Clin Med. 1993;122(4):412–20. 8. Levine GN, D’Amico AV, Berger P, Clark PE, Eckel RH, Keating NL, et al. Androgen-deprivation therapy in prostate cancer and cardiovascular risk: a science advisory from the American Heart Association, American Cancer Society, and American Urological Association: endorsed by the American Society for Radiation Oncology. CA Cancer J Clin. 2010;60(3):194–201. 32. Conteduca V, Di Lorenzo G, Tartarone A, Aieta M. The cardiovascular risk of gonadotropin releasing hormone agonists in men with prostate cancer: an unresolved controversy. Crit Rev Oncol Hematol. 2013;86(1):42–51. 9. Jones TH, Saad F. The effects of testosterone on risk factors for, and the mediators of, the atherosclerotic process. Atherosclerosis. 2009;207(2):318–27. 10. Malkin CJ, Pugh PJ, Morris PD, Asif S, Jones TH, Channer KS. Low serum testosterone and increased mortality in men with coronary heart disease. Heart. 2010;96(22):1821–5. 11. Svartberg J, von Muhlen D, Schirmer H, Barrett-Connor E, Sundfjord J, Jorde R. Association of endogenous testosterone with blood pressure and left ventricular mass in men. The Tromso Study. Eur J Endocrinol. 2004;150(1):65–71. 12. Whitsel EA, Boyko EJ, Matsumoto AM, Anawalt BD, Siscovick DS. Intramuscular testosterone esters and plasma lipids in hypogonadal men: a meta-analysis. Am J Med. 2001;111(4):261–9. 13. Azoulay L, Yin H, Benayoun S, Renoux C, Boivin JF, Suissa S. Androgen- deprivation therapy and the risk of stroke in patients with prostate cancer. Eur Urol. 2011;60(6):1244–50. 14. Alibhai SM, Duong-Hua M, Sutradhar R, Fleshner NE, Warde P, Cheung AM, et al. Received: 8 January 2015 Accepted: 28 February 2016 Received: 8 January 2015 Accepted: 28 February 2016 24. Van Hemelrijck M, Garmo H, Holmberg L, Ingelsson E, Bratt O, Bill-Axelson A, et al. Absolute and relative risk of cardiovascular disease in men with prostate cancer: results from the Population-Based PCBaSe Sweden. J Clin Oncol. 2010;28(21):3448–56. Acknowledgements h k The work was supported by the National Basic Research Program of China (grant no. 2012CB518304) and the International S&T Cooperation Program of China (ISTCP) (grant no. S2012GR0142). Thank all of the authors of primary studies included in their meta-analyses. Page 7 of 7 Page 7 of 7 Page 7 of 7 Page 7 of 7 Meng et al. BMC Cancer (2016) 16:180 Author details 1D f 21. Huang G, Yeung CY, Lee KK, Liu J, Ho KL, Yiu MK, et al. Androgen deprivation therapy and cardiovascular risk in chinese patients with nonmetastatic carcinoma of prostate. J Oncol. 2014;2014:529468. 1Department of Neurology, Tianjin Nankai Hospital, Nankai Clinical School of Tianjin Medical University, Tianjin, China. 2Department of Urology, Second Hospital of Tianjin Medical Unversity, Tianjin Institute of Urology, 23 Pingjiang Road, Tianjin 300211, China. 3Tianjin University of Traditional Chinese Medicine, Tianjin, China. 4Tianjin Institute of Medical and Pharmaceutical Sciences, Tianjin, China. 5First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China. 1Department of Neurology, Tianjin Nankai Hospital, Nankai Clinical School of Tianjin Medical University, Tianjin, China. 2Department of Urology, Second Hospital of Tianjin Medical Unversity, Tianjin Institute of Urology, 23 Pingjiang Road, Tianjin 300211, China. 3Tianjin University of Traditional Chinese Medicine, Tianjin, China. 4Tianjin Institute of Medical and Ph i l S i Ti ji Chi 5Fi T hi H i l f Ti ji 22. Jespersen CG, Norgaard M, Borre M. Reply to C. 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https://openalex.org/W2588723626	https://www.intechopen.com/citation-pdf-url/53080	English	null	Application of Optical Interferometry for Characterization of Thin-Film Adhesion	InTech eBooks	2,017	cc-by	8,900	Application of Optical Interferometry for Characterization of Thin-Film Adhesion Application of Optical Interferometry for Characterization of Thin-Film Adhesion Sanichiro Yoshida , David R. Didie , Jong-Sung Kim and Ik-Keun Park Sanichiro Yoshida, David R. Didie, Jong-Sung Kim and Ik-Keun Park Additi l i f ti i il bl t th d f Sanichiro Yoshida , David R. Didie , Jong-Sung Kim and Ik-Keun Park Sanichiro Yoshida, David R. Didie, Jong-Sung Kim and Ik-Keun Park Additi l i f ti i il bl t th d f Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/66205 http://dx.doi.org/10.5772/66205 Abstract In this chapter, application of optical interferometry for the characterization of thin-film adhesion to the substrate is discussed. The thin-film system is configured as one of the end mirrors of a Michelson interferometer and oscillated with an acoustic transducer from the substrate side. The oscillation causes sinusoidal displacement of the film surface around the initial (neutral) position, and the interferometer detects its amplitude as the relative phase difference behind the beam splitter. When the driving frequency of this oscillation is tuned to a range where the film-substrate interface is dominantly oscillated, the elasticity of the interface can be analyzed from the oscillation amplitude. The principle of this method is straightforward but in reality, fluctuation of the initial phase (the relative phase corresponding to the initial film position) compromises the signal. A technique known as the carrier fringe method along with spatial frequency domain analysis is employed to reduce the noise associated with the initial phase fluctuation. The possibility of the present method to analyze the so-called blister effect on thin-film adhesion is discussed. Keywords: optical interferometry, opto-acoustic technique, thin-film adhesion, blister effects, non-destructive evaluation Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) Interested in publishing with us? Contact book.department@intechopen.com Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com Open access books available Countries delivered to Contributors from top 500 universities International authors and editors Our authors are among the most cited scientists Downloads We are IntechOpen, the world’s leading publisher of Open Access books Built by scientists, for scientists 14% 191,000 210M TOP 1% 154 7,200 Chapter 4 al chapter 1. Introduction Thin-film systems are used in a variety of applications ranging from micro-electro-mechanical- systems (MEMS) to artificial joints. Poor adhesion of the film material to the substrate leads to delamination or other modes of coating failure, and is an important factor of quality control in © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, and reproduction in any medium, provided the original work is properly cited. Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Optical Interferometry 62 the manufacturing stage. However, detection of poor adhesion is not easy. In particular, when the film material is poorly adhered but not causing a structural abnormality, detection is very difficult. Static methods such as acoustic imaging microscopy or X-ray diffractometry cannot be used to identify the problem. Dynamic analysis capable of characterizing the elastic behavior of the interface is essential. Among dynamic techniques to evaluate the film adhesion strength, ultrasonic techniques [1–5] are the prevailing methods. In these methods, ultrasonic waves are excited in the sub- strate and film materials, and abnormality is detected from the propagation characteristics of the ultrasonic wave. The recent trend indicates that the film thickness is reduced for bet- ter performance of the thin-film system. This forces the ultrasonic wavelength to be shorter, hence the frequency to be higher. Reduction in the wavelength works well for the purpose of detecting defects or other nonuniform issues in the interface, as the spatial resolution is increased. However, for characterization of elasticity of the film-substrate interface, an in- crease in the frequency makes the analysis difficult. This is because normally, poor adhe- sion has lower elastic modulus than the healthy adhesion. Consequently, the frequency is too high to oscillate the poor adhesion effectively, and the signal representing the poor ad- hesion tends to be small. For several thin-film specimens, resonance-like behaviors of the film-substrate interface have been found [7]. In the course of this research, we have learned much about practical issues of Michelson interferometers. The operation principle of a Michelson interferometer is straightforward. However, in reality, its application to engineering is not as simple as it sounds. Especially, when the interferometer is used in air, environmental disturbance can easily affect the measurement. It is always possible to place the entire interferometric paths in a vacuum, but it causes extra costs and handling procedures. In many engineering applications, it is not favorable. In this paper, after various findings from the above research being discussed, a method is proposed to reduce environmental disturbance that compromises the optical phase signal representing the oscillation at the acoustic frequency. In this method, a known optical-path variation is introduced in a direction lateral to the interferometer axis so that the Initial Phase Difference is visualized as a pattern of mutually parallel dark and bright stripes (known as a carrier-fringe pattern [8]), and the data is processed in the spatial-frequency domain. With this configuration, a change in the relative optical path shifts the fringe locations in a plane normal to the interferometric axis. This allows us to perform two-dimensional analysis on the image plane of the imaging device, and thereby find a weakly-adhered spot. The method is especially useful when the frame rate of the imaging device is significantly lower than the acoustic frequency, as is normally in this case. Under this condition, the relative phase change at the acoustic frequency is detected as the corresponding reduction in the fringe contrast that can be related to the height of the main peak in the Fourier spectrum. Since the spatial frequency corresponding to the main peak is determined by the spacing of the carrier fringe pattern, the peak value does not depend on slow shift of the entire fringe pattern due to an environmental disturbance. In other words, the detection system tries to probe the oscillatory behavior caused by the poor adhesion at a frequency on the blue side of the spectrum, as schematically illustrated in Figure 1. The transmissibility (the transfer function) of a me- chanical oscillator decreases with a quadratic dependence on the frequency (f−2) on the high frequency side of the resonance. Figure 1. Resonance curves with different resonant frequencies. Figure 1. Resonance curves with different resonant frequencies. Considering the above situation, we have devised an optical interferometric system to characterize the adhesion of thin films to their substrate [6]. A Michelson interferometer is used to analyze harmonic response of thin-film specimens when they are oscillated with an acoustic transducer. The film surface displacement resulting from the acoustic oscillation is detected as relative optical path changes behind the beam splitter. With the assumption that the film- substrate interface has a lower elastic modulus than the film or the substrate material and by choosing the acoustic frequency appropriately, it is possible to characterize the elastic behavior Figure 1. Resonance curves with different resonant frequencies. Considering the above situation, we have devised an optical interferometric system to characterize the adhesion of thin films to their substrate [6]. A Michelson interferometer is used to analyze harmonic response of thin-film specimens when they are oscillated with an acoustic transducer. The film surface displacement resulting from the acoustic oscillation is detected as relative optical path changes behind the beam splitter. With the assumption that the film- substrate interface has a lower elastic modulus than the film or the substrate material and by choosing the acoustic frequency appropriately, it is possible to characterize the elastic behavior Considering the above situation, we have devised an optical interferometric system to characterize the adhesion of thin films to their substrate [6]. A Michelson interferometer is used to analyze harmonic response of thin-film specimens when they are oscillated with an acoustic transducer. The film surface displacement resulting from the acoustic oscillation is detected as relative optical path changes behind the beam splitter. With the assumption that the film- substrate interface has a lower elastic modulus than the film or the substrate material and by choosing the acoustic frequency appropriately, it is possible to characterize the elastic behavior Application of Optical Interferometry for Characterization of Thin-Film Adhesion http://dx.doi.org/10.5772/66205 63 63 of the interfaces. 2.1. Why optical interferometer? The substrate of typical thin film systems is of the order of 100 μm or less in thickness. In order to excite 10 waves in the substrate, the wavelength of the acoustic signal must be 10 μm or shorter. The acoustic velocity in silicon (a typical substrate) is 8 km/s. This results in the acoustic frequency higher than 800 MHz, leading to the situation where detection of elastic behavior associated with resonant frequency of the order of 100 MHz or less is difficult. Poor adhesion normally has a resonant frequency substantially lower than 100 MHz. In addition, most thin- film systems are subject to environmental disturbance of much lower frequency. Therefore, the use of high acoustic frequency is unrealistic. 2.2. Optical configurations Figure 2 illustrates the principle of operation of the present method. A Michelson interferom- eter is configured with one end mirror replaced by the thin-film specimen. Call this interfero- Optical Interferometry 64 metric arm the signal arm, and the other the reference arm. In the signal arm, the specimen is placed with the film side facing the beam splitter. The specimen is oscillated with an acoustic transducer from the rear (substrate) side, and the resultant oscillation of the film surface is detected as the corresponding change in the optical path length relative to the reference path. It is postulated that the elastic modulus of the interface is lower than that of the film or the substrate material, as the right drawing of Figure 1 illustrates. With this postulate, it follows that the resonant frequency of the interface is lower than that of the film or substrate. In other words, when the acoustic frequency is tuned in a frequency range where the interface can possibly have resonant points, both the film and substrate oscillate as rigid bodies. Therefore, it is possible to detect the differential displacement of the film surface that represents the dynamics of the interface. Figure 2. Michelson interferometer and experimental arrangement. 2.3. Optical intensity behind beam splitter The light intensity on the image plane behind the beam splitter can be expressed as follows. Figure 2. Michelson interferometer and experimental arrangement. 2.3. Optical intensity behind beam splitter ht intensity on the image plane behind the beam splitter can be expressed as follows. ( ) ( ) 0 0 0 0 0 0 0 2 2 cos sin 2 2 cos sin é ù = + - + = + + é ù ë û ë û s r I t I I k l l kd t I I t w d d w (1) (1) Here I0 is the intensity of the reference and signal beams, k is the wave number of the laser light in (rad/m), ls0 and lr0 are respectively, the initial (physical) length of the signal and reference arms, δ0 = ls0 – lr0 is the arm length difference, δ = kd and d are the oscillation amplitude of the film surface in (rad) and (m), and ω is the oscillation (driving) angular frequency of the film specimen. Here the reference and signal beams are the noninterfering light beams in the Application of Optical Interferometry for Characterization of Thin-Film Adhesion http://dx.doi.org/10.5772/66205 65 65 reference and signal arms, respectively; their intensities are assumed to be equal to each other. (In reality, they are not equal to each other but the gist of the argument here is not affected by inequality. The error associated with the inequality is discussed later in this paper.) The second term in the right-hand side of Eq. (1) with cos[δ0+ δ sin ωt] is called the interference term. This term is important as it contains the relative phase change information. I(t) is captured by a photodetector or an imaging device placed behind the beam splitter. Two methods are possible to detect the relative optical path change behind the beam splitter. Call them the total-intensity and two-dimensional methods. Both methods have advantages and disadvantages. In the total-intensity method, the total intensity I(t) is captured by a fast photodetector such as a silicon PIN photodiode. The advantage of this method is that the response time of the detector is fast enough to analyze I(t) at the same frequency as the acoustic transducer (ω in Eq. (1)). A disadvantage of this method is that it detects the interference term representing the entire cross-sectional area of the laser beam reflected off the specimen. It is unable to resolve the intensity over the plane of the specimen. 2.3. Optical intensity behind beam splitter Another disadvantage of this method is that it is vulnerable to unwanted optical path changes due to environmental disturbance. In the two-dimensional method, an imaging device (an array of photodetector such as a CCD (Charge Coupled Device)) is used for the photodetector behind the beam splitter. (Hereafter, this type of imaging device is referred to as a CCD.) A typical CCD consists of approximately 500 rows and 500 columns of pixels. It is possible to detect the relative phase change two dimensionally on a pixel-by-pixel basis. Another advantage of this method is that by intro- ducing a so-called carrier fringe system, it is possible to reduce the influence of the unwanted optical path length change due to environmental disturbance. The disadvantage of this method is that the frame rate (the sampling rate) of a CCD has normally orders of magnitude lower than the acoustic frequency. Consequently, the detected signal is greatly down-sampled. Below we discuss the two configurations in more detail. 2.3.1. Total intensity configuration In this configuration, the light beams from the two arms are aligned so that they overlap each other for the entire path they share, i.e., the optical path from the beam splitter to the photo- detector. Under these conditions, the film surface displacement due to the acoustic oscillation changes the relative path length difference commonly to all points on the x-y plane (the plane of the specimen). The photodetector signal is proportional to the total intensity expressed by Eq. (1), where the oscillation comes from the interference term. By measuring the intensity of the signal beam and the reference beam separately, we can evaluate 2I0 and express the interference as follows. ( ) 0 0 0 2 cos sin 2 - + = é ù ë û I t I t I d d w (2) (2) Optical Interferometry 66 In evaluating the oscillation amplitude δ from Eq. (2), the initial phase difference δ0 plays an important role. The phase oscillation due to the acoustic transducer occurs around δ0. Since the function cosθ has the greatest slope at θ = 0, the oscillation amplitude of Eq. (2) is maxi- mized when δ0 = 0. In other words, the amplitude of the oscillation due to δ is maximized when the initial interference is totally destructive. Figure 3 illustrates this situation for �0 = 0, � 4, � 2, when the oscillation amplitude δ is 0.2 as an example. The issue is that when an environmental factor such as changes in the refractive index of the air due to temperature fluctuations vary the value of δ0 in a random fashion, it becomes impossible to distinguish whether observed interferometric intensity variation is due to δ or δ0 in Eq. (2). Figure 3. Interference term with three different initial phases. Figure 3. Interference term with three different initial phases. 2.3.2. Two-dimensional configuration In this configuration, a CCD is used to capture the intensity represented by Eq. (1). Normally, the frame rate of a CCD is significantly lower than the acoustic frequency. A CCD with a frame rate comparable to the acoustic frequency is available but it is expensive and sometimes the number of pixels is limited in exchange for a higher frame rate. So, here we discuss image analysis for a low frame rate case. The issue of environmental relative phase fluctuation discussed above applies to the two Figure 3. 2.3.1. Total intensity configuration Interference term with three different initial phases. (a) Simple Michelson method Eq. (1) represents the instantaneous intensity observed behind the beam splitter. To discuss the case where the CCD’s frame rate is much lower than the signal frequency, it is convenient to rewrite Eq. (1) in terms of Bessel functions of first kind. Using the following identities, ( ) ( ) ( ) ( ) 0 2 4 cos sin 2 cos2 2 cos4 = + + +L t J J t J t d w d d w d w ( ) ( ) ( ) 1 3 sin sin 2 sin 2 sin3 = + +L t J t J t d w d w d w ( ) ( ) ( ) ( ) 0 2 4 cos sin 2 cos2 2 cos4 = + + +L t J J t J t d w d d w d w ( ) ( ) ( ) 1 3 sin sin 2 sin 2 sin3 = + +L t J t J t d w d w d w Eq. (1) can be rewritten as follows. Eq. (1) can be rewritten as follows. Eq. (1) can be rewritten as follows. ( ) ( ) ( ) ( ) ( ) ( ) { } ( ) ( ) ( ) 0 0 0 0 0 0 0 0 0 0 0 0 2 4 0 0 1 3 2 2 cos sin 2 2 cos cos sin 2 sin sin sin 2 2 cos 2 cos2 2 cos4 } 2 sin {2 sin 2 sin3 } = + + = + - = + + + + - + + L L I t I I t I I t I t I I J J t J t I J t J t d d w d d w d d w d d d w d w d d w d w (3) (3) In the present case, the acoustic frequency is in the range of 1–20 KHz, and the CCD has a frame rate of 30 fps (frames per second), or three orders of magnitude lower than the acoustic frequency. In other words, the data taken by the digital camera is greatly down-sampled. Under these conditions, the output of the CCD can be expressed as follows. 2.3.2. Two-dimensional configuration In this configuration, a CCD is used to capture the intensity represented by Eq. (1). Normally, the frame rate of a CCD is significantly lower than the acoustic frequency. A CCD with a frame rate comparable to the acoustic frequency is available but it is expensive and sometimes the number of pixels is limited in exchange for a higher frame rate. So, here we discuss image analysis for a low frame rate case. The issue of environmental relative phase fluctuation discussed above applies to the two- dimensional configuration as well. However, the introduction of a carrier fringe system in conjunction with frequency domain analysis greatly overcomes this issue. Below we first consider the case when a carrier fringe system is not introduced (called the simple Michelson method) followed by the carrier fringe method. Application of Optical Interferometry for Characterization of Thin-Film Adhesion http://dx.doi.org/10.5772/66205 67 (a) Simple Michelson method (a) Simple Michelson method (a) Simple Michelson method ( ) { } ( ) ( ) ( ) 0 0 0 0 0 2 1 0 0 0 0 2 1 10 2 1 cos 4 cos cos 2 4 sin sin(2 1) ¥ = ¥ - = + + - - å ò ò åò N N N N I J dt I J N t dt I J N t t t t d d d d w d d w (4) ( ) { } ( ) ( ) 0 0 0 0 0 2 1 0 0 2 1 cos 4 cos cos 2 ¥ = + + - å ò ò N N I J dt I J N t dt t t d d d d w (4 (4) ( ) 0 0 2 1 10 4 sin sin(2 1) ¥ - = - åò N N I J N t t d d w ( ) 0 0 2 1 10 4 sin sin(2 1) ¥ - = - åò N N I J N t t d d w Here τ is the exposure time of the CCD and N is an integer. Of the terms on the right-hand side of Eq. (4), those terms that contain the summation over N oscillate. On the other hand, the first integral is constant with respect to time and therefore increases in proportion to τ. Conse- quently, under the condition where the exposure time is much greater than the period of oscillation, i.e., τ ≫ 2π/ω, the signal S(τ) can be approximated by the first integral. ( ) ( ) { } 0 0 0 2 1 cos @ + S I J t t d d (5) (5) Figure 4 compares the signal evaluated by Eq. (4) and the approximate signal by Eq. (5) for the driving frequency of 11 KHz as a function of the exposure time. It is seen that for τ = 1/30 =33 Figure 4 compares the signal evaluated by Eq. (4) and the approximate signal by Eq. (5) for the driving frequency of 11 KHz as a function of the exposure time. It is seen that for τ = 1/30 =33 Optical Interferometry 68 (ms), the exposure time corresponding to the frame rate of 30 fps (the frame rate used in the present study), the approximation by Eq. (5) is accurate. Figure 4. (a) Simple Michelson method ( ) ( ) { } 0 0 2 1 cos @ + S I xJ t t a d (7) (7) Here, x is the coordinate axis set up on the specimen’s surface and α is the angle of til Here, x is the coordinate axis set up on the specimen’s surface and α is the angle of tilt. The advantage of this technique in the present context is as follows. In the simple Michelson method, if environmental disturbance changes the relative phase, it directly affects the signal expressed by Eq. (5). There is no way of knowing whether the change in the signal is due to the oscillation amplitude or the environmental noise unless the sampling rate of the optical detector is higher than the acoustic frequency. When the sampling rate is lower than the acoustic frequency, the detector’s signal passes though a number of maxima and minima corresponding to the constructive and destructive interference. On the other hand, if carrier fringes are introduced, it is always possible to capture the constructive and destructive interferences; the former corresponds to the bright fringes and the latter to the dark fringes. An optical path length change causes a shift of the fringe pattern in a lateral direction at the same frequency as the optical path length change. If the optical path length change is due to the acoustic oscillation of the specimen, the fringes move back and forth transversely to the beam (dither) at the acoustic frequency. The CCD cannot resolve this fast dithering motion. Consequently, the fringe contrast is reduced. If the optical path length change is due to an environmental effect, the fringe shift is most likely slower than the sampling rate and can be resolved as a change in the fringe location by the CCD with the fringe contrast unchanged. A slight angular misalignment changes the fringe spacing, and causes some error in the fre- quency-domain analysis as will be discussed later. However, the error is much smaller than the simple Michelson method. The carrier fringe method is especially effective if the fringe data is analyzed in the spatial- frequency domain. The reduction in the fringe contrast due to fast dithering is detected in the Fourier spectrum as a reduction in the height of the main peak at the frequency determined by the fringe spacing associated with cos αx in Eq. (7). (a) Simple Michelson method Comparison of J0(δ) approximation and complete integral. Figure 4. Comparison of J0(δ) approximation and complete integral. In Eq. (5), the first term 2I0τ is the sum of the optical intensity of the signal and reference arms. Experimentally, these intensities can be easily obtained by blocking one of the arms at a time and using the CCD behind the beam splitter. By subtracting this term from the total signal and dividing the result by 2I0τ, we can derive expression of the interference term (Eq. (2)) for the total intensity configuration as follows. ( ) ( ) 0 0 0 0 2 cos 2 - = S I J I t t d d t (6) (6) In principle, by knowing the initial relative phase δ0 from an independent experiment (such as changing the reference arm length through fringes with the acoustic transducer turned off), we can even estimate the value of δ from the known curve of J0(δ) and d = δ/k. However, in reality, environmental noise causes fluctuations in the optical path length. As will be discussed later, a temperature change of 0.1°C in the air in the beam path can cause a considerable change in the relative phase δ0. Also, angular misalignment such as the one due to seismic disturbance reduces the accuracy in the subtraction of the I0 in Eq. (6); since the intensity of the reference and signal beams is measured at different times from the total intensity, any angular misalign- ment shifts the beam center on the image plane of the CCD, and that reduces the accuracy of Eq. (6). (b) Carrier fringe method Application of Optical Interferometry for Characterization of Thin-Film Adhesion http://dx.doi.org/10.5772/66205 6 69 In this method a linear spatial phase-variation is introduced so that the relative phase changes over several periods of 2π across the cross-section of the laser beam. This technique is known as the introduction of carrier fringes and widely used in ESPI (Electronic Speckle–Pattern Interferometry) [7]. Carrier fringes can be introduced by slightly tilting the specimen or the reference mirror, or by inserting a mechanism to introduce a linear phase variation such as an optical wedge. Figure 2 shows an example where the specimen is slightly tilted. We can express the CCD’s output in this case by replacing cos δ0 with cos αx as follows. 3.1. Thin film specimens A pair of platinum-titanium (Pt-Ti) coated silicon (Si) thin film specimens is used in the present experiments. The Si substrate is cut along the [1 0 0] plane and 750 μm in thickness. The Ti layer is coated on the Si substrate and the Pt is coated over the Ti layer. The thickness of the Pt and Ti layers are 100 nm and 10 nm, respectively. In one specimen (the treated specimen) of the pair, the Ti layer is coated after the substrate surface is treated with oxygen-plasma bombardment. This treatment makes the Si surface hydrophilic, and therefore strengthens the Ti-Si bond. In the other specimen (the untreated specimen), the Ti layer is coated without a surface treatment. (a) Simple Michelson method Thus, by forming Fourier spectrum of the optical intensity profile and evaluating the peak height of the spectrum we can evaluate the fringe contrast, and in turn, estimate the oscillation amplitude. The spatial fringe shift due to environmental change in δ0 does not change the Fourier spectrum as it is not a function of x. An angular misalignment due to environmental disturbance can change the spectrum peak height, but the effect is relatively small (see below). Optical Interferometry 70 (a) Analysis with simple Michelson method Figure 5 shows the intensity profiles of the reference and signal beams captured by a CCD placed behind the beam splitter. For the measurement of each profile, the other beam is blocked. Thus, they do not include the interference term in Eq. (1). The reference beam is reflected off the mirror (the Y-end mirror in Figure 2) and therefore its profile is Gaussian. The signal beam is reflected off the specimen whose surface is not as flat as the mirror. Consequently, its profile is somewhat deformed. When these intensities are subtracted from Eq. (1) for the evaluation of the relative phase in Eq. (2), this causes some part of the interference beam in its cross- sectional area to miss interference. Figure 6 indicates those non-interfering portion as a dip. Figure 5. Reference and signal beam profiles. Figure 5. Reference and signal beam profiles. Application of Optical Interferometry for Characterization of Thin-Film Adhesion http://dx.doi.org/10.5772/66205 71 Application of Optical Interferometry for Characterization of Thin-Film Adhesion http://dx.doi.org/10.5772/66205 71 Figure 6. Beam profile of interference term. The two profiles in Figure 6 are taken from the same experiment where the specimen is driven by the acoustic transducer under the same condition; the driving frequency and amplitude being the same. The left and right profiles are obviously different. The left profile indicates that the interference term is positive and the left profile indicates that it is more negative. Since the phase change due to the film surface displacement is not large enough to change the sign of Eq. (6) (i.e., it is unlikely that J0(δ)<0), it is likely that the difference comes from a change in the reference phase difference (δ0 in Eq. (6)). http://dx.doi.org/10.5772/66205 Figure 6. Beam profile of interference term. The two profiles in Figure 6 are taken from the same experiment where the specimen is driven by the acoustic transducer under the same condition; the driving frequency and amplitude being the same. The left and right profiles are obviously different. The left profile indicates that the interference term is positive and the left profile indicates that it is more negative. Since the phase change due to the film surface displacement is not large enough to change the sign of Eq. (6) (i.e., it is unlikely that J0(δ)<0), it is likely that the difference comes from a change in the reference phase difference (δ0 in Eq. (6)). (a) Analysis with simple Michelson method The two profiles in Figure 6 are taken from the same experiment where the specimen is driven by the acoustic transducer under the same condition; the driving frequency and amplitude being the same. The left and right profiles are obviously different. The left profile indicates that the interference term is positive and the left profile indicates that it is more negative. Since the phase change due to the film surface displacement is not large enough to change the sign of Eq. (6) (i.e., it is unlikely that J0(δ)<0), it is likely that the difference comes from a change in the reference phase difference (δ0 in Eq. (6)). The two profiles in Figure 6 are taken from the same experiment where the specimen is driven by the acoustic transducer under the same condition; the driving frequency and amplitude being the same. The left and right profiles are obviously different. The left profile indicates that the interference term is positive and the left profile indicates that it is more negative. Since the phase change due to the film surface displacement is not large enough to change the sign of Eq. (6) (i.e., it is unlikely that J0(δ)<0), it is likely that the difference comes from a change in the reference phase difference (δ0 in Eq. (6)). Figure 7(a) shows the interference term as a function of time when the acoustic transducer is on and off. Apparently, the fluctuation is significantly higher when the transducer is on. Figure 7(b) is the Fourier transform of Figure 7(a). The peak frequency of the disturbance is less than 1 Hz which is lower than the frame rate of the CCD used in the experiment. This indicates that the phase fluctuation affects the approximated expression Eq. (5) when the transducer is on. Figure 7. Variation of interference term as a function of time (left) and frequency (right). Figure 7. Variation of interference term as a function of time (left) and frequency (right). It is likely that the temperature rise due to heat emitted from the transducer is one of the causes of the phase fluctuation. An independent temperature measurement indicates that the air Optical Interferometry 72 temperature easily rises over 0.1°C within 1 s after the transducer is turned on, and that the temperature fluctuates by ±0.1°C approximately every few minutes. (a) Analysis with simple Michelson method In one set of measurement in which the transducer is turned on and off every 3 min, a total temperature rise of 0.4°C is recorded over a period of 30 min. It is suspected that air convection causes the temperature fluctuation. It is informative to make a rough estimate of the phase change due to the above temperature change. The optical phase change due to the temperature dependence of the refractive index of air can be expressed as follows. 2 l n d dT T f p l ¶ = ¶ (8) (8) (8) Here λ is the wavelength, l is the path length, n is the refractive index of air and dT is the temperature change. The temperature coefficient ∂n/∂T of air is −0.87×10−6 (1/°C) [8]. The arm length of the interferometer used in this experiment is 10 (cm). The wavelength of the laser used in this study is 632.8 nm. So, the phase change due to a temperature change of ±0.1°C over the round trip in the interferometric arm is 20 (cm)/632.8 (nm) ×0.87×10−6×0.1=2.75% (of the period 2π). Accordingly, the phase error due to the air temperature change of 0.4°C observed over 30 min is 2.75×4 = 11.0% of the period. The issues of the deformed phase front and the initial phase fluctuation observed in Figures 6 and 7 make it difficult to use Eq. (5) with the total intensity method. In the next section, the carrier fringe method that greatly reduces the influence of the initial phase fluctuation is discussed. (b) Analysis with carrier fringe method (b) Analysis with carrier fringe method The fluctuation of the initial phase δ0 can be evaluated with the carrier fringe method as well. In this case, a change in δ0 causes a shift in the carrier fringe location. The top row of Figure 8 shows the spatial shift of carrier fringes at three different driving frequencies. In conducting the measurement at the three driving frequencies, the voltage input to the transducer is adjusted so that the oscillation amplitude of the transducer surface is the same for all the frequencies. It is seen that the shift, i.e., the change in the reference phase δ0 is rather random, supporting the above speculation that convection of air in the interferometric arm causes the initial phase fluctuation. The lower row of Figure 8 is the Fourier spectrum of the spatial intensity variation of the fringe pattern observed at the respective driving frequencies. The first and main spectral peak observed at the spatial frequency of 0.02 (1/pxl) represents the carrier fringe periodicity of the light intensity; cos αx in Eq. (6) or the fringe pattern (see top of Figure 9). The Fourier spectrum is computed from the intensity profile over the horizontal span of 600 pxl (across a horizontal line near row 250 in the fringe image in Figure 9). Thus, the minimum frequency is 1/600 (1/ pxl). This means that the frequency of 0.02 (1/pxl) corresponds to the 0.02/(1/600) =12th harmonics, or the periodicity of 600/12=50 (pxl). The fringe patterns in Figure 9 indicate this periodicity. Application of Optical Interferometry for Characterization of Thin-Film Adhesion http://dx.doi.org/10.5772/66205 Application of Optical Interferometry for Characterization of Thin-Film Adhesion http://dx.doi.org/10.5772/66205 73 73 Figure 8. Fluctuation of the initial phase and corresponding change in Fourier spectrum observed with carrier fringe method. Figure 8. Fluctuation of the initial phase and corresponding change in Fourier spectrum observed with carrier fringe method. Figure 9. Effect of fringe shift and intensity profile change on Fourier spectrum. Figure 9. Effect of fringe shift and intensity profile change on Fourier spectrum. The lower row of Figure 8 clearly illustrates the advantage of the carrier fringe method. The spectrum plotted for each driving frequency is superposition of 100 frames. Since the CCD frame rate is 30 fps, 100 frames correspond to approximately 3 s in time. Notice that the spectra observed at driving frequency 14.0 KHz show that the spectrum data are scattered in the frequency range left of the peak. This observation indicates that the random variation of the reference phase change is reflected in the low spatial frequency region of the spectrum; as cosδ0 changes, the total intensity detected by the CCD fluctuates, and that changes the low spatial frequency component. However, the peak height of the FFT spectrum is unaffected. This is because the peak value corresponds to the spatial dependence cos αx, not cosδ0 as a Figure 9. Effect of fringe shift and intensity profile change on Fourier spectrum. The lower row of Figure 8 clearly illustrates the advantage of the carrier fringe method. The spectrum plotted for each driving frequency is superposition of 100 frames. Since the CCD frame rate is 30 fps, 100 frames correspond to approximately 3 s in time. Notice that the spectra observed at driving frequency 14.0 KHz show that the spectrum data are scattered in the frequency range left of the peak. This observation indicates that the random variation of the reference phase change is reflected in the low spatial frequency region of the spectrum; as cosδ0 changes, the total intensity detected by the CCD fluctuates, and that changes the low spatial frequency component. However, the peak height of the FFT spectrum is unaffected. This is because the peak value corresponds to the spatial dependence cos αx, not cosδ0 as a Optical Interferometry 74 function of time. Thus, it can be said that the peak height difference comes from the oscilla- tion amplitude δ in Eq. (6). Application of Optical Interferometry for Characterization of Thin-Film Adhesion http://dx.doi.org/10.5772/66205 73 Here, the change in the total intensity depends on the initial phase δ0 ; when δ0 is closer to π/2 its change is greater as the cosine function has the greatest slope around π/2 (Figure 3). Since the initial phase changes randomly, we have no control over the total intensity. Figure 9 illustrates the advantage of the carrier fringe method more explicitly. This figure depicts two representative cases observed at driving frequency of 8.5 and 14.0 KHz. For each frequency, the first and last frames of 100 consecutive frames are shown. Figure 9(a) and (b) shows the fringe images, the upper graphs of Figure 9(c) and (d) are the spatial intensity profiles over 640 horizontal pixels, and the lower graphs of Figure 9(c) and (d) are the corresponding Fourier spectra. In the case of 8.5 KHz driving, the fringe pattern shifts approximately by 10 pxl over the 100 frames, but its profile is unchanged. In this case, the peak value of the Fourier spectrum is the same for the first and last frames. In the case of 14.0 KHz driving, the fringe shift is similar or less than the 8.5 KHz case but the intensity profiles are changed; the fringe image for the first frame (the left image of Figure 9(b)) shows that the right bright fringe is stronger than the left bright fringe in in- tensity whereas the image of the last frame (the right image of Figure 9(b)) shows that the left bright fringe is stronger in intensity. The top graph of Figure 9(d) indicates the differ- ence in the intensity patterns between the first and last frames more explicitly. In this case, the peak values of the Fourier spectrum for the first and last frames are different. As indicated above, the fringe shifts are due to the random change in the initial phase. The change in the intensity profile is most likely caused by angular misalignment of the reference and signal beams. These observations indicate that while the carrier fringe method is not affected by fluctuation of the initial phase, the angular misalignment must be suppressed as much as possible to reduce errors. 3.3. Frequency domain analysis with carrier fringe method The consistency of the spectrum peak observed in Figure 8 indicates that it is possible to evaluate the oscillation amplitude quantitatively. Consider the relation between the input voltage to the acoustic transducer and the oscillation amplitude of the film surface. At a given oscillation frequency, the electric power of the transducer is proportional to the square of the applied (input) voltage, and the mechanical power associated with the elastic motion of the film is proportional to the square of the oscillation amplitude. Thus, the in- put voltage and the film surface oscillation amplitude are proportional to each other. Un- der the condition that the oscillation frequency is orders of magnitude higher than the CCD’s frame rate, an increase in the oscillation amplitude reduces the fringe contrast (be- cause the CCD cannot follow the fast shift of the fringes). Therefore, it is expected that the blurriness of the fringe pattern increases with the input voltage to the transducer. With the reduction of fringe contrast, the peak value of the Fourier spectrum decreases. Further, by fitting the reduction in the fringe contrast with the use of Eq. (6), it is possible to estimate the oscillation amplitude accurately. Application of Optical Interferometry for Characterization of Thin-Film Adhesion http://dx.doi.org/10.5772/66205 7 75 Figure 10 compares fringe contrasts as a function of the input voltage. As expected, the fringe becomes blurrier with the increase in the input voltage. Figure 11 plots the Fourier spectra obtained at the six voltages (including 0 V) indicated in Figure 10. With the increase in the input voltage, the spectrum peak decreases. Figure 10. Fringe contrast at various input voltage to transducer. Figure 10. Fringe contrast at various input voltage to transducer. Figure 11. Fourier spectra for six input voltages shown in Figure 10. Figure 10. Fringe contrast at various input voltage to transducer. Figure 11. Fourier spectra for six input voltages shown in Figure 10. Figure 11. Fourier spectra for six input voltages shown in Figure 10. Based on the proportionality between the input voltage and the oscillation amplitude, it is possible to estimate the oscillation amplitude by fitting the experimental relation between the input voltage and the spectrum peak value to J0(δ) (Eq. (5)). When the input voltage is zero hence δ = 0, J0 takes the maximum value of unity. As the oscillation amplitude increases, J0(δ) decreases to the first root at δ = 2.4048. 3.3. Frequency domain analysis with carrier fringe method Thus, by evaluating the peak value relative to the case when the transducer is turned off, it is possible to estimate the oscillation amplitude d = δ/k. Figure 12 plots the peak values shown in Figure 11 relative to the highest value obtained with the null input voltage using a factor a in J0(aV) as the fitting parameter. Here V denotes the input voltage. The measured spectrum peak values fit to the Bessel function reasonably well. Based on the proportionality between the input voltage and the oscillation amplitude, it is possible to estimate the oscillation amplitude by fitting the experimental relation between the input voltage and the spectrum peak value to J0(δ) (Eq. (5)). When the input voltage is zero hence δ = 0, J0 takes the maximum value of unity. As the oscillation amplitude increases, J0(δ) decreases to the first root at δ = 2.4048. Thus, by evaluating the peak value relative to the case when the transducer is turned off, it is possible to estimate the oscillation amplitude d = δ/k. Figure 12 plots the peak values shown in Figure 11 relative to the highest value obtained with the null input voltage using a factor a in J0(aV) as the fitting parameter. Here V denotes the input voltage. The measured spectrum peak values fit to the Bessel function reasonably well. Optical Interferometry 76 Figure 12. Spectrum peak value as a function of input voltage to transducer. Figure 13. Oscillation amplitude estimated for each voltage input. Figure 12. Spectrum peak value as a function of input voltage to transducer. Figure 12. Spectrum peak value as a function of input voltage to transducer. Figure 13. Oscillation amplitude estimated for each voltage input. Figure 13. Oscillation amplitude estimated for each voltage input. Application of Optical Interferometry for Characterization of Thin-Film Adhesion http://dx.doi.org/10.5772/66205 77 77 77 Now that the Bessel function-like behavior of the spectrum peaks is confirmed, the value of δ can be estimated from the spectrum peak value obtained for each input voltage relative to the peak value obtained with the transducer turned off (zero input voltage). Subsequently, the oscillation amplitude can be found from d = δ/k. Figure 13 plots the value of d found in this fashion. 3.4. Driving frequency sweep By repeating the same measurement as the lower row of Figure 8, it is possible to find out the frequency dependence of the film surface. As is the case of Figure 8, the input voltage to the acoustic transducer is adjusted so that the oscillation amplitude of the transducer surface is the same for all the driving frequencies tested. Figure 14 shows the result of such a series of measurement for a driving frequency range of 8–14.5 KHz. Here, the vertical axis is the value of the Fourier spectrum peak obtained from the intensity profile across the fringe pattern averaged over five rows near the vertical center of the fringe images. In ac- cordance with the argument made above, the higher the peak, the smaller the film surface oscillation. Since the transducer surface has the same oscillation amplitude for all the driv- ing frequencies, the frequency dependence of the film surface oscillation observed in this figure represents the transducer surface to the film surface transfer function. Since the transfer function of the substrate and film materials themselves are considered to be unity Figure 14. Driving frequency sweep of Fourier spectrum peak value. Figure 14. Driving frequency sweep of Fourier spectrum peak value. Optical Interferometry 78 in this frequency range, the transfer function represents the elastic property of the film-sub- strate interface. The lower the spectrum peak the greater the oscillation on the film surface, which can be interpreted as the greater oscillation of the interface. The transfer function shown in Figure 14 is obtained for five rows near the vertical center of the fringe image. By repeating the same procedure for other rows, it is possible to draw a map of the transfer function. Figure 15 shows a three-dimensional map obtained in this fashion for four specimens; the untreated, treated, and bare silicon specimens. Here one horizontal axis is the row number and the other horizontal axis is the driving frequency. The top two plots are the cases when silicon substrates only are used (called the bare silicon specimens), and the bottom two plots are cases when the treated and untreated specimens are used. The treated and untreated specimens are attached to two different acoustic transducers. 3.4. Driving frequency sweep To eliminate the effect associated with the use of the different transducer, one bare silicon specimen is attached to the same transducer as the treated specimen and the other bare silicon specimen is attached to the other transducer used for the untreated specimen. Figure 15. Fourier spectrum peak for several rows as a function of driving frequency. Figure 15. Fourier spectrum peak for several rows as a function of driving frequency. Figure 15. Fourier spectrum peak for several rows as a function of driving frequency. The two plots for the bare silicon specimens appear to be flat, indicating that the bare silicon specimens do not have clear frequency dependence in the oscillation. On the other hand, the Application of Optical Interferometry for Characterization of Thin-Film Adhesion http://dx.doi.org/10.5772/66205 7 79 treated and untreated specimens show frequency dependences. As mentioned above, the frequency dependence represents the elastic characteristics of the substrate-film interface. It is interesting to note that the untreated specimen shows a crater-like pattern around row 250 through 280 near driving frequency of 10 KHz. It is possible to interpret this pattern as representing the so-called blister effect [9–11]; the interface has a weakly adhered spot where the film experiences membrane-like oscillation when the specimen is driven. The treated specimen does not show a crater-like pattern. Instead, there is a valley running through all rows around the driving frequency of 8 KHz. We observed similar patterns in the treated and untreated specimens for a number of times. It is possible that the precoating surface treatment makes the adhesion more uniform so that the chances of the specimen having blisters is lower. These observations particularly interest us because there is no established technique to evaluate the blister effect non-destructively. 4. Conclusion An optical interferometric method to characterize the elastic behavior of the interface of thin- film systems is discussed. The thin-film specimen is configured as one of the end-mirrors of a Michelson interferometer with the film side facing the beam splitter. The specimen is oscillated sinusoidally with an acoustic transducer. The harmonic response of the film surface to the acoustic oscillation is detected as relative optical phase difference between the two interfero- metric arms. An algorithm to estimate the amplitude of the film surface oscillation from the relative optical phase measurement is discussed. Environmental noise that compromises the relative phase measurement is analyzed. The use of a carrier fringe system in conjunction with analysis in the spatial frequency domain is proposed as a method to reduce the influence of environmental noise is discussed. Under some conditions, the effectiveness of the proposed method is demonstrated with experiment. A sample set of data obtained with Pt-Ti-Si thin-film system is presented. The three-dimen- sional mapping of the adhesion strength obtained with the carrier fringe method indicates some behavior of the film surface that can be interpreted as representing the so-called blister effect. It is interesting to note that the blister-like behaviors observed in the surface-treated and non-treated specimen are different from each other. This observation is of particular interest to us as non-destructive evaluation of the blister effect is not easy. More investigation is under way. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP), NRF-2013M2A2A9043274, NRF-2011-220-D00002, and the Louisiana Board of Regents, LEQSF(2016-17)-RD-C-13. Author details Sanichiro Yoshida1, David R. Didie1, Jong-Sung Kim2 and Ik-Keun Park2 Address all correspondence to: syoshida@selu.edu *Address all correspondence to: syoshida@selu.edu 1 Department of Chemistry and Physics, Southeastern Louisiana University, Hammond, Louisiana, USA 1 Department of Chemistry and Physics, Southeastern Louisiana University, Hammond, Louisiana, USA 2 Department of Mechanical and Automotive Engineering, Seoul National University of Sci- ence and Technology, Nowon-gu, Seoul, South Korea 2 Department of Mechanical and Automotive Engineering, Seoul National University of Sci- ence and Technology, Nowon-gu, Seoul, South Korea Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP), NRF-2013M2A2A9043274, NRF-2011-220-D00002, and the Louisiana Board of Regents, LEQSF(2016-17)-RD-C-13. Optical Interferometry 0 80 References [1] Lemons RA, Quate CF: Acoustic microscopy. In: Mason WP, Thurston RN, editors. Physical Acoustics. London: Academic Press; 1979; Vol. XIV, pp. 1–92. [2] Weglein RD: Acoustic microscopy applied to SAW dispersion and film thickness measurement. IEEE. Trans. Sonics. 1980; 27: 82–86. [3] Atalar A: An angular-spectrum approach to contrast in reflection acoustic microscopy. J. Appl. Phys. 1978; 49: 1530–1539. [4] Atalar A: A physical model for acoustic signatures. J. Appl. Phys. 1979; 50: 8237–8239. [5] Telschow KL, Deason VA, Cottle DL, Larson JD: III: Full-field imaging of gigahertz film bulk acoustic random motion. IEEE. Trans. Ultrason. 2003; 94: 79–88. [6] Yoshida S, Didie DR, Didie D, Sasaki T, Park HS, Park IK, Gurney D: Opto-acoustic method for the characterization of thin-film adhesion. Appl. Sci. 2016; 6: doi:10.3390/ app6060163. [7] Sciammarella CA, Sciammarella FM: Experimental Mechanics of Solids. Hoboken: Wiley; 2012. ISBN-10: 0470689536. [8] Effect of Temperature on Refractive Index (dn/dt). Available from: http://www.ohara- gmbh.com/e/katalog/tinfo_2_4.html [Accessed on 2016-09-11]. [9] Bedrossian J, Kohn RV: Blister patterns and energy minimization in compressed thin films on compliant substrates. Commun. Pure Appl. Math. 2015; 68: 472–510. [10] Dennenberg H: Measurement of adhesion by a blister method. J. Appl. Polym. Sci. 1961; 5: 125–134. [11] Volinsky AA, Moody NR, Gerberich WW: Interfacial toughness measurements for thin films on substrates. Act Mater. 2002; 50: 441–466.
https://openalex.org/W2010787595	https://journals.oslomet.no/index.php/formakademisk/article/download/801/1117	English	null	From Product to Service Design: A Thinking Paradigm Shift	Formakademisk	2,014	cc-by	10,412	Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Abstract Society, industry and the economy are all experiencing changes caused by a shift from products to services. While a “problem-solving” approach is commonly used for the development of products, new design approaches are needed as the primary unit of exchange moves from goods to services. This research argues that a fundamental transformation in the design world is taking place, manifested in a thinking paradigm shift from problem solving (designing products) towards systems thinking (designing services). This paper draws on design literature to identify concepts of systems thinking and problem solving to help understand core elements in the shift from product to service design. It also reports on a series of semi-structured interviews with designers working in five design consultancies that have moved from product design to services design. The results show a change in the way designers think and approach projects when facing the challenges of designing services, confirming a movement from problem solving to systems thinking. However, systems thinking is not replacing problem solving but complementing it. The results also indicate that the growing complexity of the issues designers deal with influences the adoption of systems thinking in responding to service design challenges, as well as current changes in people’s ideas about sustainability and society. Keywords: Service design, product design, systems thinking, paradigm shift, innovation, problem solving www.FORMakademisk.org Moving away from problem solving in product design Problem solving is regarded as a main driver for design activity (Lawson & Dorst, 2009). Designers tend to develop products in response to phenomena framed as “problems”. This way of dealing with a world they are supposed to modify and improve gives designers the means to focus their activities towards an end. Also, it provides a pattern to measure the success of their design proposals against, since “problems solved” can often be seen as an indicator of “good design solutions”. In addition, the problem-solving approach can also define the design process in full. Johansson-Skoldberg et al. (2013, p. 125) suggest that the design process is a problem-solving activity framed by analytical and synthetic thinking, and present Buchanan’s idea that design activity is related to a “step-by-step model of the design process with its two distinct phases: an analytical step of problem definition, followed by a synthetic sequence of problem solution”. As the problem-solving approach helps designers to focus, to measure the success of their creations and to undertake design activity following a suitable design process, it seems to be sufficient as an overarching principle for product design activity. However, the problem-solving approach seems to be associated with a model of design that may be becoming obsolete. On the one hand, as suggested by Jonas (1996), this approach seems to be based on the assumption that problems can be well-defined and solved through a good knowledge of people’s needs and desires, and that it assumes the designer’s ability to know what is “good for people”. This renders design as an activity almost exclusively driven by the designers’ own understanding of the issues they are dealing with. However, user involvement through participatory design methods is increasingly becoming standard practice in design. As a consequence of this, the problem-solving approach model coming from the “design methods movement” underpinned by cybernetic thinking from the 60’s and 70’s (Jonas, 1996), seems to be at odds with the idea of a design process in which users take an active part in the formulation and resolution of issues through design. Conversely, as social change and technological developments mean that society is becoming more interconnected, and new ways of interaction and organisation lead to new experiences and ways of being, so the nature of the issues that designers deal with are changing, as is the way in which designers deal with them. Literature review A literature review has been conducted in order to identify the two main concepts of this research, problem solving and systems thinking, and how they relate to product and service design. This has been embedded in a narrative that describes the transition from product design to service design in current design practice, articulating the adoption of systems thinking by service designers in response to the increasing complexity of services. This literature review covers the areas of design (product design and design thinking in particular), service design (mostly from the design point of view and occasionally from the management perspective), systems thinking and complexity. The narrative has been developed around 5 main headings: -Moving away from problem solving in product design -The shift from product to service design -Services as complex systems -Designers dealing with services are designers dealing with systems -Systems thinking in the design of services Introduction Society, industry and the economy are all experiencing a shift from products to services. As a result of this there is an on-going ‘conceptual shift’ in business and industry characterised by a movement from traditional goods-centred dominant logic (G-DL) to emerging service-centred dominant logic (S-DL) (Vargo & Lusch, 2008). While a “problem-solving” approach is commonly used in the design of products(Cross, 1990; Taura & Nagai, 2011; Rogers et al., 2005; Dorst & Dijkhuis, 1995) the primary unit of exchange is moving from goods to services (Vargo & Lusch, 2008) evidencing that new design approaches for the development of services are needed. This need becomes even more critical as on the one hand, the very nature of products that might be related to services has changed from being purely physical and tangible to become a mixture of both physical and virtual (or intangible) attributes (Rodriguez, 2010); while on the other hand, goods are being absorbed or replaced by services, which are now considered to be specialised competences such as knowledge and skills that people can acquire and exchange (Vargo & Lusch, 2008). These changes have direct implications for the work of designers and the way they approach problems and issues. Designers are moving from product-centred design activity to product-service or service-centred design activity. As Young (2008, p. 43) explains, “The shift in focus from product- and artefact-centred design theory to system- and service-oriented thinking has followed the advent and growth of services in our economy and society accompanied by corresponding changes in technology”. This research seeks to understand if a fundamental transformation in the design world is taking place, manifested in a thinking paradigm shift from problem solving (designing products) to systems thinking (designing services). Bearing in mind the increasingly complex issues designers face while designing services, systems thinking would appear the most appropriate approach As Hugentobler et al. (2004, p. 2) argue, while discussing the implications of systems thinking as an answer to complexity as a problem, “If we really want to support the shift from designers as executants to designers as executives, who originate ideas and plan Vol.7, Nr.3, 2014, Art. 5, 1-27 1 www.FORMakademisk.org Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift processes to put these ideas into practice, then systems thinking has to be considered an essential part of this programme.” Moving away from problem solving in product design Jonas explains how a problem-solving approach is becoming less central in design, as designers have to deal with issues that are complex, fuzzy, non-predictable and pluralistic in values. He describes them as “ill-defined” Vol.7, Nr.3, 2014, Art. 5, 1-27 www.FORMakademisk.org 2 Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift problems, arguing the need for design tools and methods for “the description and analysis of complex problem fields” (Jonas, 1996, p. 4). The shift from product to service design f f p g While dealing with increasingly complex issues, product designers have expanded the scope of their activities beyond the design of objects within their “traditional” boundaries of form, function, material and production (Miettinen, 2011, p. 56), moving into the realm of interactions, systems and environments. Consequently, the conception of product amongst product designers has departed from its material existence, becoming activities, services, and policies (Buchanan, 2001). p ( ) This expansion in the activity and scope of product designers, framed by the economic and industrial shift from G-DL to S-DL, has enabled them to move into the design of services. Additionally, specific product design perspectives such as “a user-centred design approach, a variety of qualitative and quantitative research and data gathering approaches, and visualisation techniques such as sketching, imagining and prototyping” (Miettinen, 2011, p. 60) has allowed product designers to champion the development of service design as a new design discipline heavily underpinned by research and user involvement. For example, this can be noticed in the UK in the creation of pioneering services design consultancies by product designers such as Live Work (2001), or Engine (2000), or by innovative curricular shifts in product design courses towards the design of services such as took place at the Glasgow School of Art in 2005. In this context, service design can be thought as an enquiry rather than a problem-solving activity, situated in the realm of non-engineering design and based on the conception of services as the basic unit of economic exchange instead of something distinct from goods (Kimbell, 2011). Diagram 1 shows Kimbell’s graphic synthesis of this concept. Diagram 1 (Redrawn from (Kimbell, 2011) Diagram 1 (Redrawn from (Kimbell, 2011) 3 3 Vol.7, Nr.3, 2014, Art. 5, 1-27 www.FORMakademisk.org www.FORMakademisk.org Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Designers dealing with services are designers dealing with systems Designers dealing with services are designers dealing with systems It seems that while designing services, designers need to deal with the “complexity and multiple stakeholders that are inherent in services Polaine et al., (2013, p. 187) and with ‘systems’ that “present a different type of complexity than industrial products” p. 85. Additionally, Clatworthy (2011, p. 80) identifies services as systems in which “customers and services interact through many different touch points during the customer journey”. Also, alluding to the services dynamic nature, Clatworthy (2011, p. 85) argues that “services require us to design systems that adapt well to constantly changing parts”. Following Polaine and Clathworthy’s connection between the design services and the development of systems, and their consideration of services as complex dynamic systems, it seems natural that there is a need for designers to use a systemic approach when facing the challenges of designing a service. From a different perspective, Love (2003) problematises the “uncritical conflation of the activities of designing and systems analysis” as it can create confusion in the theoretical development of both fields, as well as leading to the hampering of design and system analysis processes and outcomes. In contrast to the systems thinking approach, Love suggests that the problem-solving approach provides “information that designers and design stakeholders can draw on to make better judgments about the compositional issues that are central to the core activity of designing” (Love, 2003, p. 4). Yet Love recognises that designers use “system methods and perspectives” as sophisticated tools for gathering information. However, using “system methods and perspectives” simply for gathering information seems to be insufficient to help understand the complex and fuzzy issues designers might be trying to address, and to unravel possible undetected areas with potential and relevance for design intervention. The scope of systems thinking can be wider, and encompass critical analytical, synthetic and creative aspects of the design of services. As Nielsen & Nielsen (2009, p. 5) explain,“having detailed knowledge about the service as a system opens up for experimentation with new innovations around how the service can be designed.” www.FORMakademisk.org Services as complex systems Services often involve complex interactions between users, service providers and other stakeholders. Furthermore, they mediate the exchange of information and goods between people. Services are regarded as “complex and multifaceted phenomena” that comprise interrelated aspects as diverse as: “Environment, domain, activities, tools and artefacts, goals, agents, collaborations and group value(s) and Effectiveness” (Wild, 2009, p. 25). Making such reference to the complexity of services, Polaine et al. (2013, p. 81) justify the use of the ‘ecologies metaphor’ used to describe how services often “harbour a complexity that can be compared to systems in nature”. As systems, services can ‘interact’ with other services creating networks of services, becoming service systems. Also, as the provision of services from companies and organisations are often interlaced with the provision of products, they become product service systems (PSS) (Morelli, 2006). Systems thinking in the design of services As services can be thought of as systems, and designing systems of services might offer scope for a service designer, the adoption of systems thinking seems to become essential for the practice of service design. Thus several authors recognise the importance of systems thinking in services design. For example, while Gloppen (2009, p. 89) describes systems thinking as a research area to “further develop the service design leadership role”, Kwon & van Boeijen (2012) see systems thinking as one of the three service design focuses along with experience and time-based medium. As the service design discipline emerges, networks of services grow and evolve, generating newer and wider networks of interdependent systems. Within these systems and their complexity lie the challenges for service designers. In this respect Polaine et al. (2013, p. 85) Vol.7, Nr.3, 2014, Art. 5, 1-27 www.FORMakademisk.org 4 Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift explain, “These systems present a different type of complexity than industrial products. Products require designers to deal with many moving parts, but services require us to design systems that adapt well to constantly changing parts. Networks, organizations, and technology evolve on a daily basis, but the service still needs to deliver a robust customer experience”. explain, “These systems present a different type of complexity than industrial products. Products require designers to deal with many moving parts, but services require us to design systems that adapt well to constantly changing parts. Networks, organizations, and technology evolve on a daily basis, but the service still needs to deliver a robust customer experience”. y p Consequently, it is not surprising that systems thinking has been adopted by service designers, as the Design Council’s RED paper 2 reports that “A number of design groups have broadened the scope of design to include disciplines such as interaction, experience and service design. All of these demand a holistic approach, a level of systems thinking, a focus on individual behaviour, and the orchestration of a range of different design inputs.” (Burns et al, 2006, p. 10) Nelson & Stolterman suggest a view of systems thinking with characteristics that seem to cohere with fundamental aspects of service design, and which opens up the possibility of being used beyond Love’s concept of “gathering information”. Systems thinking in the design of services In their book “The Design Way”, they outline a list of elements that characterise the systems approach, which coincides with the holistic, dynamic and complex nature of designing services (Nelson & Stolterman, 2012, p. 60): www.FORMakademisk.org Systems approach • Interrelationships/compositions • Interconnections/emergence • Inclusive, unifying, and integrating • Holistic inquiry • Observer dependent • Multidimensional • Mutual: - Analytic and synthetic - Left and right brain - Rational and aesthetic - Objective and subjective - Individual and unified - Complex and simple - Complex and simple - Similar and different - Similar and different - Thinking and acting - Big picture and details Nelson & Stolterman argue the existence of two distinctive scholarly discourses around the idea of systems. They identify systems from an epistemological stance when they are an “embodied way of thinking” or from an ontological stance when a system is “the thing that is thought about”. The ontological stance refers to the “understanding of systems as “real things” and is located within the confines of system science and the scientific method. The epistemological refers to a “systemic inquiry approach”, which focuses on a way of thinking that enables different fields of focused enquiry to be related to each other”. These authors emphasise the idea that systems thinking is “a stance that can be assumed by a change in mindset”, one which relies less on the “mastery of a set of theories, methods, and facts”. (Nelson & Stolterman, 2012, p. 64) This study takes the view of systems thinking as a way of thinking about and understanding phenomena, embracing Nelson & Stolterman’s elements of a system approach. Their systems thinking approach can help to understand relational aspects of otherwise fuzzy and complex issues, and become an instrument of analysis and synthesis. Rather than a collection of science-based methods, systems thinking should be read in this study as a world view that sees things as being holistic and interconnected (Maani & Maharaj, 2001). Vol.7, Nr.3, 2014, Art. 5, 1-27 5 5 Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Vol.7, Nr.3, 2014, Art. 5, 1-27 Approach and methodology pp gy As the literature review provides a theoretical framework for this study, it also contextualises a series of one hour semi-structured interviews with designers from five different design consultancies (Table 1) who have moved from product design to services design. Table 1 These designers have been chosen as they are regarded as protagonists in the shift from product to service design, mainly in the UK. Table 1 shows primary information about the interviewees. Diagram 2 presents a timeline illustrating some of the interviewees’ relevant landmarks within the context of product and services design during the last 20 years. These designers have been chosen as they are regarded as protagonists in the shift from product to service design, mainly in the UK. Table 1 shows primary information about the interviewees. Diagram 2 presents a timeline illustrating some of the interviewees’ relevant landmarks within the context of product and services design during the last 20 years. Vol.7, Nr.3, 2014, Art. 5, 1-27 www.FORMakademisk.org www.FORMakademisk.org 6 Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift www.FORMakademisk.org 7 Vol.7, Nr.3, 2014, Ar Diagram 2 Diagram 2 Vol.7, Nr.3, 2014, Art. 5, 1-27 www.FORMakademisk.org www.FORMakademisk.org www.FORMakademisk.org www.FORMakademisk.org 7 Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift The interviews aimed to elicit interviewees’ perceptions of: www.FORMakademisk.org The interviews aimed to elicit interviewees’ perceptions of: Concepts: In order to gain understanding of the interviewees’ perceptions about the relevant concepts by which they might articulate their views (on products, services, service design) The shift: With the purpose of understanding interviewees’ view of the shift from product design to service design (drivers, development, influences) Design thinking/approach: To elicit interviewees’ perceptions about the potential changes in their design thinking and approach, particularly in relation to problem solving and systems thinking (in the context of the shift from designing products to designing services). Concepts: In order to gain understanding of the interviewees’ perceptions about the relevant concepts by which they might articulate their views (on products, services, service design) Concepts: In order to gain understanding of the interviewees’ perceptions about the relevant concepts by which they might articulate their views (on products, services, service design) p y y g ( p , , service design) The shift: With the purpose of understanding interviewees’ view of the shift from product design to service design (drivers, development, influences) Design thinking/approach: To elicit interviewees’ perceptions about the potential changes in their design thinking and approach, particularly in relation to problem solving and systems thinking (in the context of the shift from designing products to g The shift: With the purpose of understanding interviewees’ view of the shift from product design to service design (drivers, development, influences) To achieve this, the interview questions were divided into five main themes, normally with two questions each. Interviewees were asked to answer each question, and on occasions to draw or write some of their ideas (to facilitate eliciting their thoughts). The themes and questions were as follows: Theme 1 - Product vs. services What is the difference between a product and a service? How different is it to design a product than a service? Theme 2 - Design of Services When did you start to design services and why? (What was the first service design project you did?) Wh d i kill h d l d f d i i i ? What design skills have you developed for designing services? Theme 3 - Shift from products to services There has been a shift from product to services design. What has influenced this shift? How has professional practice changed with the design of services? The interviews aimed to elicit interviewees’ perceptions of: (Has the nature of your work changed?) me 4 - Problem solving and systems thinking Theme 4 - Problem solving and systems thinking Would you be able to write 5 words to explain your design thinking when designing products and when designing services? You have one minute for each. Would you be able to write 5 words to explain your design thinking when designing products and when designing services? You have one minute for each. Problem-solving thinking is a fundamental skill for the design of products. Do you agree? Elaborate. ems thinking is a fundamental skill for the design of services. Do you agree? Elaborate. Systems thinking is a fundamental skill for the design of services. Do you agree? Elaborate. How does problem solving weigh in comparison to systems thinking when designing products and when designing services? y g g y g How does problem solving weigh in comparison to systems thinking when designing products and when designing services? The interviews were conducted personally or via Skype. They were audio-recorded and summarised. The audio recordings were played and listened to by the researchers several times and notes of key concepts were taken on Post-its. These Post-its were subsequently organized on a grid, becoming a method of coding. Further discussions took place between the researchers in order to identify and differentiate the range of views in response to the interview questions. Diagrams and tables helped to synthesise emerging ideas and findings. The findings of this study have been synthesised following the themed structure of the interview. Each theme corresponds to individual sub-sections. The views of the interviewees have been reported, emphasising perceived commonalities and differences between the interviewees’ responses. At the end of each subsection a table or a diagram has been included, synthesising emergent ideas. Vol.7, Nr.3, 2014, Art. 5, 1-27 www.FORMakademisk.org 8 Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift The discussion section examined the findings and made comparisons with the literature review. By extrapolating theory from the literature and first-hand information from the interviews, an attempt has been made to offer a synthetic view of the extent to which a shift in design thinking has taken place in the transition from product to design services, moving from a problem solving to a systems thinking approach. The interviews aimed to elicit interviewees’ perceptions of: The validity of this research is limited, as its finding are based on the subjective perceptions of the researcher about the phenomena studied, as well as the views collected from the people interviewed. Equally, it has limitations in terms of reliability (generalisation) as the number of people interviewed does not constitute a representative sample of people involved in the issues of this study. The claims of this study are moderatum generalisations (Payne & Williams, 2005) and do not attempt to offer a comprehensive and definitive explanation of the phenomena studied. However, a start has been made to underpin further and more conclusive research on the subject of the shift from problem solving to systems thinking in services design. Findings The interviews were structured according to the main themes of the research, in order to gain understanding of (i) the interviewees’ perceptions of the differences between products and services; (ii) what the design of services entails; (iii) how the shift from product to service design developed; and (iv) their views about changes in their thinking with regard to problem solving and systems thinking. Theme 1 - Product and services This difference relates to the “manufactured” character of products and to the relationship of services with “organisational change” and “back-end systems”. For example one of the interviewees declares that while the process is the same, different “crafts” and “techniques” are employed for the design of services and products. This difference relates to the “manufactured” character of products and to the relationship of services with “organisational change” and “back-end systems”. Another interviewee emphasises the scale of products and services as a factor affecting their design process. She suggests that the design process for services is more complex as services are “bigger” and more complex than objects. One of the interviewees suggests that there is a substantial difference between the development of prototypes when designing products and services. In the first instance, the prototypes are a representation of the final product made through models, renderings and mock- ups amongst others. In the second instance, prototypes are the realisation of the entire service, only at a smaller scale in terms of duration and coverage (number of stake holders). Actually, it is a fully functional scaled down version of the service (a pilot), rather than a representation of it. Another interviewee seems to imply that there is only one design process, This process helps to understand the context in such a way that a decision can be made about whether a product or a service is the best medium to deliver a design solution. Another interviewee argues that the design process is affected by the nature of services and products in regard to their complexity and constrains. He explains how products have limited features and functions, and therefore the process to design those deals only with a defined set of constrains. In contrast, services are complex in nature and ask the design process for a broader set of deliverables. He says “the more you work on it, the more you uncover”. Yet there seems to be one single difference at a higher level in the process of designing products and services, in the form of an additional step towards the end of the process. For products, the design process finishes with the final specification for production. For services it goes beyond the specification of the service to include the implementation of it and, in some cases, the initial running of it. Theme 1 - Product and services What is the difference between a product and a service? Views on the differences between product and services amongst services designers are noticeable and not homogeneous. One of the interviewees believes that products and services relate functionally to each other, as products can deliver services and services are “ecosystems” that can contain products. Two other interviewees see the difference in terms of interaction. They explain that while a product involves a single interaction, a service consists of multiple and different interactions over time. One interviewee declares that the nature of products is static (as they remain unchanged once they have been designed) and the nature of a service is dynamic: they have an “on-going” and flexible character. He highlights the wider scope of services, which includes for example the role of people. Another interviewee makes reference to the physical character of products as opposed to the intangible character of services. This interviewee highlights the commonality of product and services as they are both means by which design solves problems. A further interviewee emphasises the academic character of any differentiation between products and services, arguing that this is not relevant for users. Six criteria emerged from the interviewees’ answers to establish differences between products and services: -Complexity and number of interactions -Static/dynamic character -Static/dynamic character Scope -Static/dynamic character -Scope -Scope -Tangibility -Relationship with problems -Relevance of differentiation Vol.7, Nr.3, 2014, Art. 5, 1-27 www.FORMakademisk.org 9 Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift According to these criteria, perceived differences between products and services are outlined in Table 2. According to these criteria, perceived differences between products and services are outlined in Table 2. Table 2 How different is it to design a product than a service? How different is it to design a product than a service? Services designers seem to agree that the process for the design of services and for the design of products is similar at a high level. However they find some differences at lower levels. Vol.7, Nr.3, 2014, Art. 5, 1-27 10 www.FORMakademisk.org www.FORMakademisk.org Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift For example one of the interviewees declares that while the process is the same, different “crafts” and “techniques” are employed for the design of services and products. Theme 1 - Product and services To summarise, the differences between designing a product and a service relate to five main areas: first, in relation to the craft and techniques employed; secondly in terms of their perceived complexity; thirdly, regarding the type of prototypes employed; fourthly, concerning the project constraints and breadth of expected deliverables; and finally, in connection with their process length, number of steps and end point. Table 3 illustrates the perceived differences between designing a product and designing a service. Vol.7, Nr.3, 2014, Art. 5, 1-27 11 www.FORMakademisk.org www.FORMakademisk.org Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Table 3 Table 3 Table 3 Theme 2 - Design of Services g f When did you start to design services and why? (What was the first service design project you did?) When did you start to design services and why? (What was the first service design project you did?) When did you start to design services and why? (What was the first service design project you ) It seems that the transition from product design to services design occurred gradually and started with the realisation of the importance of user experience and user-centred design. It seems that the transition from product design to services design occurred gradually and started with the realisation of the importance of user experience and user-centred design. One interviewee reported that his first contact with services design occurred during one of his projects when studying product design. He realised that a very important part of the product he was designing was to offer to its user a good experience. His first professional services design project was at Cisco. It was related to data storage, and he integrated work carried out by developers with the insights of users. Another interviewee was also exposed to services design for the first time during her product design course at the Glasgow School of Art. While developing a project involving a lamp shade, she went beyond it by setting up a website to allow customers to personalise their products. Later she got involved in services design through a social innovation lab organised by Nesta and from there she set up a design services consultancy. One of the interviewees started his first service-related design activities while working for the Red group at the design council, exploring new interactions for voting, citizenship ceremonies, etc. www.FORMakademisk.org Theme 1 - Product and services This made him realise the importance of experiences and behaviours in design. Vol.7, Nr.3, 2014, Art. 5, 1-27 12 Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift He then specialised in services design projects, working for the design consultancy Participle and later in social innovation design at Tacsi in Australia. Another interviewee with an educational background in product design and interaction design got involved in the design of services via his professional activity designing web pages. His first design services project was about data management. He then co-founded Livework, considered by many to be the first services design consultancy. It is noticeable that all of these designers have moved from product design to services design, but none of them have returned to product. Instead they have moved to other areas such as experience design or social innovation design. Four main reasons explain why the product designers who were interviewed moved into services design. First, the nature of their design approach “naturally” led them to the development of services rather than products as a design output; secondly, the government agenda seems to stimulate design work in the area of services; thirdly, the transferability of their product design skills to service design challenges enables them to make a swift transition; and finally, the development of new professional design areas stimulated the shift. Table 4 summarises these reasons. Table 4 Table 4 Table 4 13 Vol.7, Nr.3, 2014, Art. 5, 1-27 www.FORMakademisk.org www.FORMakademisk.org Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift What design skills have you developed for designing services? As service designers, the interviewees felt they had developed a wide set of skills. These skills have been grouped into 10 different categories: Attitude, Interpersonal, Research, Communication, Participatory Design, Aesthetics, Business, Thinking (about the situation), Knowledge and Interdisciplinary. It is noticeable that systems thinking was named by only one of the designers, while skills related to business were mentioned by three of them. The categories of Research, Communication and Thinking (about the situation) are the ones that have most items, suggesting some emerging skills in the shift from product to services design. Table 5 shows the skills developed according to the interviewees’ account. 14 Vol.7, Nr.3, 2014, Art. Theme 3 - Shift from products to services Theme 3 - Shift from products to services f f p There has been a shift from product to services design. What has influenced this shift? f f p g f f It seems that a diverse collection of factors has influenced the shift from product to services design. First, general awareness and concern amongst designers (and the general public) about environmental issues has increased: for example, one of the interviewees says that environmental concerns are moving designers to think that creating fewer objects is a positive action. He also says that as objects become multifunctional they become “dumb”, as they are difficult to use and most of their functions become redundant and easily replaceable in a service environment. As function is delivered mostly through an immaterial entity (the service), services seems a more sustainable (and designerly) option to physical products. He also mentions that this is sometimes not supported outside design, and that for example some politicians in the UK still support the development of products over services, in the hope of generating more employment in the manufacturing sector. Secondly, the worldwide expansion of the internet and the development of interactive technology has also played a fundamental influence on the shift. One of the interviewees explains it by saying that being digital enable multimodal experiences, and that this enables designers to develop services that improve user journeys. Networking technologies and the internet are also identified by other interviewees as accelerators and amplifiers of the services phenomenon. A third aspect relates to an on-going trend in social and government environments towards user and citizen centeredness. As highlighted by another interviewee, this implies the need for service design thinking rather than product design thinking, as “face to face” design interventions typical of service design practice are required, such as co-design, user conversations, etc. He also highlights that services, as opposed to products, are a better platform for inducing behavioural change, for finding new innovation methodologies and new business models in public services, and to tackle social challenges such as youth disengagement or child protection issues. While he recognises the potential of services design to bring tools to addressing these challenges, he also insists on the need for a rigorous approach that involves other disciplines such as business or social sciences in order to achieve real innovation. Theme 1 - Product and services 5, 1-27 14 www.FORMakademisk.org www.FORMakademisk.org Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Table 5 15 Vol.7, Nr.3, 2014, Art. 5, 1-27 www.FORMakademisk.org www.FORMakademisk.org Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Theme 3 - Shift from products to services Otherwise, he says, “...if we keep doing the same thing, we will keep getting what we always got.” One last aspect that influences the shift relates to the start-up phenomenon. One of the interviewees argues that this embodies the transition from product to services, as it enables the design and development of new services business models. The interviewee comments that this might be a negative development for established services design consultancies, as the start-up model removes the need for a service designer. This is becoming ever more critical, as current services design consultancies have relied too much on public funding for their work (as they mostly works for public organisations), and have developed less expertise in working with business and the private sector (at least in the UK). Diagram 3 shows a summary of factors influencing the shift from product to services design according to the interviewees. Vol.7, Nr.3, 2014, Art. 5, 1-27 16 www.FORMakademisk.org www.FORMakademisk.org Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Diagram 3 Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Diagram 3 How has professional practice changed with the design of services? (Has the nature of your work changed?) How has professional practice changed with the design of services? (Has the nature of your work changed?) As one of the interviewees explains, the manufacturing industry has been drying out in the last 30 years and this has made product designers refocus their professional practice towards the design of services. Initially, they concentrated on working for the public sector and the government, as their agenda focused on behavioural change and having people’s needs driving their activity and policies. With the financial crisis, public funding for pursuing this agenda became less abundant and a new shift towards the financial, insurance and banking services occurred. Consultancies such as IDEO exemplify this shift. In this context, there have been noticeable changes in the practice of design. For example, services designers need to invest time and energy in justifying their research methods (as that often requires investing more time and money than clients would expect). As one of the interviewees explains, this might be necessary because some of the research methods used by designers are perceived by their clients as something they have already done. www.FORMakademisk.org www.FORMakademisk.org Theme 3 - Shift from products to services One aspect raised by several interviewees highlights the increased focus of design activity on understanding people, their needs and behaviours, as the expected design output is often a user behavioural change. However, as another interviewee explains, designers need also to focus their activity beyond customer experience improvement towards the design of the business model. Another important change relates to the disciplinary identity of the designers. Services design projects often involve teamwork with people from a wide range of disciplines, and with different project stakeholders. The activity of these teams, as highlighted by one of the interviewees, is centred on the purpose rather than the participants’ skills. Therefore, designers ought to “care” less about their discipline, and be more ready to embrace other people’s approaches. One of the interviewees illustrates this by explaining how, in the interdisciplinary project sessions he holds in his services practice, he asks colleagues to “leave their profession” Vol.7, Nr.3, 2014, Art. 5, 1-27 www.FORMakademisk.org www.FORMakademisk.org 17 Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift outside the meeting room, and come in not as designer or architect, or whatever profession they have, but as a person. One last change is expressed by one of the interviewees as the need for designers to be more responsible. This refers to the idea that the design of a service does not end with the approval of a final concept (as may well happen in a product design project). Instead, the service design project includes the realisation of prototypes and the implementation of pilots for testing purposes, and can also include the operation and running of the service for an initial period. As this happens, the designers’ area of responsibility increases, as well as the provision of their design toolkit. Diagram 4 illustrates the main changes in professional design practice while doing service design identified by the interviewees. Diagram 4 Theme 4 - Problem solving and systems thinking Theme 4 - Problem solving and systems thinking Theme 4 - Problem solving and systems thinking g y g The questions relating to problem solving and systems thinking did not offer the interviewees a predefinition of these concepts or ask them directly to give their own definition. However, as inferred from their answers, it became evident that although the interviewees did not have an homogeneous understanding of problem-solving thinking and systems thinking they did share several fundamental ideas in their definitions, especially in regard to systems thinking. The first interviewee, for example, describes problem solving as a designer’s mental process for seeking, finding and creating new visions and new technological opportunities in order to solve issues. He believes that systems thinking as well as problem solving is part of design thinking and that both are fundamental to being a designer. He refers to systems thinking as architectural orchestration. He argues that since product designers need to understand various Vol.7, Nr.3, 2014, Art. 5, 1-27 18 Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift complex processes such as manufacturing processes, as well as investment and how organisations do things, they will use problem solving more than systems thinking, while in designing services he believes that the “eco-system” is much more complicated or perhaps much less mature and fuzzier, needing more of a systems thinking approach. complex processes such as manufacturing processes, as well as investment and how organisations do things, they will use problem solving more than systems thinking, while in designing services he believes that the “eco-system” is much more complicated or perhaps much less mature and fuzzier, needing more of a systems thinking approach. g y g pp The second interviewee thinks that to create new products a designer has to constantly use problem solving thinking to identify problems. She sees systems thinking as a quality that a service designer should have in order to design services and as an ability to deal with complexity. She thinks that in systems thinking the designer’s brain will zoom out from very specific things or from single things to tackle more complex situations or systems comprising various elements. Another interviewee believes that systems thinking refers to the ability to deal with complex situations, where multiple elements need to “work together”. Theme 4 - Problem solving and systems thinking It also relates to the ability to systematise and to grow or scale things and keep them working. It is also about the ability to think of multiple interrelated concepts and being able to see a situation from the outside. The fourth interviewee thinks that systems thinking is a tool to understand the impact that individual products or services have in society in the context of a complex world. Within systems thinking, problem solving can result from problem analysis. Problem analysis is the identification of the current state of affairs at the onset of the design process. The last interviewee thinks that systems thinking is a way of understanding situations structured as networks of interconnected nodes. He also regards problem solving as design, implying that problem-solving thinking is equivalent to design thinking. It is clear that all these competing views have in common the idea of systems thinking as a necessary skill to deal with complexity and fussiness, and as a design tool to enable designers to step back from specific problems and rethink them as part of wider complex systems. Even if they not agree, they do not contradict the view this study takes (as explained in the literature review) that systems thinking is a way of thinking about and understanding phenomena, and a holistic world view which helps to understand relational aspects of otherwise fuzzy and complex issues, thus becoming an instrument of analysis and synthesis. Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift www.FORMakademisk.org Would you be able to write 5 words to explain your design thinking when designing products and when designing services? Would you be able to write 5 words to explain your design thinking when designing products and when designing services? The interviewees were able to identify a wide range of words to describe their thinking while designing products and services. Even though most of them manifested at the beginning of the interview (in theme 1) that the design of services was not that different from designing products (at least at a higher levels), the results of this question seems to indicate otherwise. Out of 12 words elicited for product and 10 for service, there was only one in common: user. This leads to thinking that the user-centred approach, common amongst product designers, is transferred to the design of services. It is also noticeable that words directly associated with systems thinking, such as network, organisation, system, task, outputs and process, were only include as descriptors for the design of services. Table 6 shows the design thinking concepts elicited by the interviewees. The words in the left-hand column represent categories identified by the researchers of this study. Vol.7, Nr.3, 2014, Art. 5, 1-27 19 www.FORMakademisk.org www.FORMakademisk.org Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shi Table 6 Problem-solving thinking is a fundamental skill for the design of products. Do you agree? El b t Table 6 Problem-solving thinking is a fundamental skill for the design of products. Do you agree? Elaborate www.FORMakademisk.org Table 6 Problem-solving thinking is a fundamental skill for the design of products. Do you agree? Elaborate. The interviewees generally agreed that problem-solving thinking is a fundamental skill for the design of products. However, some of them suggested that there are equivalent but more “positive” ways of framing this skill, implying the negative character of the word problem. One interviewee equates “problem” with “opportunity”, emphasising the optimistic character of the later. Similarly, another interviewee proposes to replace problem-solving with opportunity- grasping. g p g Some additional observations were made on the usefulness of problem solving. For example, one of the interviewees stated that the problem-solving approach is useful for integrating tangible and intangible elements in design. Another highlighted its usefulness if geared towards the building of visions. Other interviewee made a distinction between design tasks which focus on altering or improving an existing product and those concerned with the creation of a “new product or innovation”. He pointed out that problem solving is more useful for the latter than for the former. Diagram 5 shows the reasons underpinning the idea that the problem-solving approach is a fundamental skill for the design of products according to the interviewees. Vol.7, Nr.3, 2014, Art. 5, 1-27 www.FORMakademisk.org 20 z & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Diagram 5 Systems thinking is a fundamental skill for the design of services. Do you agree? Elaborate. The interviewees agreed on the importance of systems thinking for designing services. A generalised conception amongst the interviewees is the usefulness of systems thinking to deal with complex issues in design services. For example, one of them explains that in dealing with complexity, systems thinking can be useful to zoom in on specific processes such as customer journeys, to understand dynamic processes such as service delivery, and to zoom out into wider contextual structures such as government. Another interviewee values systems thinking as a tool for grasping complexity, for making multiple things work together and to enable the generation of multiple interrelated concepts. He also believes that systems thinking enables the systematisation of pilot services with the purpose of making them grow and work when scaled up. Another interviewee comments on the perceived quality of systems thinking to develop services for social benefit. He claims that in order to do good for society, “systems thinking is your basic Vol.7, Nr.3, 2014, Art. 5, 1-27 21 www.FORMakademisk.org Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift toolkit”. In contrast, a further interviewee affirms that systems thinking helps to understand that the solution is not in an individual artefact, but is the combination of a network of actors coming together. Diagram 6 illustrates concepts associated with the relevance of systems thinking in the design of services according to the interviewees. toolkit”. In contrast, a further interviewee affirms that systems thinking helps to understand that the solution is not in an individual artefact, but is the combination of a network of actors coming together. Diagram 6 illustrates concepts associated with the relevance of systems thinking in the design of services according to the interviewees. Diagram 6 Theme 3 - Shift from products to services Amongst the main factors influencing the shift from product to services design are the increasing development of technology, the emergence of new business models, government agendas for the development of services, and changes in people’s mainstream thinking and values. As designers move from product design to services design, there are perceived changes in the scope of their profession, especially regarding the increasing involvement of users in the design process and the focus on user behavioural change and innovation in business models and public services. This possibly suggests that important changes in designers’ thinking are necessary, related to their ability to understand complex areas such as business and public services. However, as important as the adoption of systems thinking might appear for the satisfaction of this need, other types of thinking such as business thinking and customer service thinking might be as important as systems thinking. Diagram 6 Diagram 6 How does problem solving weigh in comparison to systems thinking, when designing products and when designing services? How does problem solving weigh in comparison to systems thinking, when designing products and when designing services? The interviewees seem to agree on the value of both problem-solving and systems thinking approaches for both product and services design. However, some of them believe that one particular approach is better suited to either product or services design. For example, one of the interviewees explains how problem solving is more appropriate to product design, while systems thinking is better for the design of services due to their fuzzy nature and greater size and complexity. He argues that systems thinking helps the designer of services to “orchestrate” multiple variables and stakeholders within a service. Other interviewees believe that systems thinking and problem solving can be integrated to address complex or “wicked” problems in product and service design. Additionally, one of them explains that both approaches are necessary and not mutually exclusive. Furthermore, systems thinking serves to sketch the landscape in which problems are identified and solved: systems thinking overarches problem solving. Vol.7, Nr.3, 2014, Art. 5, 1-27 www.FORMakademisk.org www.FORMakademisk.org 22 Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Another interviewee describes how in services design is possible to use systems thinking without needing to employ problem solving; for example, to understand a network of mutually interacting elements. Yet, he explains, as solving a problem in a network brings repercussions to the whole network, a systems thinking approach allows designers to understand these repercussions. He summarises this concept by explaining that service design is solving a problem (using problem-solving thinking) in the context of systems (understood by using systems thinking). Table 7 summarises when a problem-solving and/or a systems thinking approach is appropriate in product and service design according to the interviewees. Six different factors determine the level of appropriateness of each approach, and help to define the weight of each approach in product design and service design. It is noticeable that in regard to two of these factors (“integration” and “function of approach”) both problem solving and systems thinking have similar weight. Table 7 Table 7 23 Vol.7, Nr.3, 2014, Art. 5, 1-27 www.FORMakademisk.org Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Theme 2 - Design of Services It is apparent that product designers have been able to move into the design of services thanks mainly to the transferability of their skills, and that this process has been also stimulated by government and social drive. Systems thinking is recognised as an important skill, but others such as research skills, collaborative skills, etc. are considered important too. It might be the case that the thinking shift in service design is not necessarily towards the paradigm of systems thinking, but to another one and yet to be identified. Discussion Based on the findings from the interviews, it is apparent that the shift from problem-solving thinking to systems thinking as a consequence of a shift from product to services design is not as simple as the literature review suggests, and systems thinking appears to be only one of the aspects of this change in design thinking. To develop this idea, each of the finding themes of the previous “finding” section will be discussed. Theme 1 - Product vs. services It seems that differences between products and services are related to their dynamicity, scope width and tangibility. However, product and services seem to be integrateable, as products can be accompanied by or be part of services, and contingent, as neither one is mutually necessary for the other’s existence. Designing services is regarded as a more complex activity than designing products. The design process nature changes for services as its end becomes fuzzy and entangled with the implementation and running of the service. If there is an identifiable factor related to the difference between products and services and their design processes which indicates a shift in design thinking, it must be the dynamic and intangible nature of services, as well as its higher complexity. These differences might be an indication that a new type of thinking is required by product designers for the design of services. Theme 4 - Problem solving and systems thinking Theme 4 Problem solving and systems thinking It is apparent that design thinking changes noticeably from product design to service design, but regardless of this the focus on the user remains a key consideration in both cases. It seems that systems thinking is not regarded as a very important feature in product design, but i i d i it i i ll b f it f l t d l ith l it t d It is apparent that design thinking changes noticeably from product design to service design, but regardless of this the focus on the user remains a key consideration in both cases. It seems that systems thinking is not regarded as a very important feature in product design, but in service design it is, especially because of its usefulness to deal with complexity, as suggested in the literature. In contrast, problem solving is perceived as equally useful in the design of both Vol.7, Nr.3, 2014, Art. 5, 1-27 www.FORMakademisk.org 24 Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift products and services (although it has been suggested that problem solving is sometimes counterproductive in service design). This may imply that, rather than causing a shift from problem-solving thinking to systems thinking, the transition from product to service design has produced an “addition”, bringing systems thinking to the design of services. Actually, in service design, problem solving and systems thinking are not opposite, competing or mutually exclusive ways of thinking, but complementary. This research suggests that the need for problem-solving and systems thinking approaches in design activity is not determined by the desired design output (products and/or services). Instead, it seems to be individually dictated by the particular and individual design process adopted by an individual designer in each project undertaken. Their likelihood of being employed mostly depends on how general (holistic) or specific is the view that the designer takes at the moment of application, as well as whether the reason for adopting either approach is geared more towards understanding or resolution. Conclusions This paper has presented the results of a study examining how design activity has changed for product designers who have become service designers. In particular, it has explored the adoption of systems thinking as an approach to the design of services, in relation to the problem- solving approach commonly utilised by product designers. First, it has examined relevant literature, explaining how the problem-solving approach has become insufficient to deal with the fuzzy nature of the issues designers face nowadays. It has also explored how product design has shifted to service design, explaining why the systems thinking approach can be useful to deal with the complex nature of services and how it has been adopted by service designers. Secondly, it has presented the results of a series of semi-structured interviews with designers working in five design consultancies that have moved from product design towards services design. It has reported the interviewees’ views about differences and commonalities between product design and service design, about their experiences while shifting from product to service design, and about their design approach in relation to problem solving and systems thinking approaches. The literature review and the interviews results have been compared, showing that a change in the way designers think and approach projects has taken place, demonstrating that the design of products requires a different approach to the design of services. The weight of the evidence seems to indicate that a movement from problem solving to systems thinking takes place when designers are faced with the challenges of designing a service. However it seems that the systems thinking approach does not necessarily replace the problem-solving approach but complements it. The results also indicate that the growing complexity of the issues designers have to deal with influences the adoption of systems thinking in response to service design challenges. It also shows that current changes in ideas about sustainability, society, etc. have also an impact on this. This paper does not claim to be definitive and can be taken only as initial exploration of the subject. Further study will be needed to establish exactly how the systems thinking approach is utilised in the design of services, and to examine how it compares with other alternative design approaches employed in services design. Vol.7, Nr.3, 2014, Art. Conclusions 5, 1-27 25 www.FORMakademisk.org www.FORMakademisk.org Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Liliana Rodriguez PhD student Loughborough Design School, Loughborough University Email address: lulugaia01@yahoo.co.uk Liliana Rodriguez PhD student Loughborough Design School, Loughborough University Email address: lulugaia01@yahoo.co.uk Carlos Peralta Senior Lecturer in Design. School of Art, Design and Media, University of Brighton Email address: purplecamaleon@yahoo.co.uk Vol.7, Nr.3, 2014, Art. 5, 1-27 26 www.FORMakademisk.org Liliana Rodriguez & Carlos Peralta From Product to Service Design: A Thinking Paradigm Shift Bibliography Buchanan, R. (2001). Design Research and the New Learning. Design Issues, 17(4), pp. 3-23. Burns, C., Cottam, H., Vanstone, C. & Winhall, J. (2006). Transformation Design. Red Paper 02, London: Design Council. Clatworthy, S. (2011). Interaction Design: Services as a Series of Interactions. 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https://openalex.org/W4287111926	https://iris.unica.it/bitstream/11584/320237/1/journal.pcbi.1009045.pdf	English	null	Thalamo-cortical spiking model of incremental learning combining perception, context and NREM-sleep	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	18,353	OPEN ACCESS Citation: Golosio B, De Luca C, Capone C, Pastorelli E, Stegel G, Tiddia G, et al. (2021) Thalamo-cortical spiking model of incremental learning combining perception, context and NREM- sleep. PLoS Comput Biol 17(6): e1009045. https:// doi.org/10.1371/journal.pcbi.1009045 Citation: Golosio B, De Luca C, Capone C, Pastorelli E, Stegel G, Tiddia G, et al. (2021) Thalamo-cortical spiking model of incremental learning combining perception, context and NREM- sleep. PLoS Comput Biol 17(6): e1009045. https:// doi.org/10.1371/journal.pcbi.1009045 Editor: Maxim Bazhenov, University of California San Diego, UNITED STATES Received: April 27, 2020 Accepted: May 5, 2021 Published: June 28, 2021 Editor: Maxim Bazhenov, University of California San Diego, UNITED STATES Editor: Maxim Bazhenov, University of California San Diego, UNITED STATES Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pcbi.1009045 Thalamo-cortical spiking model of incremental learning combining perception, context and NREM-sleep Bruno GolosioID1,2☯, Chiara De LucaID3,4☯, Cristiano CaponeID4, Elena PastorelliID3,4, Giovanni StegelID5, Gianmarco TiddiaID1,2, Giulia De BonisID4, Pier Stanislao PaolucciID4 Bruno GolosioID1,2☯, Chiara De LucaID3,4☯, Cristiano CaponeID4, Elena PastorelliID3,4, Giovanni StegelID5, Gianmarco TiddiaID1,2, Giulia De BonisID4, Pier Stanislao PaolucciID4 1 Dipartimento di Fisica, Università di Cagliari, Cagliari, Italy, 2 Istituto Nazionale di Fisica Nucleare (INFN), Sezione di Cagliari, Cagliari, Italy, 3 Ph.D. Program in Behavioural Neuroscience, “Sapienza” Università di Roma, Rome, Italy, 4 Istituto Nazionale di Fisica Nucleare (INFN), Sezione di Roma, Rome, Italy, 5 Dipartimento di Chimica e Farmacia, Università di Sassari, Sassari, Italy 1 Dipartimento di Fisica, Università di Cagliari, Cagliari, Italy, 2 Istituto Nazionale di Fisica Nucleare (INFN), Sezione di Cagliari, Cagliari, Italy, 3 Ph.D. Program in Behavioural Neuroscience, “Sapienza” Università di Roma, Rome, Italy, 4 Istituto Nazionale di Fisica Nucleare (INFN), Sezione di Roma, Rome, Italy, 5 Dipartimento di Chimica e Farmacia, Università di Sassari, Sassari, Italy a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. * Chiara.DeLuca@roma1.infn.it Abstract The brain exhibits capabilities of fast incremental learning from few noisy examples, as well as the ability to associate similar memories in autonomously-created categories and to com- bine contextual hints with sensory perceptions. Together with sleep, these mechanisms are thought to be key components of many high-level cognitive functions. Yet, little is known about the underlying processes and the specific roles of different brain states. In this work, we exploited the combination of context and perception in a thalamo-cortical model based on a soft winner-take-all circuit of excitatory and inhibitory spiking neurons. After calibrating this model to express awake and deep-sleep states with features comparable with biological measures, we demonstrate the model capability of fast incremental learning from few exam- ples, its resilience when proposed with noisy perceptions and contextual signals, and an improvement in visual classification after sleep due to induced synaptic homeostasis and association of similar memories. Citation: Golosio B, De Luca C, Capone C, Pastorelli E, Stegel G, Tiddia G, et al. (2021) Thalamo-cortical spiking model of incremental learning combining perception, context and NREM- sleep. PLoS Comput Biol 17(6): e1009045. https:// doi.org/10.1371/journal.pcbi.1009045 1 Introduction Increasing experimental evidence is mounting for both the role played by the combination of bottom-up (perceptual) and top-down/lateral (contextual) signals [1] and for the beneficial effects of sleep as key components of many high-level cognitive functions in the brain. In the following, we give an overview of some aspects, driven from experimental observations, that we have taken as fundamental building blocks for the construction of the model we present. g p It is known that the cortex follows a hierarchical structure [2]; starting from this, Larkum et al. [1] propose an associative mechanism built-in at a cellular level into the pyramidal neu- ron (see Fig 1B), exploiting the cortical architectural organization (see Fig 1C). Long-range connectivity in the cortex follows the basic rule that sensory input (i.e., the feed-forward stream) terminates in the middle cortical layers, whereas information from other parts of the cortex (i.e., the feedback stream) mainly projects to the outer layers. This also applies to projec- tions from the thalamus, a structure that serves as both a gateway for feed-forward sensory information to the cortex and a hub for feedback interactions between cortical regions. Indeed, only 10% of the synaptic feedback inputs to the apical tuft come from nearby neurons, and the missing 90% arise from long-range feedback connections. This feedback information stream is vitally important for cognition and conscious perception: this picture leads to the suggestion that the cortex operates via an interaction between feed-forward and feedback information. Larkum et al. [1] highlight that, counter-intuitively, distal feedback input to the tuft dendrite could dominate the input/output function of the cell: short high-frequency bursts would be produced on a combination of distal and basal input. As a consequence, although small (under-threshold) signals contribute only to their respective spike initiation zones, the fact that input has reached the threshold in one zone is quickly signalled to other zones. This pro- vides the possibility for a contextual prediction: the activity in the apical tuft of the cell can lower the activity threshold driven by the basal region, the target of the specific nuclei in the thalamus that projects there the perceptual and feed-forward streams. In summary, this mech- anism is ideally suited to associating feed-forward and feedback cortical pathways. Thus, they propose a conceptual interpretation of these biological pieces of evidence: the feedback signal aims at predicting whether a particular pyramidal neuron could or should be firing. PLOS COMPUTATIONAL BIOLOGY PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles Funding: This work has been supported by the European Union Horizon 2020 Research and Innovation program under the FET Flagship Human Brain Project (grant agreement SGA3 n. 945539 and grant agreement SGA2 n. 785907; recipient Pier Stanislao Paolucci) and by the INFN APE Parallel/Distributed Computing laboratory. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. fights noise in perceptual and internal knowledge and it supports the categorical associa- tion of examples belonging to the same digit class, through reinforcement of class-specific cortico-cortical synapses. The distributions of pre-sleep and post-sleep firing rates during classification change in a manner similar to those of experimental observation. These changes promote energetic efficiency during recall of memories, better representation of individual memories and categories and higher classification performances. Competing interests: The authors have declared that no competing interests exist. Author summary We created a thalamo-cortical spiking model (ThaCo) with the purpose of demonstrating a link among two phenomena that we believe to be essential for the brain capability of effi- cient incremental learning from few examples in noisy environments. Grounded in two experimental observations—the first about the effects of deep-sleep on pre- and post-sleep firing rate distributions, the second about the combination of perceptual and contextual information in pyramidal neurons—our model joins these two ingredients. ThaCo alter- nates phases of incremental learning, classification and deep-sleep. Memories of hand- written digit examples are learned through thalamo-cortical and cortico-cortical plastic synapses. In absence of noise, the combination of contextual information with perception enables fast incremental learning. Deep-sleep becomes crucial when noisy inputs are con- sidered. We observed in ThaCo both homeostatic and associative processes: deep-sleep Copyright: © 2021 Golosio et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The model and data used to draw the results outlined in this manuscript are available at https://github.com/APE-group/ ThaCo2.git and 10.5281/zenodo.4769175. 1 / 26 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 1 Introduction Moreover, any neuron can fire only if it receives enough feed-forward input. Resulting from this interpre- tation, the internal representation of the world by the brain can be matched at every level with ongoing external evidence via a cellular mechanism, allowing the cortex to perform the same operation with massively parallel processing power. Soft Winner-Take-All (WTA) plays an important role in many high-level cognitive func- tions such as decision making [3–5] classification and pattern recognition [6, 7]. Under a rough simplification, this mechanism can be realized through the competition among groups of excitatory neurons connected towards the same population of inhibitory neurons, which in turn is connected towards the excitatory groups it arbitrates [8–11]. Within appropriate condi- tions, the inhibitory signal will be sufficiently high to suppress the signal of all the low-firing PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 2 / 26 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles Fig 1. Thalamo-cortical spiking model (ThaCo). Panels B and C to compare the architecture of our model with the biological principles described in [1]. A) Scheme of the Thalamo-cortical spiking model (ThaCo). Input images, passed through a filter (HOG) are projected (blue arrow) to thalamic excitatory neurons (tc), mimicking the mechanism of the retinal visual stimulus. Thalamic neurons stimulate cortical excitatory neurons (cx) with a perceptual feedforward excitation (blue). Cortico- cortical and cortico-thalamic are considered as top-down prediction connections (red). (Red arrows—context/prediction) Currents coding for higher abstraction features incoming from other cortical areas. Cortical inhibitory neurons (in) arbitrate competition among cortical groups in a soft Winner-Take-All mechanism (WTA). Inhibitory reticular neurons (re) control the thalamic firing rate. The cortical layer is in turn connected to readout neurons (ro) B) A cellular mechanism for associating feed-forward and feedback signals. Low-level features are encoded in primary sensory regions and this signal propagates up the visual hierarchy (e.g. striate cortex (V1) sensitive to orientation, V4 sensitive to colour, V5 sensitive to motion, and inferior temporal (IT) cortex sensitive to shapes and objects). Higher-level areas provide feedback information (context or expectation) to lower areas. The ThaCo model presented in this paper is a single area model and the contextual signal is assumed to collect during training the knowledge carried by all other areas in the hierarchy (see red arrows in panel A). 1 Introduction C) Conceptual representation of the back- propagation activated calcium (BAC) firing hypothesis supporting efficient binding of features and recognition. Pyramidal neurons receiving predominantly feed- forward information are likely to fire steadily at low rates, whereas the simultaneous presence of contextual and perceptual streams changes the mode of firing to bursts (BAC firing). This coincidence mechanism is mimicked in our ThaCo model. D) During training (left), the injection of contextual signal, plays the role of internal prediction and increases the perceptual threshold of a subset of cortical neurons. The simultaneous presence of perceptual and contextual promotes a high firing rate in h i i ki h BAC h i Al h i l f h i l f h i l l d f h l i l hi h Fig 1 Thalamo-cortical spiking model (ThaCo) Panels B and C to compare the architecture of our model with the biological principles described in [1] A) Scheme Fig 1. Thalamo-cortical spiking model (ThaCo). Panels B and C to compare the architecture of our model with the biological principles described in [1]. A) Scheme of the Thalamo-cortical spiking model (ThaCo). Input images, passed through a filter (HOG) are projected (blue arrow) to thalamic excitatory neurons (tc), mimicking the mechanism of the retinal visual stimulus. Thalamic neurons stimulate cortical excitatory neurons (cx) with a perceptual feedforward excitation (blue). Cortico- cortical and cortico-thalamic are considered as top-down prediction connections (red). (Red arrows—context/prediction) Currents coding for higher abstraction features incoming from other cortical areas. Cortical inhibitory neurons (in) arbitrate competition among cortical groups in a soft Winner-Take-All mechanism (WTA). Inhibitory reticular neurons (re) control the thalamic firing rate. The cortical layer is in turn connected to readout neurons (ro) B) A cellular mechanism for associating feed-forward and feedback signals. Low-level features are encoded in primary sensory regions and this signal propagates up the visual hierarchy (e.g. striate cortex (V1) sensitive to orientation, V4 sensitive to colour, V5 sensitive to motion, and inferior temporal (IT) cortex sensitive to shapes and objects). Higher-level areas provide feedback information (context or expectation) to lower areas. The ThaCo model presented in this paper is a single area model and the contextual signal is assumed to collect during training the knowledge carried by all other areas in the hierarchy (see red arrows in panel A). 1 Introduction C) Conceptual representation of the back- propagation activated calcium (BAC) firing hypothesis supporting efficient binding of features and recognition. Pyramidal neurons receiving predominantly feed- forward information are likely to fire steadily at low rates, whereas the simultaneous presence of contextual and perceptual streams changes the mode of firing to bursts (BAC firing). This coincidence mechanism is mimicked in our ThaCo model. D) During training (left), the injection of contextual signal, plays the role of internal prediction and increases the perceptual threshold of a subset of cortical neurons. The simultaneous presence of perceptual and contextual promotes a high firing rate in such neurons, mimicking the BAC mechanism. Also, the simulataneous presence of the signal from the cortical layer and of the contextual signal promotes a high Fig 1. Thalamo-cortical spiking model (ThaCo). Panels B and C to compare the architecture of our model with the biological principles described in [1]. A) Scheme of the Thalamo-cortical spiking model (ThaCo). Input images, passed through a filter (HOG) are projected (blue arrow) to thalamic excitatory neurons (tc), mimicking the mechanism of the retinal visual stimulus. Thalamic neurons stimulate cortical excitatory neurons (cx) with a perceptual feedforward excitation (blue). Cortico- cortical and cortico-thalamic are considered as top-down prediction connections (red). (Red arrows—context/prediction) Currents coding for higher abstraction features incoming from other cortical areas. Cortical inhibitory neurons (in) arbitrate competition among cortical groups in a soft Winner-Take-All mechanism (WTA). Inhibitory reticular neurons (re) control the thalamic firing rate. The cortical layer is in turn connected to readout neurons (ro) B) A cellular mechanism for associating feed-forward and feedback signals. Low-level features are encoded in primary sensory regions and this signal propagates up the visual hierarchy (e.g. striate cortex (V1) sensitive to orientation, V4 sensitive to colour, V5 sensitive to motion, and inferior temporal (IT) cortex sensitive to shapes and objects). Higher-level areas provide feedback information (context or expectation) to lower areas. The ThaCo model presented in this paper is a single area model and the contextual signal is assumed to collect during training the knowledge carried by all other areas in the hierarchy (see red arrows in panel A). C) Conceptual representation of the back- propagation activated calcium (BAC) firing hypothesis supporting efficient binding of features and recognition. 1 Introduction Pyramidal neurons receiving predominantly feed- forward information are likely to fire steadily at low rates, whereas the simultaneous presence of contextual and perceptual streams changes the mode of firing to bursts (BAC firing). This coincidence mechanism is mimicked in our ThaCo model. D) During training (left), the injection of contextual signal, plays the role of internal prediction and increases the perceptual threshold of a subset of cortical neurons. The simultaneous presence of perceptual and contextual promotes a high firing rate in such neurons, mimicking the BAC mechanism. Also, the simulataneous presence of the signal from the cortical layer and of the contextual signal promotes a high PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 3 / 26 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles firing rate in readout neurons. In the classification phase (centre) the contextual signal is turned off. In the sleeping phase (right), the sensory pathways are turned off, and all the activity is generated spontaneously. https://doi.org/10.1371/journal.pcbi.1009045.g001 firing rate in readout neurons. In the classification phase (centre) the contextual signal is turned off. In the sleeping phase (right), the sensory pathways are turned off, and all the activity is generated spontaneously. https://doi.org/10.1371/journal.pcbi.1009045.g001 https://doi.org/10.1371/journal.pcbi.1009045.g001 https://doi.org/10.1371/journal.pcbi.1009045.g001 https://doi.org/10.1371/journal.pcbi.1009045.g001 excitatory groups of neurons whereas the high-firing ones survive. Such conditions can be achieved thanks to synaptic plasticity, which strengthens the connections among neurons of the same group and weakens those among competing groups, coupled with homeostatic mech- anisms [12, 13]. Spike-timing-dependent plasticity (STDP) has been proposed as one of the essential learn- ing ingredients in the cortex [14–18]. According to this plasticity rule, if the postsynaptic neu- ron fires an action potential just after a presynaptic spike, the synaptic weight will increase, whereas in the opposite case it will decrease. Through this mechanism, the synapses connect- ing neurons correlated by a principle of causality are metabolically rewarded. Chen et al. [19] have shown that networks of excitatory and inhibitory spiking neurons with either STDP or short-term plasticity can generate dynamically-stable WTA behaviour under certain condi- tions on initial synaptic weights. Another key aspect that we consider in this study is the role of sleep during learning. Sleep is essential in all animal species, and it is believed to play a crucial role in memory consolida- tion [20, 21], in the creation of novel associations, as well as in the preparation of tasks expected during the next awake periods. 1 Introduction Indeed, young humans pass the majority of time sleeping, and the youngest are the subjects that have to learn at faster rates. In adults, sleep deprivation is detrimental for cognition [22] and it is one of the worst tortures that can be inflicted. Among the multiple effects of sleep on the brain and body, we focus here on the con- solidation of learned information [23]. Homeostatic processes could normalize the representa- tion of memories and optimize the energetic working point of the system by recalibrating synaptic weights [24] and firing rates [25]. Specifically, Watson et al. [25] show that fast-firing pyramidal neurons decrease their firing rates over sleep, whereas slow-firing neurons increase their rates, resulting in a narrower population firing rate distribution after sleep. Also, sleep should be able to select memories for association, promoting higher performance during the next awake phases [26]. Indeed, Capone et al. [27] demonstrate the beneficial effects of sleep- wake phases involving homeostatic and associative processes in a visual classification task. Indeed, in [27], some of us illustrated how to assemble a simplified thalamo-cortical spiking model that can both express deep-sleep-like oscillations (in the form of an emergent, self- induced network phenomenon) and enter an awake-like asynchronous regime. This dynam- ical behaviour has been obtained by changing a few parameters in the equation that describes the dynamics of excitatory neurons in the spiking model, and thus exploiting a well established modelling principle that represents a few prominent features of brain-state acetylcholine- mediated neuromodulation, able to induce in the model the transition between awake-like asynchronous and deep-sleep-like oscillatory regimes [28]. However, this neuromodulation modelling principle has neither been previously applied to the study of the deep-sleep cogni- tive effects nor to simulations of learning-sleep cycles (such as in our previous work [27] and in this study). Specifically, the spiking model we propose is trained on a set of training patterns (here, on images of handwritten digits) and then exposed to never-seen examples to be classi- fied (here, among human-assigned digit classes). Also, the model structure proposed in [27] and adopted in this work, is able to perform an asynchronous awake-like state of the network, by acting on the neural dynamics parameters. When the prescribed changes in the neural parameters induce the network to express deep-sleep-like oscillations, STDP is observed to produce in the model the spontaneous emergence of a differential homeostatic process. 1 Introduction First, a down-regulation emerges of the stronger synapses created by the STDP during the training, Another key aspect that we consider in this study is the role of sleep during learning. Sleep is essential in all animal species, and it is believed to play a crucial role in memory consolida- tion [20, 21], in the creation of novel associations, as well as in the preparation of tasks expected during the next awake periods. Indeed, young humans pass the majority of time sleeping, and the youngest are the subjects that have to learn at faster rates. In adults, sleep deprivation is detrimental for cognition [22] and it is one of the worst tortures that can be inflicted. Among the multiple effects of sleep on the brain and body, we focus here on the con- solidation of learned information [23]. Homeostatic processes could normalize the representa- tion of memories and optimize the energetic working point of the system by recalibrating synaptic weights [24] and firing rates [25]. Specifically, Watson et al. [25] show that fast-firing pyramidal neurons decrease their firing rates over sleep, whereas slow-firing neurons increase their rates, resulting in a narrower population firing rate distribution after sleep. Also, sleep should be able to select memories for association, promoting higher performance during the next awake phases [26]. Indeed, Capone et al. [27] demonstrate the beneficial effects of sleep- wake phases involving homeostatic and associative processes in a visual classification task. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 4 / 26 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles those synapses that connect the best-tuned neurons during the training phase on each training example. At the same time, we observe that STDP increases the strength of synapses among neurons tuned on patterns belonging to the same class. Such hierarchical, spontaneous reorga- nization promotes better post-sleep classification performances. In short, the underlying mechanism is based on the similarity among thalamic coding of training examples belonging to the same class. During deep-sleep oscillations, such similarity supports the preferential acti- vation of thalamo-cortico-thalamic connection paths among neural groups tuned to training examples belonging to the same class, and the consequent coactivation and unsupervised strengthening of class-specific synapses. This point has been illustrated in [27]. Combining the above-described set of cortical principles, we aimed at creating a simplified, yet biologically-plausible, thalamo-cortical spiking model (ThaCo, see Fig 1A). 1 Introduction ThaCo exploits the combination of contextual and perceptual signals to construct a soft Winner-Take-All mechanism (WTA) capable of fast learning from few examples [29] in a synaptic matrix shaped by spike-timing-dependent plasticity (STDP). ThaCo has been calibrated to express deep-sleep-like activity and to induce modifications to the distributions of pre- and post-sleep firing rates comparable to biological measures like those carried out by Watson et al. [25] for an investigation of the deep-sleep effects on learning and classification (another beneficial aspect, the recovery and restoration of bio-chemical optimality, is not considered at this level of abstraction). In the context of machine learning, a distinction is made between instance- incremental methods, which learn from each training example as it arrives, and batch-incre- mental methods, in which the training data are organized in groups of examples, called batches, and the model is trained only on complete batches [30]. Depending on how different classes are represented by the examples in the batches, there are three training schemes [31]: new instances (NI), in which each new batch contains different instances of the same classes represented in previous batches, new classes (NC) in which examples belonging to novel clas- ses become available in subsequent batches, and new instances and classes (NIC) in which sub- sequent training batches contain examples from both known and new classes. However, it should be noted that when it is necessary to constantly evaluate the performance of incremen- tal learning, for reasons of computational efficiency the training set is divided into batches even for models capable of instance-incremental learning. Shimizu et al. [32] propose a train- ing method based on balanced mini-batches, which reduces the effect of imbalanced data in supervised training. Our work is focused on instance incremental learning, and the training scheme is based on balanced mini-batches. Specifically, with ThaCo we investigated several brain aspects and learning capabilities: 1- incremental learning from few examples; 2- resil- ience to noise when trained over degraded-quality examples and asked to classify corrupted images; 3- comparison with the performances of knn algorithms; 4- the ability to fight noise in the contextual signal thanks to the introduction of a biologically-plausible deep-sleep-like state, inducing beneficial homeostatic and associative synaptic effects. 2 Results In this work we test the capability of the implemented thalamo-cortical network model (ThaCo) of expressing incremental learning when trained to learn and recall images (from the MNIST dataset), and we investigated the role and the mechanisms of the occurrence of biolog- ical-like deep-sleep dynamics. First, we present a comparison of the ThaCo model behaviour with the biological observations made by Watson et al. [25] on the changes of firing rate distri- butions in awake, sleep and post-sleep phases (see Fig 2). Indeed, since one of the goals of this work is to implement a biologically-plausible model capable to display different “cognitive states”, the comparison with experimental outcomes is important to question its plausibility. 5 / 26 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 Fig 2. Sleep-like features. A) State-wise differences of average firing rate, to be compared with Fig 2A by Watson et al. [25]. Cumulative distribution of the firing rates of individual cortical neurons (log scale); note the brain-state dependent differences (colour). Vertical lines separate neurons sorted by AWAKE firing rates into six subgroups (sextiles) with an equal number of elements. B) Firing rate changes across sleep in each of the six groups defined by the awake firing rates, to be compared with Fig 3B by Watson et al. [25]. High firing rate neurons show decreasing activity; low firing rate neurons do not increase their activity over sleep. C) Opposite modulation of neurons of different firing rates, to be UTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles nal Biology \| https://doi org/10 1371/journal pcbi 1009045 June 28 2021 6 / 26 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles Fig 2. Sleep-like features. A) State-wise differences of average firing rate, to be compared with Fig 2A by Watson et al. [25]. Cumulative distribution of the firing rates of individual cortical neurons (log scale); note the brain-state dependent differences (colour). Vertical lines separate neurons sorted by AWAKE firing rates into six subgroups (sextiles) with an equal number of elements. B) Firing rate changes across sleep in each of the six groups defined by the awake firing rates, to be compared with Fig 3B by Watson et al. [25]. High firing rate neurons show decreasing activity; low firing rate neurons do not increase their activity over sleep. 2 Results C) Opposite modulation of neurons of different firing rates, to be PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 6 / 26 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles compared with Fig 3D by Watson et al. in [25]. Comparison of individual neuron firing rates during the first and last packet of sleep. The regression line is significantly different from unity, showing that high and low firing rate neurons are oppositely modulated over sleep. D) Cortical neuron population mean firing rate changes across sleep, to be compared with Fig 3B by Watson et al. in [25]. E) Awake firing rate distribution of cortical neurons pre-sleep (upper plot) and plot-sleep (lower plot). Solid lines depict descriptive statistics parameters: Q1, 25% quartile; Q2, 50% quartile (median); Q3, 75% quartile. Middle plot: boxplots of the distributions. The central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. https://doi.org/10.1371/journal.pcbi.1009045.g002 https://doi.org/10.1371/journal.pcbi.1009045.g002 After the validation obtained against experimental results, we demonstrate the capability of the model to learn incrementally, i.e. to continuously extend its knowledge by learning from new training examples while retaining most of the previously acquired memories. The learning ability of the model was assessed using an approach that alternates incremental training with tests meant to evaluate the pre-sleep and post-sleep classification performance. During the training phase, samples are randomly extracted from the training set of the MNIST database; they are given in input to the system together with example-specific contextual signals that reach the cortical neurons, and a digit-class-specific contextual signal that reaches only the read-out neurons. Notably, to stress out the difference with respect to the training phase, dur- ing the classification phase no contextual signal is transmitted: the response of the network is recorded, based on the firing rates of the excitatory neurons of the cortex. It should be empha- sized that the proposed model is not an engineering solution to the problem of incremental learning in pattern classification, but a simplified model of the low-level processes that sup- ports and emulates the ability to learn incrementally in the biological brain. Indeed, the size of the training set is relatively small compared to those often used in machine learning, and some issues that are of primary importance for both artificial and biological incremental learning, such as catastrophic forgetting, go beyond the aims of this paper. 2 Results Then, We measure the model incremental classification performance and we compare it to that expressed by the K-Nearest neighbour family of artificial incremental learning algorithms (specifically Knn-1, Knn-3, Knn-5). Knn is an extensively used classification algorithm, which has been succesfully applied to a wide range of problems in different fields. Furthermore, unlike many other classi- fication systems used in machine learning, the Knn family is suitable for incremental learning and also it works relatively well even with few training examples and, for large enough training sets, the Knn algorithm is guaranteed to yield an error rate no worse than twice the Bayes error rate, which is the minimum achievable given the distribution of the data [33]. For these rea- sons, the Knn classifier has been chosen as reference for the evaluation of the classification ability of the proposed system. We show that—even without the beneficial contribution of sleep—this model shows higher resilience to noisy inputs than Knn. Finally, we demonstrate the beneficial effects of deep-sleep-like cortical slow oscillations on the post-sleep classification accuracy of MNIST characters when a noisy contextual signal is injected during the awake training (a situation that could be interpreted both as the case of different levels of prior knowledge about the correct classification label of the current example during the training, and as related to the largely stochastic nature of cortical organisation and of the activity of other cortical areas). 2.1 ThaCo model pre- and post-sleep firing rates and comparison with the experiments We compare the network behaviour of the ThaCo model during three simulated phases (pre- sleep awake-like, deep-sleep-like and post-sleep awake-like, see Fig 2) with those observed in rats by Watson et al. in [25]. When approaching the design of the ThaCo spiking model, an improvement of what some of us presented in [27], we relied on the well-established frame- work of Mean-Field theories [34], [35] to construct a network capable of spontaneously 7 / 26 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles displaying two different dynamical regimes. This is obtained by acting on some parameters of the excitatory neurons (specifically, the spike frequency adaptation (SFA) and the excitatory synaptic conductance), to model acetylcholine-mediated neuromodulation on neural dynam- ics that supports the transitions between awake-like asynchronous activity and deep-sleep-like slow oscillations [28]. Specifically, in Fig 3A we show the incoming current to each cortical neuron versus its adaptation current during different network stages (pre- and post-sleep clas- sification, beginning and end of the sleeping phase). In particular, sleep states are characterized by high levels of spike frequency adaptation currents (obtained through a modulation of the SFA parameter), inducing oscillations. Moreover, late sleep and after-sleep classification have low levels of input currents, due to a sleep-mediated synaptic depression leading to a reduction in the current circulating in the network. When set in the deep-sleep state, a non-specific stim- ulus, administered at a low steady firing rate to cortical neurons, is sufficient to elicit the emer- gence of cortically-generated Up-states and of thalamo-cortical Slow Oscillations (SO). As shown in top lines of Fig 3B and 3C, in the SO regime, the thalamo-cortical spiking network displays a firing rate oscillation frequency between 0.25Hz and 1.0Hz and durations of Up- states (a few hundreds of ms) comparable with experimental observations in deep-sleep recordings. During the initial stages of SO, Up-states are independently sustained by neuron populations tuned to specific images memorized during the training phase, and tend to reacti- vate thalamic neuron coding for the memorized images. Then, thanks to the similarity among training instances, the recruitment of other neural groups in the cortex is promoted. This cre- ates preferential cortico-talamo-cortical excitatory pathways, inducing an STDP-mediated association of cortical neurons previously tuned to training instances that expressed similar thalamic representation (see Fig 3B and 3C). 2.1 ThaCo model pre- and post-sleep firing rates and comparison with the experiments Sleep-like activity, on the other hand, affects the network status during the following awake classification phase 4: the effect of STDP during sleep is a general reduction and homogenization of input current distribution, as shown in a comparison between the pre-sleep stage 1 and post-sleep stage 4 in the diagram. B and C) Spiking cortical and thalamic activity produced during training (10 examples, one per digit class), classification (20 images) and sleeping phase for two consecutive sets respectively. First row: mean firing rate of the cortical neurons trained over a set of 10 examples (each set is used to independently train 200 cortical neurons, 20 per digit example); during the sleeping phase, the slow oscillation frequency trend in time is also depicted. Second row: raster plot of the first 400 cortical neurons. Third row: mean firing rates of thalamic neurons. Once recruited in the training phases, the cortical neurons participate in classification and sleeping phases. https://doi.org/10.1371/journal.pcbi.1009045.g003 Fig 3. Incremental learning with alternation of awake training and sleep in ThaCo. A) Dots: incoming current to each cortical neuron versus its adaptation current during different network stages of the activity presented in B. Pre-sleep classification phase (represented in blue, number 1), early sleep- like phase (in red, number 2), late sleep-like phase (in orange, number 3) and post-sleep classification phase (in green, number 4). Ellipses: Areas in the plot associated with different network stages, estimated from data through a Gaussian Mixture Model with a full covariance matrix. During sleep, the total input current to the cortical neurons decreases due to the sleep-induced homeostatic effect, that reduces recurrent connections weights in the cortical layer (see the transition from number 2 to number 3 in the diagram), notwithstanding the constant external aspecific stimulus. Sleep-like activity, on the other hand, affects the network status during the following awake classification phase 4: the effect of STDP during sleep is a general reduction and homogenization of input current distribution, as shown in a comparison between the pre-sleep stage 1 and post-sleep stage 4 in the diagram. B and C) Spiking cortical and thalamic activity produced during training (10 examples, one per digit class), classification (20 images) and sleeping phase for two consecutive sets respectively. 2.1 ThaCo model pre- and post-sleep firing rates and comparison with the experiments We name top-down prediction such cortico-tha- lamic activation that spontaneously occurs during SO. During the sleep period, thanks to cor- tico-cortical plasticity, the coactivation of neurons originally tuned to training instances of the same class becomes a typical feature of each Up-state: the WTA mechanism cooperates in selecting different neuron codings for different classes during each Up-State. Another key aspect is the generalized homeostatic depression, which is known to happen during deep-sleep and serves as a protection, to prevent Up-state-mediated associations that could drive towards a fully associated network. This effect is modelled thanks to the Non-Linear Temporal Asym- metric Hebbian (NLTAH) learning rule of the STDP we used [36], which reduces the strength among the most frequently coactivated neurons, leading to a progressive reduction of mean firing rates and frequency of the Up-States (see Fig 3B and 3C, top rows) which is consistent with experimental observations, in particular for what concerns the decrease of SO frequency during the night course [37]. The first noteworthy result presented in this paper is that this new calibration of the model greatly enhances the match with experimental data, as detailed in the following. Indeed, while the model [27] was already able to express the transition between states [38] such as sleep-like slow oscillations activity and awake-like classification, the refine- ments of its parameters here introduced make ThaCo more biologically plausible, leveraging as calibration tool the accurate comparison with experimental observations of differential changes in firing rates. In their work, Watson et al. [25] used large-scale recordings to examine the activity of neurons in the frontal cortex of rats and observe the distributions of pyramidal cell firing rates in different brain states: Awake, REM, nonREM and Microarousals. They found that periods of nonREM sleep reduced the post-sleep awake activity of neurons with high pre-sleep firing rate while up-regulating the firing of slow-firing neurons. Moreover, in PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 8 / 26 Fig 3. Incremental learning with alternation of awake training and sleep in ThaCo. A) Dots: incoming current to each cortical neuron versus its adaptation current during different network stages of the activity presented in B. Pre-sleep classification phase (represented in blue, number 1), early sleep- like phase (in red, number 2), late sleep-like phase (in orange, number 3) and post-sleep classification phase (in green, number 4). 2.1 ThaCo model pre- and post-sleep firing rates and comparison with the experiments Ellipses: Areas in the plot associated with different network stages, estimated from data through a Gaussian Mixture Model with a full covariance matrix. During sleep, the total input current to the cortical neurons decreases due to the sleep-induced homeostatic effect, that reduces recurrent connections weights in the cortical layer (see th transition from number 2 to number 3 in the diagram), notwithstanding the constant external aspecific stimulus. Sleep-like activity, on the other hand, affec the network status during the following awake classification phase 4: the effect of STDP during sleep is a general reduction and homogenization of input current distribution, as shown in a comparison between the pre-sleep stage 1 and post-sleep stage 4 in the diagram. B and C) Spiking cortical and thalamic activity produced during training (10 examples, one per digit class), classification (20 images) and sleeping phase for two consecutive sets respectively. First row: mean firing rate of the cortical neurons trained over a set of 10 examples (each set is used to independently train 200 cortical neurons, 20 per digit example); during the sleeping phase the slow oscillation frequency trend in time is also depicted Second row: raster plot of the first 400 cortical neurons COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycle PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles Fig 3. Incremental learning with alternation of awake training and sleep in ThaCo. A) Dots: incoming current to each cortical neuron versus its adaptation current during different network stages of the activity presented in B. Pre-sleep classification phase (represented in blue, number 1), early sleep- like phase (in red, number 2), late sleep-like phase (in orange, number 3) and post-sleep classification phase (in green, number 4). Ellipses: Areas in the plot associated with different network stages, estimated from data through a Gaussian Mixture Model with a full covariance matrix. During sleep, the total input current to the cortical neurons decreases due to the sleep-induced homeostatic effect, that reduces recurrent connections weights in the cortical layer (see the transition from number 2 to number 3 in the diagram), notwithstanding the constant external aspecific stimulus. 2.1 ThaCo model pre- and post-sleep firing rates and comparison with the experiments As shown in Fig 2B, the sextile with the highest firing rates significantly decreased its activity over sleep, in accordance with results obtained by Watson et al. [25] (see Fig 3B of their work). Finally, we evaluated the impact of sleep on the cortical firing rates distribution during awake states. In Fig 2E, we compare firing rates distribution pre- and post-sleep depicting the homeostatization effect of sleep. An interesting feature is that lognormal distributions spontaneously emerge from our sim- ulations. This result is coherent with experimental observations and with theoretical consider- ations showing that the lognormal distribution of activities in randomly connected recurrent networks is a natural consequence of the non-linearity of the input-output gain function [39]. In agreement with Watson et al. [25], we also found that the arithmetic mean of the population firing rates declined throughout sleep, as visible using a test of correlation of spike rate versus time (see Fig 2D to be compared with Fig 3B by [25], the slope of the rate change within time- normalized sleep from all cx neurons in all recordings is R = −0.10, p = 10−3). In order to dem- onstrate that sleep brings varying differential effects across the rate spectrum, we compared mean firing rates in the first and the last 100s of sleep. As depicted in Fig 2C, fast-firing neu- rons decreased their rates over sleep, whereas slow-firing neurons increased their rates (to be compared with Fig 3D by [25]). To quantify this observation, we assessed spike rates of the same neurons in the first versus the last nonREM 100s of sleep and found the slope of this cor- relation significantly departed from unity (slope, 95% confidence interval 0.6015 −0.6130). F h f ll i [ ] di id d Th C i i l i i il Furthermore, following [25], we divided ThaCo excitatory cortical neurons into six sextile groups sorted by their awake firing rates (Fig 2A). As shown in Fig 2B, the sextile with the highest firing rates significantly decreased its activity over sleep, in accordance with results obtained by Watson et al. [25] (see Fig 3B of their work). Finally, we evaluated the impact of sleep on the cortical firing rates distribution during awake states. In Fig 2E, we compare firing rates distribution pre- and post-sleep depicting the homeostatization effect of sleep. 2.1 ThaCo model pre- and post-sleep firing rates and comparison with the experiments First row: mean firing rate of the cortical neurons trained over a set of 10 examples (each set is used to independently train 200 cortical neurons, 20 per digit example); during the sleeping phase, the slow oscillation frequency trend in time is also depicted. Second row: raster plot of the first 400 cortical neurons. Third row: mean firing rates of thalamic neurons. Once recruited in the training phases, the cortical neurons participate in classification and sleeping phases. https://doi.org/10.1371/journal.pcbi.1009045.g003 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 9 / 26 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles distribution of neuronal mean firing rates for both awake and nonREM states of our model, to be compared with Fig 2A of Watson et al. [25] (REM not included in ThaCo). Median rates (± SD) of excitatory cortical neurons in ThaCo in each state are: awake, 1.2 ± 1.1Hz and nonREM, 0.6 ± 0.3Hz. 0.6 ± 0.3Hz. An interesting feature is that lognormal distributions spontaneously emerge from our sim- ulations. This result is coherent with experimental observations and with theoretical consider- ations showing that the lognormal distribution of activities in randomly connected recurrent networks is a natural consequence of the non-linearity of the input-output gain function [39]. In agreement with Watson et al. [25], we also found that the arithmetic mean of the population firing rates declined throughout sleep, as visible using a test of correlation of spike rate versus time (see Fig 2D to be compared with Fig 3B by [25], the slope of the rate change within time- normalized sleep from all cx neurons in all recordings is R = −0.10, p = 10−3). In order to dem- onstrate that sleep brings varying differential effects across the rate spectrum, we compared mean firing rates in the first and the last 100s of sleep. As depicted in Fig 2C, fast-firing neu- rons decreased their rates over sleep, whereas slow-firing neurons increased their rates (to be compared with Fig 3D by [25]). To quantify this observation, we assessed spike rates of the same neurons in the first versus the last nonREM 100s of sleep and found the slope of this cor- relation significantly departed from unity (slope, 95% confidence interval 0.6015 −0.6130). Furthermore, following [25], we divided ThaCo excitatory cortical neurons into six sextile groups sorted by their awake firing rates (Fig 2A). 2.2 The ThaCo network model and the training protocol signal, leading to an enhancement of connections between the cortical neurons trained over the presented example and the subgroup of readout neurons associated with the correct class. The simultaneous stimulation by perceptual signals and contextual signals emulates the orga- nizing principle of the cerebral cortex as described by Larkum et al. [1], approximating the effects of the dendritic apical amplification mechanism at the cellular level. It is worth noting that this is the only phase when a category-specific (rather than an example-specific) signal is given to the network: protocols concerning this ro layer are supervised training protocols, We set the network parameters in an under threshold regime that enables the training above described through the selected STDP model on the single-compartment standard Adaptive Exponential (AdEx) integrate-and-fire neuron that would not otherwise distinguish among basal and apical stimuli. See details about the model construction, the presentation of visual stimuli and the addition of noise in the Material and Methods, Section 4 and in S1 Text. 2.2 The ThaCo network model and the training protocol It is worth noting that this is the only phase when a category-specific (rather than an example-specific) signal is given to the network: protocols concerning this ro layer are supervised training protocols, whereas those for the other layers can be referred as unsupervised training protocols. During the classification phase, signals resulting from preprocessed images are again transmitted to the thalamus analogously to the training phase, however, no contextual signal is transmitted to either cortical and readout neurons. During this stage, the neuronal activation results from the combination of the current injected by perceptual signals and the one injected by recurrent interconnections strengthened by the synaptic STDP dynamics during the training and modi signal, leading to an enhancement of connections between the cortical neurons trained over the presented example and the subgroup of readout neurons associated with the correct class. The simultaneous stimulation by perceptual signals and contextual signals emulates the orga- nizing principle of the cerebral cortex as described by Larkum et al. [1], approximating the effects of the dendritic apical amplification mechanism at the cellular level. It is worth noting that this is the only phase when a category-specific (rather than an example-specific) signal is given to the network: protocols concerning this ro layer are supervised training protocols, whereas those for the other layers can be referred as unsupervised training protocols. During the classification phase, signals resulting from preprocessed images are again transmitted to the thalamus analogously to the training phase, however, no contextual signal is transmitted to either cortical and readout neurons. During this stage, the neuronal activation results from the combination of the current injected by perceptual signals and the one injected by recurrent interconnections strengthened by the synaptic STDP dynamics during the training and modi- fied by STDP during sleep cycles. We infer the network answer to the classification task in two different ways: first, unsupervised, taking the class of the example over which the most active subgroup of cortical neurons has been trained; second, supervised, taking the class associated to the most active subgroup of readout neurons. Specifically, the readout layer performs the integration of signals coming from the subgroups of cortical neurons trained over different examples belonging to the same class (see Section 4.1 for a more detailed representation of the learning process). The activity produced by the cortical neurons during training, classification and sleeping phases is depicted in Fig 3B and 3C. 2.2 The ThaCo network model and the training protocol The proposed ThaCo circuit is organized into three layers, as shown in Fig 1A: an input layer, the thalamus, which consists of an excitatory population (tc) whose firing rate is under the control of a reticular inhibitory fully-connected population (re); the cortex, consisting of an excitatory population (cx) and an inhibitory population (in), both fully connected as well; a readout (ro) layer, to which the cortex is also fully connected, composed of subgroups of neu- rons of neurons associated to each class. The learning protocol is organized in alternation of training phases—when the internal structure of the network is shaped according to the learnt examples—and testing phases—when the classification performance of the network is evalu- ated (see Section 4.1). In both training and classification phases, the network is provided with sample images drawn from the MNIST dataset. The sample images are pre-processed to pro- duce stimulus signals that are transmitted to the excitatory neurons of the thalamus (see para- graphs “The datasets of handwritten characters” and “Thalamic coding of visual stimuli” in S1 Text for more details). During the training phase, simultaneously with the input sensory-like stimulus, contextual signals are transmitted to the excitatory neurons of the cortex and to the readout neurons. The observed bursting behaviour of the neurons is a consequence of the tem- poral coincidence between impinging perceptual and contextual signals. Specifically, for each example to learn, an example-specific group of excitatory neurons in cx is facilitated through the presentation of a contextual signal. This induces a higher activity in these neurons causing a strengthening of both thalamo-cortical synapses and recurrent synapses. This example-spe- cific tuning involves each neuron with a single training example only, whose category defines (in an unsupervised manner) a natural category for which the neuron is better tuned. Mean- while, a subgroup of readout neurons (ro) is stimulated by a digit-class specific contextual PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 10 / 26 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles signal, leading to an enhancement of connections between the cortical neurons trained over the presented example and the subgroup of readout neurons associated with the correct class. The simultaneous stimulation by perceptual signals and contextual signals emulates the orga- nizing principle of the cerebral cortex as described by Larkum et al. [1], approximating the effects of the dendritic apical amplification mechanism at the cellular level. 2.3 Incremental learning: Performances We trained the proposed network over an incremental number of training examples and eval- uated its classification performances on a set of images never shown. We also compared the average accuracy of our thalamo-cortical spiking model with that obtained using standard Knn-x classification systems for different numbers of training examples per digit category. See Fig 4 and Table 1. The model presented in this work enables instance-incremental as well as class-incremental learning. The training protocol we adopted for the results presented here was based on the balanced-mini-batches scheme proposed by [32]. More specifically, the train- ing set of hand-written digits was divided into mini-batches of 10 examples each, in which each class was represented by just one example. In S1 Text, we include a comparison of perfor- mance obtained using different training protocols. MNIST images have been presented to the ThaCo th layer using the improved pre-process- ing protocol described in paragraph Thalamic coding of visual stimuli of the S1 Text. The accuracy has been evaluated over classification trials, each one including 500 images, and the classification accuracy has been averaged over 20 trials. Fig 4A shows the accuracy for incre- mental learning as a function of the number of training examples per class. Fig 4B and 4C, on the other hand, depict the average accuracy of the compared training algorithms for the last 10 to 20 and the first 1 to 5 training examples per class respectively, to better show their different behaviour at different stages of the learning process. For the MNIST dataset, higher-order Knn algorithms surpass the performance of Knn-1 only when the training set includes more than 10 examples per digit class. It is worth noting that the soft WTA mechanism of ThaCo can PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 11 / 26 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles Fig 4. Comparison of the average accuracy of the proposed thalamo-cortical spiking model compared to artificial K-nearest-neighbour incremental algorithms in absence of noise (solid lines) and with noisy inputs(dotted lines). A) We infer the network answer to the classification task in two different ways: 1) Digit-Class Readout, as the class associated to the most active subgroup of readout neurons in ThaCo (supervised approach); 2) Example-Specific Group, by mapping the class over which the most active subgroup of cortical neurons has been trained (unsupervised approach). 2.3 Incremental learning: Performances Solid lines depict accuracy in absence of noise, dotted lines depict accuracy with “Salt and Pepper” noise (density = 0.2) injected into the unprocessed MNIST images for both training and classification phases. Accuracy is assessed on an independent test set, consisting of 500 examples; the average and standard error of the mean (SEM) are evaluated on 20 different trials using independent training sets in each. B), C) represent the same plots shown in A) on different scales, for visualization purposes and for highlighting selected features. Specifically, B) ThaCo behaves like Knn-k1 for a small number of examples; C) as the number of training examples increases, ThaCo Digit-Class-Readout exhibits performances that are comparable with higher-order Knn algorithms. https://doi.org/10.1371/journal.pcbi.1009045.g004 Fig 4. Comparison of the average accuracy of the proposed thalamo-cortical spiking model compared to artificial K-nearest-neighbour incremental algorithms in absence of noise (solid lines) and with noisy inputs(dotted lines). A) We infer the network answer to the classification task in two different ways: 1) Digit-Class Readout, as the class associated to the most active subgroup of readout neurons in ThaCo (supervised approach); 2) Example-Specific Group, by mapping the class over which the most active subgroup of cortical neurons has been trained (unsupervised approach). Solid lines depict accuracy in absence of noise, dotted lines depict accuracy with “Salt and Pepper” noise (density = 0.2) injected into the unprocessed MNIST images for both training and classification phases. Accuracy is assessed on an independent test set, consisting of 500 examples; the average and standard error of the mean (SEM) are evaluated on 20 different trials using independent training sets in each. B), C) represent the same plots shown in A) on different scales, for visualization purposes and for highlighting selected features. Specifically, B) ThaCo behaves like Knn-k1 for a small number of examples; C) as the number of training examples increases, ThaCo Digit-Class-Readout exhibits performances that are comparable with higher-order Knn algorithms. learn incrementally and has comparable performances to the best Knn-n algorithm for a given number of training examples. Specifically, the supervised ThaCo is proven to be able to per- form the integration of signals coming from subgroups of cortical neurons trained over differ- ent examples belonging to the same class and its performances are comparable to higher-order learn incrementally and has comparable performances to the best Knn-n algorithm for a given number of training examples. 2.3 Incremental learning: Performances Specifically, the supervised ThaCo is proven to be able to per- form the integration of signals coming from subgroups of cortical neurons trained over differ- ent examples belonging to the same class and its performances are comparable to higher-order PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 12 / 26 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles Knns, whereas the unsupervised ThaCo performances are proven to be comparable with Knn- 1 performances when few examples are presented. Table 1. Accuracy achieved by the different learning algorithms over a different number of training examples. The accuracy has been computed over a test set of 500 examples, and the average is done over 20 trials. Accuracy (%) Training examples per class Algorithm 1 2 3 5 10 20 Knn, k = 1 68.0 ± 1.0 76.1 ± 1.0 80.6 ± 0.8 84.5 ± 0.4 88.3 ± 0.4 90.4 ± 0.3 Knn, k = 3 37.2 ± 1.3 66.0 ± 1.1 75.4 ± 0.9 82.7 ± 0.4 88.3 ± 0.3 91.0 ± 0.3 Knn, k = 5 23.9 ± 1.5 62.0 ± 1.3 72.2 ± 0.9 81.9 ± 0.4 88.2 ± 0.3 91.2 ± 0.3 ThaCo—Digit class readout 65.7 ± 1.0 74.8 ± 0.8 80.4 ± 0.7 84.8 ± 0.6 88.6 ± 0.5 91.1 ± 0.3 ThaCo—Example specific group 65.7 ± 0.9 73.6 ± 0.9 78.1 ± 0.6 82.7 ± 0.4 86.4 ± 0.4 88.6 ± 0.4 https://doi.org/10.1371/journal.pcbi.1009045.t001 Knns, whereas the unsupervised ThaCo performances are proven to be comparable with Knn- 1 performances when few examples are presented. Knns, whereas the unsupervised ThaCo performances are proven to be comparable with Knn- 1 performances when few examples are presented. 2.4 Classification of noisy input We evaluated the network behaviour within a noisy input environment and compared it to the Knn performances. For this, we injected a ‘Salt and Pepper’ noise [40] (density = 0.2) into the unprocessed MNIST images. Noisy images are then pre-processed (see 1) and presented to the network in both training and classification phases. Fig 4A depicts the average accuracy of the network trained incrementally over a total number of 20 noisy examples per class and com- pares it to performances of Knn-n algorithms (as in section 2.3, both Knn and ThaCo algo- rithms are trained incrementally). It is worth noting that in this scenario the ThaCo algorithm has better performances than the Knn-n algorithms. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 2.5 Beneficial effect of deep-sleep in compensating the impact of noisy contextual labels some examples are associated with stronger synapses), leading to a drop in performances (comparison between blue and violet line). Still, the interposition deep-sleep-like phases between noisy-training and classification phases recovers the performances of the network trained with a non-noisy protocol. B) Sleep-induced homeostatic and associative effects on cortico-cortical synaptic-weight distributions. Pre-sleep (violet), post-sleep (green). Solid lines: mean and standard deviation. a) Intra-group connections: weight distributions of synapses connecting neurons trained over the same example (i.e. that during the training stage were triggered by the same contextual stimulus, thus activated simultaneously during a specific training p g g p y Fig 5. A) Sleep mitigates the effects of noisy contextual signals on the classification performances of ThaCo Classification performances evaluated over the Readout layer (supervised learning protocol, left) and Cortical layer (unsupervised learning protocol, right). Comparison among the network trained over non noisy examples (blue), the network trained over noisy examples without any deep-sleep like phase (violet), and the network trained over noisy examples interposing a deep-sleep-like activity between the training and the classification phases (green). The contextual signal provided in the training phase is corrupted by noise (i.e. some examples are associated with stronger synapses), leading to a drop in performances (comparison between blue and violet line). Still, the interposition deep-sleep-like phases between noisy-training and classification phases recovers the performances of the network trained with a non-noisy protocol. B) Sleep-induced homeostatic and associative effects on cortico-cortical synaptic-weight distributions. Pre-sleep (violet), post-sleep (green). Solid lines: mean and standard deviation. a) Intra-group connections: weight distributions of synapses connecting neurons trained over the same example (i.e. that during the training stage were triggered by the same contextual stimulus, thus activated simultaneously during a specific training example); b) Intra-class connections: weight distributions of synapses connecting neurons trained over different examples belonging to the same class (i.e. that have not been simultaneously triggered by the contextual stimulus in the training phase, but still have been triggered by a sensorial thalamic signal associated to images belonging to the same class) c) Inter-class connections: Connections among groups trained over different classes (i.e. triggered by the contextual stimulus together with a sensorial thalamic signal associated to images belonging to different classes). We note the homeostatic effect of sleep (in A) leading to a general reduction of weights associated to example-specific synapses and a reinforcement of the intra-class connections (in B). Synapses Fig 5. 2.5 Beneficial effect of deep-sleep in compensating the impact of noisy contextual labels For the aim of introducing more biologically-plausible elements regarding the combination of contextual and perceptual signals, we slightly modify the ThaCo training protocol: the magni- tude of the contextual signal given to both the cortex and the readout layer trained over a new learning example is now randomly extracted from a Gaussian distribution. As a consequence, some of the presented examples are better represented than others, resembling a more realistic situation in the cortex in which both the degree of knowledge projected by other areas and the number and strength of apical synapses carrying the contextual information and raising the perceptual thresholds during learning are not exactly equal for all the presented examples and all the neurons in the selected group. We introduce the deep-sleep state in our training protocol, as follows: after each training phase, we disconnect ThaCo from external inputs and induce deep-sleep-like oscillations, fol- lowing the method described in [27] and here described in Section 2.1 and in S1 Text, para- graph Sleep-like oscillatory dynamics. As expected, noise in the contextual signal leads to a drop in performance, compared to the idealized situation presented in the previous section (i.e. the careful equalization of contextual signal), but such drop can be reduced by sleep, as shown in Fig 5A. At the synaptic level, it is possible to observe how deep-sleep-like slow oscillations induce in the current ThaCo model both a regularisation of the strength of the memories of individual learned examples through homeostasis and an association between groups of neurons trained over different examples of the same class. Figs 5B and 6 report such sleep-induced optimiza- tion of the synaptic representation of memories. Specifically, within neurons belonging to the PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 13 / 26 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles Fig 5. A) Sleep mitigates the effects of noisy contextual signals on the classification performances of ThaCo Classification performances evaluated over the Readout layer (supervised learning protocol, left) and Cortical layer (unsupervised learning protocol, right). Comparison among the network trained over non noisy examples (blue), the network trained over noisy examples without any deep-sleep like phase (violet), and the network trained over noisy examples interposing a deep-sleep-like activity between the training and the classification phases (green). The contextual signal provided in the training phase is corrupted by noise (i.e. 2.5 Beneficial effect of deep-sleep in compensating the impact of noisy contextual labels The inset, showing part of the same plot in a lin-lin scale, is added to illustrate the shape of the pre-sleep distribution, and the difference in the mean values before and after sleep. https://doi.org/10.1371/journal.pcbi.1009045.g005 same example-specific group, the synaptic weights distribution decreases its mean and coeffi- cient of variation (from μ = 74, μ/σ = 0.3, skewness 0.55 pre-sleep to μ = 60, μ/σ = 0.2, skewness −0.26 post-sleep) whereas within neurons belonging to different example-specific group but coding for the same class, the synaptic weights distribution increases its mean and coefficient same example-specific group, the synaptic weights distribution decreases its mean and coeffi- cient of variation (from μ = 74, μ/σ = 0.3, skewness 0.55 pre-sleep to μ = 60, μ/σ = 0.2, skewness −0.26 post-sleep) whereas within neurons belonging to different example-specific group but coding for the same class, the synaptic weights distribution increases its mean and coefficient same example-specific group, the synaptic weights distribution decreases its mean and coeffi- cient of variation (from μ = 74, μ/σ = 0.3, skewness 0.55 pre-sleep to μ = 60, μ/σ = 0.2, skewness −0.26 post-sleep) whereas within neurons belonging to different example-specific group but coding for the same class, the synaptic weights distribution increases its mean and coefficient same example-specific group, the synaptic weights distribution decreases its mean and coeffi- cient of variation (from μ = 74, μ/σ = 0.3, skewness 0.55 pre-sleep to μ = 60, μ/σ = 0.2, skewness −0.26 post-sleep) whereas within neurons belonging to different example-specific group but coding for the same class, the synaptic weights distribution increases its mean and coefficient Fig 6. Effects of sleep on intra-class and example-specific synapses after training with a noisy contextual signal. Comparison of synaptic-weight matrices, pre-sleep (Left) vs post-sleep (Right). Training over 5 examples per class (20 neurons per example). 2.5 Beneficial effect of deep-sleep in compensating the impact of noisy contextual labels A) and B) depict all cortico-cortical synaptic weights connecting the full set of trained neurons (colour bar, logarithm scale), black lines separate neurons solicited by contextual signal together with thalamic sensorial signal pointing to images belonging to the same digit class in the training phase; C) and D) focus on the synaptic weights connecting groups of cortical neurons simultaneously solicited by a contextual signal in the training phase (thus stimulated over the same sensorial signal identifying images belonging to the same class) (colour bar, linear scale), vertical black lines separate neurons trained over different categories, horizontal black lines separate cortical groups of neurons solicited during the presentation of the same 10 examples (one per digit class). The post-sleep intra-class cooperation is evident in B and in agreement with Fig 5B.b, while the homeostatic effect over example-specific synapses is manifest in D as already suggested by Fig 5B.a. The strong, example-specific differences in synaptic weights (e.g. third example of class 4) are due to the noisy training protocol that introduces randomness in the magnitude of the contextual signal that reaches the example-specific group. https://doi.org/10.1371/journal.pcbi.1009045.g006 Fig 6. Effects of sleep on intra-class and example-specific synapses after training with a noisy contextual signal. Comparison of synaptic-weight matrices, pre-sleep (Left) vs post-sleep (Right). Training over 5 examples per class (20 neurons per example). A) and B) depict all cortico-cortical synaptic weights connecting the full set of trained neurons (colour bar, logarithm scale), black lines separate neurons solicited by contextual signal together with thalamic sensorial signal pointing to images belonging to the same digit class in the training phase; C) and D) focus on the synaptic weights connecting groups of cortical neurons simultaneously solicited by a contextual signal in the training phase (thus stimulated over the same sensorial signal identifying images belonging to the same class) (colour bar, linear scale), vertical black lines separate neurons trained over different categories, horizontal black lines separate cortical groups of neurons solicited during the presentation of the same 10 examples (one per digit class). The post-sleep intra-class cooperation is evident in B and in agreement with Fig 5B.b, while the homeostatic effect over example-specific synapses is manifest in D as already suggested by Fig 5B.a. The strong, example-specific differences in synaptic weights (e.g. 2.5 Beneficial effect of deep-sleep in compensating the impact of noisy contextual labels A) Sleep mitigates the effects of noisy contextual signals on the classification performances of ThaCo Classification performances evaluated over the Readout layer (supervised learning protocol, left) and Cortical layer (unsupervised learning protocol, right). Comparison among the network trained over non noisy examples (blue), the network trained over noisy examples without any deep-sleep like phase (violet), and the network trained over noisy examples interposing a deep-sleep-like activity between the training and the classification phases (green). The contextual signal provided in the training phase is corrupted by noise (i.e. some examples are associated with stronger synapses), leading to a drop in performances (comparison between blue and violet line). Still, the interposition deep-sleep-like phases between noisy-training and classification phases recovers the performances of the network trained with a non-noisy protocol. B) Sleep-induced homeostatic and associative effects on cortico-cortical synaptic-weight distributions. Pre-sleep (violet), post-sleep (green). Solid lines: mean and standard deviation. a) Intra-group connections: weight distributions of synapses connecting neurons trained over the same example (i.e. that during the training stage were triggered by the same contextual stimulus, thus activated simultaneously during a specific training example); b) Intra-class connections: weight distributions of synapses connecting neurons trained over different examples belonging to the same class (i.e. that have not been simultaneously triggered by the contextual stimulus in the training phase, but still have been triggered by a sensorial thalamic signal associated to images belonging to the same class) c) Inter-class connections: Connections among groups trained over different classes (i.e. triggered by the contextual stimulus together with a sensorial thalamic signal associated to images belonging to different classes). We note the homeostatic effect of sleep (in A) leading to a general reduction of weights associated to example-specific synapses and a reinforcement of the intra-class connections (in B). Synapses PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 14 / 26 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles connecting groups trained on different examples, on the other hand, are much less affected by sleep. The inset, showing part of the same plot in a lin-lin scale, is added to illustrate the shape of the pre-sleep distribution, and the difference in the mean values before and after sleep. https://doi.org/10.1371/journal.pcbi.1009045.g005 connecting groups trained on different examples, on the other hand, are much less affected by sleep. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 2.5 Beneficial effect of deep-sleep in compensating the impact of noisy contextual labels third example of class 4) are due to the noisy training protocol that introduces randomness in the magnitude of the contextual signal that reaches the example-specific group. https://doi.org/10.1371/journal.pcbi.1009045.g006 https://doi.org/10.1371/journal.pcbi.1009045.g006 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 15 / 26 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles of variation (from μ = 0.005, μ/σ = 0.001, skewness 0.003 pre-sleep to μ = 0.5, μ/σ = 1.6, skew- ness −2.9 post-sleep). Specifically, the homeostatic effect of deep-sleep-like SOs can be identified comparing Fig 6C and 6D: the distribution of synaptic weights sharpens (i.e. it exhibits smaller post-sleep σ and μ) and presents a general depression of synaptic weights. These two variations combine to produce beneficial effects. First, this leads to a lowering of the heterogeneity of representa- tion of learned examples, a reduction of the energetic cost of memory recall (reduced synap- tic strength is associated with a lower metabolic cost of synaptic activity) and lower post- sleep spiking rates (see section 2.1). Moreover, deep-sleep-like oscillations affects categorical association and is depicted in Fig 5B.b: synapse weights connecting groups of neurons trained over different examples but belonging to the same digit class increase from a nearly zero pre-sleep value, while synapses connecting representations of memories belonging to different classes are much less affected. This effect is also visible comparing Fig 6A and 6B, where synapses connecting representations of memories belonging to the same digit class light up (big squares along the diagonal). Asymmetric STDP induces, on one hand, the depression of strong synapses, on the other the association among neuronal groups coding for the same class (i.e. trained over similar stimuli), through a mechanism of resemblance in their thalamic representation. 3 Discussion PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 16 / 26 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles We stress that, even though we showed to be successful in reproducing specific experimen- tal observations, the aim of this work is not to exactly reproduce a biological network (for instance, the emulation of metabolic processes goes beyond the scope of this work), but to develop a simplified task-specific spiking neural network able to express biological features, and receive an indication on how even an approximated emulation of deep-sleep and of the combination of contextual and perceptual information can positively affect the network per- formances. Specifically, without any pretence to be biologically realistic, the neuron model used in these simulations is a point-like AdEx (see section The neuron model), yet we are able to emulate a compartment neuron behaviour (described in [1]) without introducing more complex morphological units in the network. For this, we approximated the coincidence mechanism by setting both the contextual and sensory inputs impinging on cortical neurons in a subthreshold point (see 4.1). Another major aspect of our work is the effect of sleep on the network and on the memories stored in it. The role played by sleep in memory consolidation has been widely studied from an experimental point of view [46, 47], but only recently it has become the object of theoretical and computational modelizations [27, 48–50]. In our work, we investigated computationally the effect of slow oscillations on the structure and the performances of the network when the STDP plasticity is turned on. We proved that deep-sleep-like slow oscillations can be beneficial to equalize the memories stored in a cortico-thalamic structure when learned in noisy condi- tions. Indeed, slow oscillations can compensate for the contextual noise through homeostasis, equalizing synaptic weights and creating beneficial associations that improve classification performance. The predictions of our model are also a first step toward the reconciliation of recent experi- mental observations about both an average synaptic down-scaling effect (synaptic homeostatic hypothesis—SHY [51]) and a differential modulation of firing rates [25] induced by deep- sleep, which is believed to be a default state mode for the cortex [52]. As mentioned above, we focused on the role of NREM-sleep for memory consolidation. The simulation of a complete sleep cycle that includes REM and micro-arousal phases goes beyond the scope of this paper and is currently under investigation. 3 Discussion One more limitation of this work is that it does not take into account the role of synchronization among different brain regions. Actually, assuming a typical neural density in the range of 5 104 neurons per mm2 of the cortex, and considering that the maximum size of the proposed model rises up to 5000 cortical neurons, such a number is equivalent to a small cortical area with a dimension of about 300μm, that is well below the size of a single cortical area. To overcome this limitation, we are extending the model to multi-layers and multi-area descriptions. Finally, this work represents an additional contribution in understanding sleep mechanisms and functions, in line with the efforts we are carrying out in data analysis [53, 54] and in large- scale simulations [55], aimed at bridging different elements in a multi-disciplinar approach. In particular, it hints to a careful balance between architectural abstraction and experimental observations as a valid methodology for the description of brain mechanisms and of their links with cognitive functions. 3 Discussion We propose a simplified thalamo-cortical spiking model (ThaCo) that exploits the combina- tion of context and perception to build a soft-WTA circuit, and that is able to express sleep- like slow oscillations. In order to be compliant with biological rhythms, we first verified that the proposed network is able to reproduce the experimental measures of neuronal firing rates during awake and deep-sleep states performed by Watson et al [25]. The agreement with the experiments has been achieved by further developing the thalamo-cortical spiking model pro- posed in [27] and by setting the model parameters to better fit the experimental recordings. The model we propose is capable of fast incremental learning from few examples (its perfor- mances are comparable to those expressed by Knn, of rank increasing with the number of examples) and of alternating several learning-sleep phases; moreover, it demonstrates resil- ience when subjected to noisy perceptions with better performances than Knn algorithms; these three facts constitute significant extensions to the previous study [27]. In recent years, there has been growing interest in the development of artificial neural net- works (ANNs) or deep neural networks inspired by features found in biology, yet still using mechanisms for learning and inference which are fundamentally different from what is actu- ally observed in biology. On the other hand, there is also a plenty of computational models aiming at reproducing biological proprieties in an exact way. Many models have been pro- posed for pattern recognition tasks that use biologically-plausible mechanisms, combining spiking networks and STDP plasticity [41–43]. The ThaCo model has been developed in line with this philosophy, delivering a spiking neural network which relies on a combination of biologically plausible mechanisms. It uses conductance-based AdEx neurons, STDP and lateral inhibition. A crucial ingredient, which mostly differentiates our approach from previous works, is the introduction of a contextual signal which drives the training procedure, making it similar to a target-based approach [44, 45] and enabling huge advantages in terms of training velocity and precision. Such mechanism was inspired by the work done by Larkum [1] sug- gesting that the activity of a neuron is amplified when it receives a coincidence of signals from both lower and higher levels of abstraction. This allows the recruitment of new neurons to learn novel examples through the incremental building of a soft-WTA mechanism. 4.1 Winner-take-all mechanisms by combining context and perception We set the network parameters to induce the creation of WTA mechanisms by emulating the organizing principle of the cortex described by Larkum et al. [1]. During the training, the network is set in a hard-WTA regime (firing rate different from zero only on a selected example-specific sub-set of neurons), while during classification it works in a soft-WTA regime (i.e. the firing rate can be different from zero in multiple groups of neurons, and the winner group is assumed to be the one firing at the higher rate). Specifi- cally, during the training, we set our parameters to be so that the thalamic signal alone is not sufficient to make neurons spike. This is reported in Fig 7C that represents the mean firing rate and the membrane potential over time for a group of cortical neurons stimulated to encode for a training example in three different contextual scenarios: Fig 7C-center shows the network behaviour in the absence of a thalamic signal; Fig 7C-left shows the network response without the contextual signal; Fig 7C-right shows the network behaviour with both the contex- tual and the thalamic signal. The cortical activity in the absence of contextual signal is null and it is really low when only the stimulation of the contextual signal is present. The combined action of the two, on the other hand, yields a higher spiking activity. We can therefore con- clude that we put the network in what we named an under-threshold regime. Moreover, to bet- ter show the implemented soft Winner-take-all dynamics, we present the mean firing rate of three subgroups of cortical neurons trained over different examples belonging to different cat- egories during both retrieval (i.e. training examples are presented again to the network without any contextual signal) and classification phases. The implementation of WTA dynamics is depicted in Fig 7D. 4.1.1 Simple mathematical model of soft-WTA creation. In this section, we discuss the capability of our model to learn over a few examples through soft-WTA mechanisms. First, we demonstrate how the network is surely endowed with the capability to behave like a Knn clas- sifier. In the first training step, the network is exposed to one example for each of ten digit class (L = 10). Let D(l) = {1 + (l −1)K, . . 4 Materials and methods The results of the ThaCo model (Section 2) have been obtained thanks to fine implementations of several features. Such fine-tuning is presented in this Section and in the S1 Text. In particu- lar, Section 4.1 addresses the crucial point of the model calibration, aimed at inducing a soft- WTA mechanism by combining context and perception, achieved by setting the network parameters in what we call an under-threshold regime, that enables a training through the 17 / 26 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles selected STDP model on single-compartment standard Adaptive Exponential integrate-and- fire neuron (AdEx) that would not otherwise distinguish among basal and apical stimuli (see S1 Text). MNIST characters are coded by the thalamus according to the scheme presented in S1 Text that preserves a notion of distance among visual features. Simulation reported were executed on dual-socket servers with eight-core Intel(R) Xeon(R) E5–2620 v4 CPU per socket. The cores are clocked at 2.10GHz with HyperThreading enabled, so that each core can run 2 processes, for a total of 32 processes per server. The ThaCo model has been implemented using the NEST 2.12.0 [56] simulation engine. 4.1 Winner-take-all mechanisms by combining context and perception Here, the WTA mechanism is essential to decide the classification answer; the network decision can be evaluated measuring the activation level of the groups, either in the cortex or in the readout layer. C) Combination of contextual and perceptual signals to create one group of cortical neurons sensible to a specific example in a soft winner-take-all mechanism. Three examples are presented to three cortical groups for 2s (start and stop presentation time marked by green and red dashed lines). High firing rate is induced only when the example-specific cortical group is reached by both the thalamic (perceptual) stimulus and the contextual (example-specific) signal. Upper row: mean firing rates of a cortical subgroup of cortical neurons; lower row: mean membrane potentials (the black dotted line depicts the firing threshold potential). Left column: Neuron activity when stimulated by the thalamic signal only (perception): null firing rate and under-threshold membrane potential. Central column: Neuron activity when stimulated by contextual signal only (internal prediction): a moderate firing rate is induced. Right column: Simultaneous perceptual and contextual signals induce a high firing rate in the example-specific group. D) Soft Winner-take-all dynamics among example-specific groups of cortical neurons during retrieval and classification phases. Mean firing rates of three groups trained to be sensitive to three different examples. D-Rows) Firing rates of the firs (blue), second(orange), third (green) neural group. D-Retrieval phase column) Exactly the three learnt examples (belonging to three different digit classes) are re-presented to the network without any contextual signal, resulting in an almost hard- WTA dynamics among the three groups. D-Classification phase column) Three novel images, for which the network has not been trained, are presented to the network without any hint from the contextual signal; a soft-WTA dynamics is emerging, rewarding for each presentation the neuron group with the highest firing rate and still allowing all the other groups to fire with non-zero probability. Here read-out neurons are not represented and the figure demonstrates how it is possible to extract a classification answer looking at the cortical layer only. https://doi org/10 1371/journal pcbi 1009045 g007 PLOS COMPUTATIONAL BIOLOGY Thalamo cortical spiking model of incremental learning/sleep cycles Fig 7. Training and classification phases. A) Training Phase: the sensorial perception (blue arrow) is encoded into the thalamic excitatory neurons. 4.1 Winner-take-all mechanisms by combining context and perception A time-specific contextual signal is delivered to a subset of cortical excitatory neurons (red arrow), raising their perceptual threshold on the example-specific thalamic activity and inducing in such group of neurons a high firing rate during training (represented with red flames drawing). STDP induces group-specific connectivity in the subset of facilitated cortical neurons, and the thalamic pattern is sculptured into synapses that connect the thalamus with the example-specific group. The readout layer, made of as many groups of cortical neurons as the number of classification classes, is also trained through the simultaneous administration of a class-specific contextual signal addressing the subset associated to the correct class label (red arrow). B) Classification Phase: no contextual signal is given to the cortex. One or more subgroups of excitatory cortical neurons reach a high (red flames), intermediate (yellow flames) or low (no flames) level of activity, depending on the similarity between the stimulating thalamic pattern and the training set. Here, the WTA mechanism is essential to decide the classification answer; the network decision can be evaluated measuring the activation level of the groups, either in the cortex or in the readout layer. C) Combination of contextual and perceptual signals to create one group of cortical neurons sensible to a specific example in a soft winner-take-all mechanism. Three examples are presented to three cortical groups for 2s (start and stop presentation time marked by green and red dashed lines). High firing rate is induced only when the example-specific cortical group is reached by both the thalamic (perceptual) stimulus and the contextual (example-specific) signal. Upper row: mean firing rates of a cortical subgroup of cortical neurons; lower row: mean membrane potentials (the black dotted line depicts the firing threshold potential). Left column: Neuron activity when stimulated by the thalamic signal only (perception): null firing rate and under-threshold membrane potential. Central column: Neuron activity when stimulated by contextual signal only (internal prediction): a moderate firing rate is induced. Right column: Simultaneous perceptual and contextual signals induce a high firing rate in the example-specific group. D) Soft Winner-take-all dynamics among example-specific groups of cortical neurons during retrieval and classification phases. Mean firing rates of three groups trained to be sensitive to three different examples. D-Rows) Firing rates of the firs (blue), second(orange), third (green) neural group. 4.1 Winner-take-all mechanisms by combining context and perception ., lK} be the set of indices of the K excitatory cortical neurons that are induced to fire by the simultaneous presence of the contextual stimulation and the thalamic input, carried by T thalamic neurons (see Fig 7C) when presented with one of the training examples l 2 {1, ‥, L}. Also, starting from an initial value wth!cx 0 , let wth!cx eq be the final average weight induced by STDP on the connections between the thalamic excitatory neurons that are active during the learning of the training example l and the K excitatory corti- cal neurons that are induced to activity. Finally, let x ðlÞ th be the binary feature vector of the train- ing example l. The average weight at equilibrium of the connections between the thalamic neurons activated by the example l and the excitatory cortical neurons can be written as: wth!cx nj ¼ ðwth!cx eq wth!cx 0 Þx ðlÞ th;j þ wth!cx 0 j 2 f1; ::; Tg; n 2 DðlÞ ¼ f1 þ ðl 1ÞK; ::; lKg; l 2 f1; . . . ; Lg ð1Þ ð1Þ PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 18 / 26 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles Fig 7. Training and classification phases. A) Training Phase: the sensorial perception (blue arrow) is encoded into the thalamic excitatory neurons. A time-specific contextual signal is delivered to a subset of cortical excitatory neurons (red arrow), raising their perceptual threshold on the example-specific thalamic activity and inducing in such group of neurons a high firing rate during training (represented with red flames drawing). STDP induces group-specific connectivity in the subset of facilitated cortical neurons, and the thalamic pattern is sculptured into synapses that connect the thalamus with the example-specific group. The readout layer, made of as many groups of cortical neurons as the number of classification classes, is also trained through the simultaneous administration of a class-specific contextual signal addressing the subset associated to the correct class label (red arrow). B) Classification Phase: no contextual signal is given to the cortex. One or more subgroups of excitatory cortical neurons reach a high (red flames), intermediate (yellow flames) or low (no flames) level of activity, depending on the similarity between the stimulating thalamic pattern and the training set. https://doi.org/10.1371/journal.pcbi.1009045.g007 4.1 Winner-take-all mechanisms by combining context and perception D-Retrieval phase column) Exactly the three learnt examples (belonging to three different digit classes) are re-presented to the network without any contextual signal, resulting in an almost hard- WTA dynamics among the three groups. D-Classification phase column) Three novel images, for which the network has not been trained, are presented to the network without any hint from the contextual signal; a soft-WTA dynamics is emerging, rewarding for each presentation the neuron group with the highest firing rate and still allowing all the other groups to fire with non-zero probability. Here read-out neurons are not represented and the figure demonstrates how it is possible to extract a classification answer looking at the cortical layer only. 19 / 26 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles After the training on the first set of L examples, a total of C = kL cortical neurons will have been exposed to the combination of contextual and thalamic stimulation (see Fig 3A). During the classification phase, represented in Fig 3B, when a never seen stimulus (the image S to be classified) is presented to the network, the average signal from the thalamic layer (composed of T neurons) to the excitatory cortical neurons (in all the L trained cortical groups) is: g ðSÞ n;th!cx ¼ X j¼1;T wth!cx nj rthx ðSÞ th;j j 2 f1; ::; Tg n 2 DðlÞ; l 2 f1; . . . ; Lg ð2Þ ð2Þ where ρth is the rate of the active thalamic neurons, and x ðSÞ th is the binary thalamic feature vec- tor of the novel image S to be classified. Assuming w0 to be much smaller than weq, the average signal from thalamic neurons to each cortical neuron belonging to D(l) group can thus be writ- ten as: g ðSÞ n;th!cx ’ X j¼1;T wth!cx eq rthx ðlÞ th;jx ðSÞ th;j ¼ wth!cx eq rthx ðlÞ th x ðSÞ th for n 2 DðlÞ ð3Þ ð3Þ where S is the novel stimulus presented during the classification phase and l is the learning example over which the set of neurons D(l) have been trained. The vectors of thalamic features can be normalized (u = x/N(x), using their Euclidean norm (NðxÞ ¼ kxk2 ¼ ðP ix2 i Þ 1 2). 4.1 Winner-take-all mechanisms by combining context and perception The Euclidean distance among each training example (l) and the images (S) to be classified can be written as dl,S = ku(l) −u(S)k2. It follows that d2 l;S ¼ 2 2uðlÞ uðSÞ, where we used the normali- zation condition for both u(l) and u(S). In this way Eq 3 can be rewritten as: can be normalized (u = x/N(x), using their Euclidean norm (NðxÞ ¼ kxk2 ¼ ðP ix2 i Þ 1 2). The Euclidean distance among each training example (l) and the images (S) to be classified can be written as dl,S = ku(l) −u(S)k2. It follows that d2 l;S ¼ 2 2uðlÞ uðSÞ, where we used the normali- zation condition for both u(l) and u(S). In this way Eq 3 can be rewritten as: g ðSÞ ðlÞ;th!cx ’ wth!cx eq rthNðlÞNðSÞuðlÞ uðSÞ ¼ wth!cx eq rthNðlÞNðSÞð1 1 2 d2 l;SÞ ð4Þ ð4Þ Eq 4 tells us that the thalamic signal is a decreasing function of the distance dl,S, if all training examples are equally normalized(kx(i)k2 = kx(j)k28i, j 2 1, ‥L) and neglecting for a while the possible changes in the thalamic rate ρth, that in our model can be mediated by the existing cortico-thalamic feedback path. Under the approximation of constant ρth we can immediately show that, after having being exposed to the first set of training examples, the soft-WTA ThaCo excitatory network is at least endowed with the capability to behave as a nearest neigh- bour classifier of the first order (Knn-1 classifier). The winning candidate K among the L com- peting cortical groups is initially suggested to the network as the one reached by the strongest thalamic stimulus when presented with the never seen image S: Eq 4 tells us that the thalamic signal is a decreasing function of the distance dl,S, if all training examples are equally normalized(kx(i)k2 = kx(j)k28i, j 2 1, ‥L) and neglecting for a while the possible changes in the thalamic rate ρth, that in our model can be mediated by the existing cortico-thalamic feedback path. Under the approximation of constant ρth we can immediately show that, after having being exposed to the first set of training examples, the soft-WTA ThaCo excitatory network is at least endowed with the capability to behave as a nearest neigh- bour classifier of the first order (Knn-1 classifier). 4.1 Winner-take-all mechanisms by combining context and perception Assuming that wcx!cx 0 wcx!cx eq , after learn- ing we have: wcx!cx l1;l2 ¼ wcx!cx eq dl1;l2 þ wcx!cx 0 ð1 dl1;l2Þ wcx!cx eq dl1;l2 l1; l2 2 1; ::; L ð7Þ ð7Þ Under the assumption that the activities of the K neurons belonging to the same subgroup (l) are similar to each other, the second term in Eq 6 reduces to the recurrent intra-group excit- atory contribution: Under the assumption that the activities of the K neurons belonging to the same subgroup (l) are similar to each other, the second term in Eq 6 reduces to the recurrent intra-group excit- atory contribution: g ðSÞ cx!cxðlÞ g ðSÞ cxðlÞ!cxðlÞ ¼ ðk 1Þ wcx!cx eq r ðSÞ ðlÞ ð8Þ ð8Þ where r ðSÞ ðlÞ is the average firing rate reached by the cortical group l when activated by the novel stimulus S. In our simplified mode, all wcx!inh and winh!cx synapses are non-plastic and set to an iden- tical value. Therefore, the third term, the input signal from cortico-cortical inhibition, is in our architecture equal to: g ðSÞ inh!cxðlÞ ¼ g ðSÞ inh!cx ¼ Ninh winh!cxr ðSÞ inh ð9Þ ð9Þ where Ninh is the number of cortical inhibitory neurons and ρinh is the inhibitory neurons activity. In summary, Eq 6, i.e. the total current stimulating each of the L groups of cortical neurons responding to the thalamic stimulus S can be reformulated: g ðSÞ ðlÞ;tot g ðSÞ ðlÞ;th!cx þ g ðSÞ cxðlÞ!cxðlÞ þ g ðSÞ inh!cx ¼ ¼ wth!cx eq rthNðlÞNðSÞð1 1 2 d2 l;SÞ þ ðk 1Þ wcx!cx eq r ðSÞ ðlÞ þ Ninh winh!cxr ðSÞ inh ð10Þ ð10Þ When the average rate is well below saturation, its relationship to the total input signal is well described by a threshold-linear function: r ðSÞ ðlÞ ¼ aðg ðSÞ ðlÞ;tot gthreshÞHðg ðSÞ ðlÞ;tot gthreshÞ ð11Þ ð11Þ where α is a constant coefficient, H is the Heaviside function and gthresh is the firing threshold. 4.1 Winner-take-all mechanisms by combining context and perception The winning candidate K among the L com- peting cortical groups is initially suggested to the network as the one reached by the strongest thalamic stimulus when presented with the never seen image S: ThaCo excitatory network is at least endowed with the capability to behave as a nearest neigh- bour classifier of the first order (Knn-1 classifier). The winning candidate K among the L com- peting cortical groups is initially suggested to the network as the one reached by the strongest thalamic stimulus when presented with the never seen image S: initial candidate KðSÞ ¼ argl max½g ðSÞ ðlÞ;th!cx ¼ argl min½dl;S for l 2 1; ::; L ð5Þ ð5Þ Indeed, under the assumption that the neuron activity depends on the incoming signal (both excitatory and inhibitory) through a transfer function FðgÞ monotonically increasing over the total incoming current g, we will now show that: 1) the role of inhibition will be to help the computation of (a soft) argmax; 2) the recurrent intra-group cortical excitation provides an additional boost to the selection of the winner. To confirm this, we shall now consider explic- itly the contribution of both recurrent and inhibitory contributions. The total average input signal to each cortical neuron depends on the group l the neuron belongs to and on the the stimulus S to be classified: g ðSÞ ðlÞ;tot ¼ g ðSÞ ðlÞ;th!cx þ g ðSÞ cx!cxðlÞ þ g inh!cxðlÞ ð6Þ ð6Þ Under the approximation of constant ρth, the first term in Eq 6 is provided by Eq 3. 20 / 26 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles Concerning the second term, the training protocol illustrated by Fig 7 creates cortico-corti- cal synapses of strength wcx!cx eq only among neurons belonging to the same group l, i.e among neurons trained on the same example, while connections among neurons selective for different training examples are left to the initial value wcx!cx 0 . 4.1 Winner-take-all mechanisms by combining context and perception Therefore, assuming that the input signal is above threshold (g ðSÞ ðlÞ;tot > gthresh), we have that g ðSÞ ðlÞ;tot ¼ wth!cx eq rthNðlÞNðSÞð1 1 2 d2 l;SÞ þ Ninh winh!cxr ðSÞ inh aðk 1Þ wcx!cx eq gthresh 1 aðk 1Þ wcx!cx eq ð12Þ ð12Þ we also require that aðk 1Þ wcx!cx eq < 1, i.e. self feedback should be smaller than one, other- wise the system would become unstable. Considering that the inhibitory signal is equal for all L groups under the provisional assumption of constant ρth, i.e. no cortico-thalamic feedback), Eq 12 tells us that the final choice of the network would confirm the initial guess of Eq 5: winning KðSÞ ¼ argl max½g ðSÞ ðlÞ;tot ¼ argl min½dl;S l 2 1; ::; L ð13Þ ð13Þ i.e. the network tends to a stationary condition in which the L groups of K neurons can be set at different firing rates that decrease with the distance dl,S. Moreover, the readout layer PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009045 June 28, 2021 21 / 26 PLOS COMPUTATIONAL BIOLOGY Thalamo-cortical spiking model of incremental learning/sleep cycles combines signal coming from groups of cortical neurons trained over different examples yet belonging to the same class: thus, the network expresses a behaviour similar to that of a a higher-order Knn—n,. 5 Supporting information S1 Text. Mechanisms and implementation details. The reader can find in this section details about the mechanisms in action in ThaCo during the Training and Classification phases (see the ‘Training and classification phases’ and ‘Balanced mini-batch training’ paragraphs, specifi- cally Fig A providing a comparison of incremental learning protocols) and details concerning the implementation and effects of deep sleep dynamics (see ‘Sleep-like oscillatory dynamics’). The specific form of STDP plasticity is described in paragraph ‘Spike-Timing-Dependent Plas- ticity’ (and depicted in Fig B showing the effects of deep-sleep on network performances) and the neuronal model in ‘The neuron model’. Details about the handwritten digits datatsets are presented in the ‘The datasets of handwritten characters’ paragraph, while paragraph ‘Tha- lamic coding of visual stimuli’ describes the fuzzy-logic-inspired pre-processing algorithm, adopted for a tuning of thalamic activity that preserves a notion of distance among visual fea- tures. 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https://openalex.org/W4221136009	http://cds.cern.ch/record/2789647/files/Fulltext from publisher.pdf	English	null	Prospects in the search for a new light <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:mrow><mml:msup><mml:mrow><mml:mi>Z</mml:mi></mml:mrow><mml:mrow><mml:mo>′</mml:mo></mml:mrow></mml:msup></mml:mrow></mml:math> boson with the <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:mrow><mml:mi>NA</mml:mi><mml:mrow><mml:mn>64</mml:mn><mml:mi>μ</mml:mi></mml:mrow></mml:mrow></mml:math> experiment at the CERN SPS	Physical review. D/Physical review. D.	2,022	cc-by	8,734	Prospects in the search for a new light Z0 boson with the NA64μ experiment at the CERN SPS H. Sieber ,1 D. Banerjee ,2 P. Crivelli ,1 E. Depero ,1 S. N. Gninenko ,3 D. V. Kirpichnikov ,3 M. M. Kirsanov V. Poliakov ,4 and L. Molina Bueno 1,5,* 1ETH Zürich, Institute for Particle Physics and Astrophysics, CH-8093 Zürich, Switzerland 2CERN, European Organization for Nuclear Research, CH-1211 Geneva, Switzerland 3Institute for Nuclear Research of the Russian Academy of Sciences, 117312 Moscow, Russia 4State Scientific Center of the Russian Federation Institute for High Energy Physics of National Research Center Kurchatov Institute (IHEP), 142281 Protvino, Russia 5CSIC—Universitat de Val`encia, Instituto de Física Corpuscular (IFIC), E-46980 Paterna, Spain (Received 4 November 2021; accepted 31 January 2022; published 22 March 2022) Banerjee ,2 P. Crivelli ,1 E. Depero ,1 S. N. Gninenko ,3 D. V. Kirpichnikov ,3 M. M. Kirs V. Poliakov ,4 and L. Molina Bueno 1,5,* (Received 4 November 2021; accepted 31 January 2022; published 22 March 2022) A light Z0 vector boson coupled to the second and third lepton generations through the Lμ −Lτ current with mass below 200 MeV provides a very viable explanation in terms of new physics to the recently confirmed ðg −2Þμ anomaly. This boson can be produced in the bremsstrahlung reaction μN →μNZ0 after a high energy muon beam collides with a target. NA64μ is a fixed-target experiment using a 160 GeV muon beam from the CERN Super Proton Synchrotron accelerator looking for Z0 production and its subsequent A light Z0 vector boson coupled to the second and third lepton generations through the Lμ −Lτ current with mass below 200 MeV provides a very viable explanation in terms of new physics to the recently confirmed ðg −2Þμ anomaly. This boson can be produced in the bremsstrahlung reaction μN →μNZ0 after a high energy muon beam collides with a target. NA64μ is a fixed-target experiment using a 160 GeV muon beam from the CERN Super Proton Synchrotron accelerator looking for Z0 production and its subsequent decays, Z0 →invisible. In this paper, we present the study of the NA64μ sensitivity to search for such a boson. This includes a realistic beam simulation, a detailed description of the detectors and a discussion μ with mass below 200 MeV provides a very viable explanation in terms of new physics to the recently confirmed ðg −2Þμ anomaly. This boson can be produced in the bremsstrahlung reaction μN →μNZ0 after a high energy muon beam collides with a target. Prospects in the search for a new light Z0 boson with the NA64μ experiment at the CERN SPS NA64μ is a fixed-target experiment using a 160 GeV muon beam from the CERN Super Proton Synchrotron accelerator looking for Z0 production and its subsequent decays, Z0 →invisible. In this paper, we present the study of the NA64μ sensitivity to search for such a boson. This includes a realistic beam simulation, a detailed description of the detectors and a discussion about the main potential background sources. A pilot run is scheduled in order to validate the simulation results. If those are confirmed, NA64μ will be able to explore all the remaining parameter space which could provide an explanation for the g −2 muon anomaly in the Lμ −Lτ model. Corresponding author. laura.molina.bueno@cern.ch Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Funded by SCOAP3. PHYSICAL REVIEW D 105, 052006 (2022) PHYSICAL REVIEW D 105, 052006 (2022) DOI: 10.1103/PhysRevD.105.052006 2006-1 Published by the American Physical Society I. INTRODUCTION ð5Þ It is worth noting that adding to the minimal Uð1ÞLμ−Lτ gauge extension of the SM a dark current interaction of the type L ⊃Z0αJDM α makes it possible to also probe light thermal dark matter and the dark matter (DM) relic abun- dance (see e.g., Ref. [37,38]). It is worth noting that adding to the minimal Uð1ÞLμ−Lτ gauge extension of the SM a dark current interaction of the type L ⊃Z0αJDM α makes it possible to also probe light thermal dark matter and the dark matter (DM) relic abun- dance (see e.g., Ref. [37,38]). p The experimental setup for the feasibility studies to look for a light Z0 boson is sketched in Fig. 1. The experiment will use the high-energy M2 muon beam at the CERN SPS [39,46] with momentum ≃160 GeV=c produced by a 450 GeV=c primary proton beam (intensity 1012–1013 protons=spill). Within this context, muons are dumped against an active target, which is a 40 radiation lengths (40X0) lead-scintillator sandwich electromagnetic calorimeter (ECAL), with a 6 × 5 cell matrix structure. While the scattered muon carries away a fraction E0μ ¼ fEμ of the primary muon energy Eμ, the other fraction of the energy, ð1 −fÞEμ, is carried away by the bremsstrahlung dark boson Z0 and its decay products resulting in missing energy Emiss ¼ Eμ −E0μ. The subdetectors downstream of the target include, in particular, a five interaction lengths (5λI) copper-scintillator veto calorimeter (VHCAL) seg- mented with a 4 × 4 matrix of cells and a hole in the middle, to veto charged secondaries produced by upstream muon nuclear interactions. Then, a series of two large 120 × 60 cm2 (6 × 3 matrix) hadronic calorimeter (HCAL) modules, with a 7.5λI steel-scintillator longitudinal seg- mentation, ensure maximal hermeticity. The experiment will use two magnet spectrometers in order to reconstruct the incoming and outgoing muon momentum. The initial muon beam momentum will be measured by the existing beam momentum stations (BMS) from the COMPASS experiment [47]. A set of micromesh gaseous structure (Micromegas or MM) tracking detectors will be located next to the stations to have a second measurement of the incoming momentum. The scattered muon momentum is reconstructed through a second MBPL magnetic spectrom- eter (a single dipole magnet with 1.4 T · m, MS2) with a set of six Micromegas tracking detectors. I. INTRODUCTION mZ0 ≤Oð1 GeVÞ, and interacts to the second and third generations of leptons through The recently confirmed 4.2σ deviation of the muon magnetic moment [1] with respect to its Standard Model (SM) prediction [2–22] might be an indication of physics beyond the SM: ð2Þ L ¼ g0ð¯μγαμ þ ¯νμγανμ −¯τγατ þ ¯ντγαντÞZ0α; ð2Þ where Z0α is the leptophilic boson field and g0 is its coupling to SM leptons. Within this model, the Z0 contribution to the muon vertex function, and thus the muon ðg −2Þμ, is calculated at one loop [36]: Δaμ ≡aμðexpÞ −aμðthÞ ¼ ð251 59Þ × 10−11: ð1Þ Interactions between muons and new physics sectors have been suggested in many models [23–26]. In particular, models with Uð1Þ gauge extension to the SM are well motivated since they are anomaly free and provide an explanation to the ðg −2Þμ anomaly through a loop con- tribution to the muonvertex function [27–32]. In the Lμ −Lτ model, with SM gauge extension SUð3Þc ⊗SUð2ÞL ⊗ Uð1ÞY ⊗Uð1ÞLμ−Lτ [30,33–35], the massive gauge vector boson Z0 acquires its mass through symmetry breaking, ΔaZ0 μ ¼ g02 4π2 Z 1 0 dx x2ð1 −xÞ x2 þ ð1 −xÞm2 Z0=m2μ : ð3Þ ð3Þ The Z0 vector boson decays invisibly to SM neutrinos in the case mZ0 < 2mμ, with decay width ΓðZ0 →¯νfνfÞ ¼ αμmZ0 3 ; ð4Þ ð4Þ Corresponding author. laura.molina.bueno@cern.ch Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Funded by SCOAP3. where f ¼ μ, τ, and αμ ¼ g02=4π. For larger Z0 masses, namely mZ0 ≥2mμ, the gauge boson also decays visibly to one of the charged components of the SUð2ÞL lepton doublets, Lμ, Lτ, with partial decay width: 52006-1 Published by the American Physical Society 052006-1 Published by the American Physical Society 2470-0010=2022=105(5)=052006(9) 052006-1 Published by the American Physical Society 052006-1 PHYS. REV. D 105, 052006 (2022) H. SIEBER et al. ΓðZ0 →¯ffÞ ¼ αμmZ0 3 · 1 þ 2m2 f m2 Z0 · ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 − 4m2 f m2 Z0 s : ð5Þ particles [44] and could also be used to test lepton flavor violation in μN →τX conversion in flight [45]. A second phase of the experiment will be devoted to exploring these processes [37]. I. INTRODUCTION The Z0 vector boson can be produced through muon bremsstrahlung μN →μNZ0 after a high energy muon beam impinges on a target. Within this context, the NA64μ experiment [37] has been designed to search for Z0 production and its subsequent invisible decay using the 160 GeV M2 beam line at the CERN Super Proton Synchrotron (SPS) accelerator [39]. Detailed computations of the differential and total cross sections for this process have been recently performed in Ref. [40]. Another experi- ment, M3, with a similar working principle, has been proposed at Fermilab [38]. A pilot run of the NA64μ experiment is planned in the fall of 2021 to study the feasibility of the technique. In this paper, we discuss the experiment prospects in terms of the main background sources and the expected trigger rate. We also study the projected sensitivities for future physics runs to allow to probe the region of parameter space suggested by the ðg −2Þμ anomaly in the context of current and future searches. II. THE METHOD OF SEARCH The NA64μ experiment [37] is a complementary experi- ment to NA64e [41,42] aiming to look for dark sectors weakly coupled to muons. The experiment is foreseen in two phases. Its first phase physics goal is to search for invisible decays of the Z0 boson, produced in the muon scattering process μ−N →μ−NZ0. Additionally, similarly to the electron mode, the experiment also explores the production of dark photons, A0, through the bremsstrahlung μ−N →μ−NA0, allowing to enlarge the parameter space of interest towards large masses [43]. NA64μ can also probe scalar, axionlike particles (ALPs), millicharged A signal event, i.e., the production of a Z0 boson, is defined as a scattered muon after the target with momentum about half of the beam nominal energy, i.e., E0μ ≲0.5E0 ≃ 80 GeV (see Fig. 1). The muon missing momentum will be FIG. 1. Schematic top view of the 2021 muon pilot run experimental setup to search for Z0 →invisible production from 160 GeV=c muon bremsstrahlung. The dash-dotted line corresponds to the path of a deflected muon after the Z0 bremsstrahlung. FIG. 1. Schematic top view of the 2021 muon pilot run experimental setup to search for Z0 →invisible production from 160 GeV=c muon bremsstrahlung. The dash-dotted line corresponds to the path of a deflected muon after the Z0 bremsstrahlung. 052006-2 PROSPECTS IN THE SEARCH FOR A NEW LIGHT … PHYS. REV. D 105, 052006 (2022) 3 − 10 2 − 10 1 − 10 1 [GeV] Z' m 0 0.1 0.2 0.3 0.4 Trigger efficiency Beam energy 160 GeV 100 GeV FIG. 2. Trigger efficiency as a function of the Z0 vector boson mass. The beam nominal energy is both (blue) 160 GeV and (green) 100 GeV, for which the scattered muon deflection is larger. 3 − 10 2 − 10 1 − 10 1 [GeV] Z' m 0 0.1 0.2 0.3 0.4 Trigger efficiency Beam energy 160 GeV 100 GeV To further suppress background coming from SM events, a set of selection criteria (cuts) is applied as follows: (i) an initial beam momentum reconstructed in the energy win- dow [140, 180] GeV; (ii) a single track in the tracking detectors with reconstructed momentum smaller than half of the beam energy; (iii) no energy deposit in the VHCAL; and (iv) no energy deposit in the HCAL modules [i.e., compatible with the one of a minimum ionizing particle (MIP)]. III. SIMULATIONS FRAMEWORK An additional counter in front of the HCAL, S4, shifted from the beam axis guarantees that only muons with enough deflection hit this counter. The signal efficiency for different Z0 masses at this trigger level is illustrated in Fig. 2. A set of veto counters, before the HCAL modules (V1), and within MS2 (Vm1;2), are used to further veto undeflected beam muons and veto charged secondaries produced in upstream interactions. The trigger rate after this selection is 0.1% of the primary beam intensity. A. The beam profile at M2 location Because of the importance of an accurate knowledge of the initial muon momentum and beam spatial distribu- tion for the trigger criteria, the M2 beam line optics is fully simulated using the TRANSPORT [50], TURTLE [51] and HALO [52] software [46] and made compatible with GEANT4 through the HEPMC software [53]. This yields realistic beam profiles as shown in Fig. 3. Simulations reproduce the large contribution of low-energy halo muons around the beam spot (about 20% of the full beam intensity [37]) that are populating the low-energy tail of the beam energy distribution. Such muons can be efficiently removed using the beam-defining counters S0 and S1, leaving 78% of the full beam intensity, with muons energy ≥100 GeV, with beam spot divergency σx ∼0.9 cm and σy ∼1.9 cm. B. Signal III. SIMULATIONS FRAMEWORK Detailed Monte Carlo (MC) simulations are performed using the GEANT4 toolkit [48] and the GEANT4-compatible DM package DMG4 [49] aiming at realistically reproducing the beam line, detectors and physics, as well as estimating the background and signal topology within the setup. FIG. 2. Trigger efficiency as a function of the Z0 vector boson mass. The beam nominal energy is both (blue) 160 GeV and (green) 100 GeV, for which the scattered muon deflection is larger. used to keep the trigger rate at the required level. Using the deflection of the scattered muon in the transverse plane, a simplified trigger system has been considered for the initial phase to run the experiment at the low beam intensity of 107 μ=spill. This trigger option is based on the selection of the well-defined primary muon beam within a small beam lateral size and divergency, and momentum spread by using scintillator counters, the BMS stations and the trackers next to it. The initial muon is tagged with a set of three counters, two of them before the target (S0 and S1), and one ensuring the presence of the muon at the end of the setup (Sμ). An additional counter in front of the HCAL, S4, shifted from the beam axis guarantees that only muons with enough deflection hit this counter. The signal efficiency for different Z0 masses at this trigger level is illustrated in Fig. 2. A set of veto counters, before the HCAL modules (V1), and within MS2 (Vm1;2), are used to further veto undeflected beam muons and veto charged secondaries produced in upstream interactions. The trigger rate after this selection is 0.1% of the primary beam intensity. used to keep the trigger rate at the required level. Using the deflection of the scattered muon in the transverse plane, a simplified trigger system has been considered for the initial phase to run the experiment at the low beam intensity of 107 μ=spill. This trigger option is based on the selection of the well-defined primary muon beam within a small beam lateral size and divergency, and momentum spread by using scintillator counters, the BMS stations and the trackers next to it. The initial muon is tagged with a set of three counters, two of them before the target (S0 and S1), and one ensuring the presence of the muon at the end of the setup (Sμ). B. Signal This result is obtained assuming that in particular selection criterium (ii) holds, i.e., a single hit per tracking detectors. In the case of more than one hit per tracker, the Micromegas detector inefficiency should be taken into account. An example of such physics processes is highly energetic secondaries produced by muons in the tracker material through ioniza- tion, μe →μe, accompanied by a poorly detected parent muon, and thus yielding a reconstructed momentum with energy <100 GeV. The probability for such an event to happen is estimated from the full sample of simulated muons, taking into account the values for Micromegas trackers inefficiency (∼0.02) extracted by previous NA64e run data, and assuming that in the second tracker downstream MS2 there is more than one hit. From the simulations, a conservative value of ≤10−11 per MOT is obtained, and can be further reduced by placing additional n trackers downstream, with a factor ∼0.02n. FIG. 4. Normalized energy spectra of the Z0 vector boson for different masses obtained from GEANT4 [48] using the DMG4 [49] package. The mixing strength is ϵ ¼ g0= ﬃﬃﬃﬃﬃﬃﬃﬃ 4πα p ¼ 10−4, where α ¼ 1=137. See Ref. [40] for more details. the Z0 vector boson is simulated using the fully GEANT4- compatible DMG4 package [49]. Dark matter observables, such as the total cross-section production and the differ- ential cross sections are correspondingly implemented according to the Weiszäcker-Williams (WW) phase-space approximation as discussed in Ref. [40]. The program selects an event where the Z0 production should occur according to the total cross section, and then both its fractional energy, x, and emission angle, θ, are accurately sampled using a Von-Neumann accept-reject sampling algorithm. The typical energy spectra of a Z0-strahlung vector boson is shown in Fig. 4 for the mass range 10 MeV to 1 GeV. In the case of NA64μ, the level of hermeticity of the detectors is inferred in the plane defined by the muon energy after ECAL and the total energy deposited in the calorimeter (ECAL, VHCAL and HCAL), ðE0μ; ECALÞ, as shown in Fig. 5. Whereas region A corresponds to events with large energy deposit in the HCAL and the diagonal B to events with energy deposited in the ECAL, the bulk C is associated to events with energy deposition in the calo- rimeters compatible with a MIP. B. Signal To estimate both the signal yield and signal topology, and thus the choice of adequate selection criteria, 1000 − 500 − 0 500 1000 x [mm] 1000 − 500 − 0 500 1000 y [mm] 1 10 2 10 3 10 4 10 0 50 100 150 200 Beam energy [GeV] 1 10 2 10 3 10 4 10 5 10 6 10 Normalised entries / 1 GeV Trigger none μ +S 1 +S 0 S FIG. 3. Beam profiles at the entrance of the NA64μ setup as obtained through the TRANSPORT [50], TURTLE [51] and HALO [52] software [46] for (left) beam spatial distribution and (right) beam energy spectrum with no trigger (blue) and (green) trigger S0 þ S1 þ Sμ. 1000 − 500 − 0 500 1000 x [mm] 1000 − 500 − 0 500 1000 y [mm] 1 10 2 10 3 10 4 10 0 50 100 150 200 Beam energy [GeV] 1 10 2 10 3 10 4 10 5 10 6 10 Normalised entries / 1 GeV Trigger none μ +S 1 +S 0 S FIG. 3. Beam profiles at the entrance of the NA64μ setup as obtained through the TRANSPORT [50], TURTLE [51] and HALO [52] software [46] for (left) beam spatial distribution and (right) beam energy spectrum with no trigger (blue) and (green) trigger S0 þ S1 þ Sμ. 052006-3 PHYS. REV. D 105, 052006 (2022) H. SIEBER et al. 0 0.2 0.4 0.6 0.8 1 x 10 2 10 3 10 Normalised dN/dx / 0.01 in MeV Z' m 10 100 500 1000 FIG. 4. Normalized energy spectra of the Z0 vector boson for different masses obtained from GEANT4 [48] using the DMG4 [49] package. The mixing strength is ϵ ¼ g0= ﬃﬃﬃﬃﬃﬃﬃﬃ 4πα p ¼ 10−4, where α ¼ 1=137. See Ref. [40] for more details. 0 0.2 0.4 0.6 0.8 1 x 10 2 10 3 10 Normalised dN/dx / 0.01 in MeV Z' m 10 100 500 1000 precision on momentum reconstruction is estimated using the Kalman-filter-based GENFIT toolkit [54] to be Δp=p ∼3%. The mismeasurement, and thus misidentifi- cation between Z0-strahlung and SM muons, is extracted from the exponential tails of the momentum residuals distribution. Through extrapolation, the probability of a 160 GeV SM muon to be reconstructed with momentum ≤80 GeV is ≲10−12 per muon on target (MOT). B. Signal Thus a high level of hermeticity is reached for all events lying within those three regions. On the other side, poor detector hermeticity due to geometrical acceptance or dead material can lead to events with large missing energy, and thus leakage towards the signal box defined in the region D (red box) of Fig. 5 ðECAL ≤20 GeV; E0μ ≤80 GeVÞ. From the distribution extrapolation in the plane ðE0μ; ECALÞ, the probability of leakage in region D due to non-hermeticity (i.e., detector acceptance) is estimated to be ≲10−12 per MOT. C. Background Missing energy/momentum experiments such as NA64μ rely not only on a robust detector hermeticity in order to avoid events with large missing energy appearing because some particles escaped detection due to acceptance, but also on a precise momentum reconstruction. Many proc- esses, such as hard muon nuclear interactions in the ECAL, hadron admixture in the M2 beam line, or mismatch in momentum reconstruction, can affect the likelihood to truly observe a Z0-strahlung process. In the following para- graphs, the full study of those main background contribu- tions is covered, being carried out through detailed MC simulations. Muons usually behave as MIPs, thus most of them traverse the whole setup with nominal momentum ∼160 GeV=c, with small energy deposit in the calorimeters (EECAL ∼0.5 GeV, EHCAL ∼2.3 GeV). On the opposite, scattered muons accompanied by Z0 emission are identified with energy E0μ ≤0.5E0 ≃80 GeV. Accurate momentum reconstruction thus allows to discriminate between possible signal candidates and SM muon events. The muon momen- tum is reconstructed both upstream of the target and downstream of the ECAL through a series of magnetic spectrometers. To account for multiple scattering (material) effects as well as tracking detector resolution, the Apart from geometrical properties of the detectors, and thus acceptance, two main sources of physical background contribute to events with large missing energy. The first one arises from hadron admixture in the M2 beam line, typically charged and neutral hadrons, such as π−, K− and K0 LðsÞ, and their subsequent (semi)leptonic decays along the setup. The level of contamination is measured with a set of beryllium absorbers in the beam line, and found to be Ph ¼ π=μ ∼10−6, with K=π ∼0.03 [39]. To estimate the hadron decay probability, Ph→X, and the 052006-4 PROSPECTS IN THE SEARCH FOR A NEW LIGHT … PHYS. REV. D 105, 052006 (2022) 0 50 100 150 200 ' [GeV] μ Reconstructed muon energy E 0 50 100 150 200 [GeV] CAL Sum of energy deposited E 1 10 2 10 3 10 4 10 5 10 A) B) C) D) 0 50 100 150 200 ' [GeV] μ Reconstructed muon energy E 0 50 100 150 200 [GeV] CAL Sum of energy deposited E 1 10 2 10 FIG. 5. C. Background The total number of simulated muons corresponds to NMOT ¼ 108. Whereas region A, B and C correspond to regions compatible with SM-related energy deposit, region D defines the interesting parameter space compatible with Z0-strahlung events (see text). related level of background, hadrons are simulated at the end of the COMPASS BMS to account for particle misidentification through momentum reconstruction. From MC simulations, along the typical distance to the active target of ∼36 m, it is found that PK−→X ∼Oð10−3Þ, whereas Pπ−;K0 L→X ∼Oð10−4Þ. Thus, the total number of decay hadrons before the entrance of the setup is estimated through Nh→X ¼ NMOT × Ph × Ph→X, which is ∼Oð10−10–10−11Þ per MOT. From those in-flight decays, background is associated to events with final state muons in the decay products, namely h →μ−X, where X is mostly associated to a neutrino in the case of π−and K−, susceptible to carry away a large fraction of its parent hadron energy, thus mimicking a Z0-strahlung event with missing energy. For such a distance, the probability of kaons decaying to muons through the purely leptonic channel, K−→μ−¯νμ, is about PK−→μ−≃0.018. For final state muons with energy Eμ < 100 GeV, this probability reduces to PK−→μ−ðEμ < 100 GeVÞ ≃0.011. In the case of pion decays, this probability is strongly reduced because of related level of background, hadrons are simulated at the end of the COMPASS BMS to account for particle misidentification through momentum reconstruction. From MC simulations, along the typical distance to the active target of ∼36 m, it is found that PK−→X ∼Oð10−3Þ, whereas Pπ−;K0 L→X ∼Oð10−4Þ. Thus, the total number of decay hadrons before the entrance of the setup is estimated through Nh→X ¼ NMOT × Ph × Ph→X, which is ∼Oð10−10–10−11Þ per MOT. From those in-flight decays, background is associated to events with final state muons in the decay products, namely h →μ−X, where X is mostly associated to a neutrino in the case of π−and K−, susceptible to carry away a large fraction of its parent hadron energy, thus mimicking a Z0-strahlung event with missing energy. For such a distance, the probability of kaons decaying to muons through the purely leptonic channel, K−→μ−¯νμ, is about PK−→μ−≃0.018. For final state muons with energy Eμ < 100 GeV, this probability reduces to PK−→μ−ðEμ < 100 GeVÞ ≃0.011. C. Background Hermeticity plane defined by the reconstructed muon energy after the ECAL and the total energy deposit in the calorimeters, ðE0μ; ECAL ¼ EECAL þ EVHCAL þ EHCALÞ, for SM muons with a single reconstructed track (one hit per Micromegas) assuming (top) trigger in counters S0, S1 and Sμ and (bottom) trigger in all counters, no energy deposit in the vetos, VHCAL and HCAL. The total number of simulated muons corresponds to NMOT ¼ 108. Whereas region A, B and C correspond to regions compatible with SM-related energy deposit, region D defines the interesting parameter space compatible with Z0-strahlung events (see text). 0 50 100 150 200 ' [GeV] μ Reconstructed muon energy E 0 50 100 150 200 [GeV] CAL Sum of energy deposited E 1 10 2 10 3 10 4 10 5 10 A) B) C) D) the kinematics of the process. The overall probability for such a process, given the kaon contamination of the beam, is estimated at the level of 3.3 × 10−10 per MOT. For the full set of selection criteria, this background reduces to 1.1 × 10−11 per MOT. Such background can be further reduced by the mean of additional absorbers in the M2 beam line. For a 3.8λI ¼ 150 cm aluminum absorber, this probability is reduced by a factor e−3.7 ≃0.023. Additionally, using a dedicated magnetic spectrometer just before the active target [28,37] reduces the effective hadron decay length to 4 m, thus suppressing further this back- ground by at least an order of magnitude according to simulations. 0 50 100 150 200 ' [GeV] μ Reconstructed muon energy E 0 50 100 150 200 [GeV] CAL Sum of energy deposited E 1 10 2 10 The second important source of background contributing to missing energy events originates from leading hadron production in the target. Those arise from muon nuclear interactions, μ−N →μ−hX, within the ECAL material, with the outgoing hadron carrying away a significant fraction of the primary muon energy (Eh ≥80 GeV). C. Background Such events can then leak through [punch through (PT)] the detector elements downstream of the target, with two possible scenarios mimicking a signal event: (i) the low energy outgoing muon is poorly detected and the leading charged hadrons deposit an energy compatible with the one of a MIP (EHCAL ∼2.5 GeV) in the HCAL module; (ii) the outgoing muon is reconstructed with low energy and the neutral hadron traverses the HCAL modules undetected (EHCAL ∼0.1 GeV). The probability for leading hadron production in the target is estimated through MC simu- lations to be 10−6 per MOT. Similarly, the probability for punching through a single or multiple HCAL module(s) is estimated with simulations and compared to available experimental data [55–57]. In the case of a single module, the effect of punch-through charged/neutral high energy hadrons—mostly π, K and neutrons n—appears as a peak in the low-energy end of the HCAL energy deposited spectrum. By extrapolating the low-end region of the spectrum, the PT probability for a single module corre- sponds to ≲10−2 per incoming hadron. A similar analysis extended to two and four modules yields a probability of ≲10−6 and ≲10−11 respectively. The overall total proba- bility of producing a leading hadron which subsequently punches through two HCAL modules is thus estimated to be ≲10−12 per MOT. ' [GeV] μ Reconstructed muon energy E FIG. 5. Hermeticity plane defined by the reconstructed muon energy after the ECAL and the total energy deposit in the calorimeters, ðE0μ; ECAL ¼ EECAL þ EVHCAL þ EHCALÞ, for SM muons with a single reconstructed track (one hit per Micromegas) assuming (top) trigger in counters S0, S1 and Sμ and (bottom) trigger in all counters, no energy deposit in the vetos, VHCAL and HCAL. The total number of simulated muons corresponds to NMOT ¼ 108. Whereas region A, B and C correspond to regions compatible with SM-related energy deposit, region D defines the interesting parameter space compatible with Z0-strahlung events (see text). FIG. 5. Hermeticity plane defined by the reconstructed muon energy after the ECAL and the total energy deposit in the calorimeters, ðE0μ; ECAL ¼ EECAL þ EVHCAL þ EHCALÞ, for SM muons with a single reconstructed track (one hit per Micromegas) assuming (top) trigger in counters S0, S1 and Sμ and (bottom) trigger in all counters, no energy deposit in the vetos, VHCAL and HCAL. C. Background Source of background Level per MOT Hadron in-flight decay ≲10−11 Momentum mismatch ≲10−12 Detector non-hermeticity ≲10−12 Single-hadron punch through ≲10−12 Dimuon production < 10−12 Total (conservatively) ≲10−11 of ðme=mμÞ5 compared to electron bremsstrahlung, is estimated through MC simulations to be ∼10−7 per MOT. Because of the phase space associated to this process, final state muons can be efficiently rejected through the double/triple MIP signature in the HCAL modules, as well as within the tracking detectors. For the typical set of cuts used within this framework, possible background due to dimuons production is rejected at the level of <10−12 per MOT. FIG. 6. Projected sensitivities for the invisible mode Z0 →¯νν in the ðmZ0; g0Þ parameter space for a total of 1011 MOT with the NA64μ experiment. The limits are given using the selection criteria (i)–(iv) and the requirement of the event lying in the signal box. The necessary ðmZ0; g0Þ values to explain the ðg −2Þμ anomaly are shown within the 2σ green band, as well as the possible parameters to cover the XENON1T excess [62]. Also shown are current experimental constraints from CCFR [63,64], BOREXINO [65,66], BABAR [67] and Belle-II [68], together with the cosmological constraints from the big bang nucleosyn- thesis (BBN) [69–71]. Projected sensitivities from Dune [72,73], Belle-II [74] and M3 [38] are also plotted. FIG. 6. Projected sensitivities for the invisible mode Z0 →¯νν in the ðmZ0; g0Þ parameter space for a total of 1011 MOT with the NA64μ experiment. The limits are given using the selection criteria (i)–(iv) and the requirement of the event lying in the signal FIG. 6. Projected sensitivities for the invisible mode Z0 →¯νν in the ðmZ0; g0Þ parameter space for a total of 1011 MOT with the NA64μ experiment. The limits are given using the selection criteria (i)–(iv) and the requirement of the event lying in the signal box. The necessary ðmZ0; g0Þ values to explain the ðg −2Þμ anomaly are shown within the 2σ green band, as well as the possible parameters to cover the XENON1T excess [62]. Also shown are current experimental constraints from CCFR [63,64], BOREXINO [65,66], BABAR [67] and Belle-II [68], together with the cosmological constraints from the big bang nucleosyn- thesis (BBN) [69–71]. Projected sensitivities from Dune [72,73], Belle-II [74] and M3 [38] are also plotted. C. Background In the case of pion decays, this probability is strongly reduced because of For completeness to this study, muon electromagnetic interactions within the target also constitute a possible source of background, especially if visible decays of Z0 are inferred (typically Z0 →μþμ−). The main process is dimuons production through the emission of a real photon (Bethe-Heitler mechanism), μ−N →μ−Nγ; γ →μþμ−. Other mechanisms responsible for such dilepton produc- tion, although more suppressed, are the production of dimuon through a virtual photon (Trident process) or through highly energetic knock-on electrons (see e.g., Ref. [58–61]). The dimuon yield, suppressed by a factor 052006-5 PHYS. REV. D 105, 052006 (2022) H. SIEBER et al. 3 − 10 2 − 10 1 − 10 1 [GeV] Z' m 5 − 10 4 − 10 3 − 10 2 − 10 1 − 10 g' MOTs 11 10 μ NA64 Phase 1 3 M CCFR BBN BaBAR Borexino σ 2 ± μ (g-2) Belle II -1 Belle II 50 fb - μ + μ Dune XENON1T FIG. 6. Projected sensitivities for the invisible mode Z0 →¯νν in the ðmZ0; g0Þ parameter space for a total of 1011 MOT with the NA64μ experiment. The limits are given using the selection criteria (i)–(iv) and the requirement of the event lying in the signal box. The necessary ðmZ0; g0Þ values to explain the ðg −2Þμ anomaly are shown within the 2σ green band, as well as the possible parameters to cover the XENON1T excess [62]. Also shown are current experimental constraints from CCFR [63,64], BOREXINO [65,66], BABAR [67] and Belle-II [68], together with the cosmological constraints from the big bang nucleosyn- thesis (BBN) [69–71]. Projected sensitivities from Dune [72,73], Belle-II [74] and M3 [38] are also plotted. 3 − 10 2 − 10 1 − 10 1 [GeV] Z' m 5 − 10 4 − 10 3 − 10 2 − 10 1 − 10 g' MOTs 11 10 μ NA64 Phase 1 3 M CCFR BBN BaBAR Borexino σ 2 ± μ (g-2) Belle II -1 Belle II 50 fb - μ + μ Dune XENON1T TABLE I. Main sources of background and expected back- ground level per muons on target (MOT). C. Background Combining all of the above main sources of background, the experiment is expected to be background free at the level of ∼1011 MOTs (see Table I). IV. FUTURE SENSITIVITY TO THE ðg −2Þμ colliders with the BABAR [67] and Belle-II [68] experi- ments. As a comparison, projected sensitivities for Dune [72,73], Belle-II [74] and M3 [38] are plotted alongside our estimated limits. colliders with the BABAR [67] and Belle-II [68] experi- ments. As a comparison, projected sensitivities for Dune [72,73], Belle-II [74] and M3 [38] are plotted alongside our estimated limits. The signal yield of invisible decay of Z0 to SM neutrinos, Z0 →¯νν, can be estimated according to [37,40] such that Nð¯ννÞ Z0 ¼ NMOT · ρN A A · X i σtotðEiμÞΔLi · BrðZ0 →¯ννÞ; ð6Þ V. CONCLUSION In this work, we presented the expected sensitivity of the NA64μ experiment to a light Z0 boson coupled to muons as a remaining low mass explanation of the ðg −2Þμ muon anomaly. The minimal model based on the broken Uð1ÞLμ−Lτ symmetry is used as a benchmark in these studies. We focused on the optimization and design of the experimental setup for the first phase of the experiment dedicated to demonstrating the feasibility of the technique. The trigger efficiency and the detector hermeticity have been studied using a dedicated GEANT4-based MC simu- lation framework and a realistic M2 beam-optics simula- tion. A trigger using the scattered muon deflection after traversing the magnet spectrometer has been designed to keep the primary beam below 0.1% and a mass-dependent signal efficiency between 15%–35%. The low trigger efficiency for smaller masses is compensated by the mass dependence of the cross section. The main expected background sources arise from momentum misreconstruc- tion in the two magnet spectrometers, hadron contamina- tion and detector hermeticity. The results obtained from the simulation show that the background level is below 10−11 per MOT being dominated by the decay in flight of the remaining hadron contamination in the M2 beam line. where N A is the Avogadro number, ρ is the target density and A the target atomic weight, ΔLi is the ith step length of the muon with energy Eiμ within the target, and σtot the total cross section for Z0 emission. The 90% confidence level upper limit on g0 is calculated using Eq. (6), thus requiring Nð¯ννÞ Z0 > 2.3 events, in the ðmZ0; g0Þ parameter space. The corresponding results are shown in Fig. 6 for NMOT ¼ 1011 assuming the selection criteria (i)–(iv) and a single scat- tered muon with energy E0μ ≤80 GeV. Also shown is the values of g0 and mZ0 necessary to explain the muon ðg −2Þμ within the 2σ band. It can be seen that for NMOT ≥1011, the parameter space necessary to explain the muon anomalous magnetic moment is fully covered. Additionally, it is also shown that with 1011 MOT, NA64μ is also sensitive to the parameters space region explaining the XENON1T excess [62]. ACKNOWLEDGMENTS We also showed two methods which can potentially reduce this background by at least an order of magnitude. Finally, we studied the experiment projected sensitivities compared to present and future experiments aiming to perform similar searches. These first estimates based on simulations reveal that with 1011 MOT we can probe the region relevant to the ðg −2Þμ anomaly, obtaining the most sensitive coverage for masses below 200 MeV. The presented simulation results and calculations are to be validated in the scheduled pilot run. We acknowledge the members of the NA64 collabora- tion for fruitful discussions, in particular, N. V. Krasnikov. The work of P. Crivelli, E. Depero, L. Molina Bueno and H. Sieber is supported by ETH Zürich and SNSF Grants No. 169133, No. 186181, No. 186158 and No. 197346 (Switzerland). The work of D. V. 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Additionally, it is also shown that with 1011 MOT, NA64μ is also sensitive to the parameters space region explaining the XENON1T excess [62]. Nð¯ννÞ Z0 > 2.3 events, in the ðmZ0; g0Þ parameter space. The corresponding results are shown in Fig. 6 for NMOT ¼ 1011 assuming the selection criteria (i)–(iv) and a single scat- tered muon with energy E0μ ≤80 GeV. Also shown is the values of g0 and mZ0 necessary to explain the muon ðg −2Þμ within the 2σ band. It can be seen that for NMOT ≥1011, the parameter space necessary to explain the muon anomalous magnetic moment is fully covered. Additionally, it is also shown that with 1011 MOT, NA64μ is also sensitive to the parameters space region explaining the XENON1T excess [62]. For completeness, existing experimental bounds on Z0 from the gauge extension Lμ −Lτ theory are shown for neutrino trident production, νN →νNμμ, with the CCFR experiment [63,64], as well as from neutrino-electron scattering with the BOREXINO experiment [65,66]. 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Scr. 96, 084005 (2021). μ [67] J. P. Lees et al. (BABAR Collaboration), Search for a muonic dark force at BABAR, Phys. Rev. D 94, 011102 (2016). 052006-9
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GF CSANE Y L EAREN - - - - - - - - L I K F T TG S VQL DQL LQ - GGV E TGN I T E I FGE F R TGK T QL CH T L AV T C QL P V ECSGGE GK - C LW I D T E GT F - GF T T A T E F H QRRS E - - - - - - - - I I Q I T TG SKE L DK L LQ - GG I E TGS I T E MFGE F R TGK T Q I CH T L AV T C QL P I DRGGGE GK - AMY I D T E GT F - GF V T AAD F H MRRS E - - - - - - - - L I C L T TG SKN L D T L LG - GGV E TGS I T E L FGE F R TGKS QL CH T L AV T C Q I P L D I GGGE GK - C L Y I D T E GT F - GF CNA I DYH DARQN - - - - - - - - L I K F T TG SKQL DA L L K - GG I E TGG I T E L FGE F R TGKS QL CH T L A I T C QL P I EQSGGE GK - C LW I D T E GT F - GF CSAQE Y L EAREN - - - - - - - - L I R F T TG S VQL DS L L K - GG I E TGN L T E L FGE F R TGK T QL CH T L AV T C QL P I EQAGGE GK - C LW I D T E GT F - GF T RA T V FQ EQRKE - - - - - - - - T I MV T TG SRE VDK L LG - GG I E VGS I T E L FGE F R TGK T QL CH T L CV T C QL P L SQGGGE GM - A L Y I D T E GT F - GF CNA I DYH DARQN - - - - - - - - L I K F T TG SKQL DA L L K - GG I E TGG I T E L FGE F R TGKS QL CH T L A I T C QL P I EQSGGE GK - C LW I D T E GT F - GF CNA I DYH DARQN - - - - - - - - L I K F T TG SKQL DS L L K - GG I E TGG I T E L FGE F R TGKS QL CH T L A I T C QL P I EQSGGE GK - C LW I D T E GT F - GF CNA I DYH DARQN - - - - - - - - L I K F T TG SKQL DS L L K - GGV E TGG I T E L FGE F R TGKS QL CH T L A I T C QL P I EQSGGE GK - C LW I D T E GT F - GF CNA I DYH DARQN - - - - - - - - L I K F T TG SKQL DA L L K - GG I E TGG I T E L FGE F R TGKS QL CH T L A I T C QL P I EQSGGE GK - C LW I D T E GT F - GF CNA I DYH DARQN - - - - - - - - L I K F T TG SKQL DA L L K - GG I E TGG I T E L FGE F R TGKS QL CH T L A I T C QL P I EQSGGE GK - C LW I D T E GT F - GF CNA I DYH DARQN - - - - - - - - L I K F T TG SKQL DA L L K - GG I E TGG I T E L FGE F R TGKS QL CH T L A I T C QL P I EQSGGE GK - C LW I D T E GT F - GF CNA I DYH DARQN - - - - - - - - L I K F T TG SKQL DA L L K - GG I E TGG I T E L FGE F R TGKS QL CH T L A I T C QL P I EQSGGE GK - C LW I D T E GT F - GF CNAVDYH DARQN - - - - - - - - L I K F T TG SKQL DS L L K - GG I E TGG I T E L FGE F R TGKS QL CH T L A I T C QL P I EQSGGE GK - C LW I D T E GT F - GF CSAQE Y L EAREN - - - - - - - - L I R F T TG S VQL DS L L K - GG I E TGN L T E L FGE F R TGK T QL CH T L AV T C QL P I EQAGGE GK - C LW I D T E GT F - GF CSAQE Y L EAREN - - - - - - - - L I R F T TG S VQL DS L L K - GG I E TGN L T E L FGE F R TGK T QL CH T L AV T C QL P I EQAGGE GK - C LW I D T E GT F - GF CNA I DYH DARQN - - - - - - - - L I K F T TG SKQL DS L L K - GG I E TGG I T E L FGE F R TGKS QL CH T L A I T C QL P I EQSGGE GK - C LW I D T E GT F - GF CSGQDY L EARGN - - - - - - - - L I K F T TG S SQL DK L LQ - GGV E TGS I T E I I GE F K TGKS QL CH T L AV T C QL P V EQSGGE GK - C LWVDS E GT F - GF CSGQDY L EARGN - - - - - - - - L I K F T TG SAQL DK L LQ - GGV E TGS I T E I I GE F K TGKS QL CH T L AV T C QL P V EQSGGE GK - C LWVDS E GT F - GF CSGQDY L QARGN - - - - - - - - L I K F T TG S VQF D T L LQ - GG I E TGS I T E V I GE F K TGKS QL CH T L AV T C QL P V EQSGGE GK - C LW I DS E GT F - GF CSAHDY L EARGN - - - - - - - - L I K F T TG S VQL DV L L K - GG I E TGS I T E I I GE F K TGKS QL CH T L S V T C QL P V EQSGGE GK - C LW I D T E GT F - GF CSGT E Y L EAR T N - - - - - - - - L I K F T TG S SQL DR L LQ - GG I E TGS I T E I FGE F R TGK T QL CH T L AV T C QL P V EHKGGE GK - C LW I D T E GT F - GF CSGT E Y L EAR T N - - - - - - - - L I K F T TG S SQL DR L LQ - GG I E TGS I T E I FGE F R TGK T QL CH T L AV T C QL P V EHKGGE GK - C LW I D T E GT F - GF CSGS E Y L QAR T N - - - - - - - - L I R F T TG SKQL DR L LQ - GG I E TGN I T E I FGE F R TGK T QL CH T L AV T C QL P V EHNGGE GK - C LW I D T E GT F - GF C L A T N F L EARQN - - - - - - - - L I R F T TG S VD L DR I LQ - GG I E SGS I T E I FGE F R TGK T QL CH T L AV T C QL P VD L AGGD GR - C LW I D T E GT F - D FQT AAE Y Y VKRQS - - - - - - - - V I N L T TG S T E L DK L LG - GGF E TGS L T E I FGE F R TGK T Q I CH T L C I T C QL PKEKGGGE GK - AMY I D T E GT F - QF KPA T DV L KQRER - - - - - - - - I VH I S TG S T K F DK L L R - GG I E TGG I T E I FGE F R TGKS Q I CH T L AV T C QMNDGKGRPG GK - C L Y I D T E GT F - GF T SASQL H AQR L E - - - - - - - - I I QV T TG SRE L DK I L D - GG I E TGS I T E I YGE F RSGK T QL CH T L CV T C QL P L DQGGGE GK - A L Y I DAE GT F - GF T T AAAVA EQRKE - - - - - - - - V I S I T TG CKE L D T I L E - GG I E TGS I T E I YGE YRCGK T QL CH T L CV T C QL P V EMGGGE GK - AMY I D T E GT F - GF CSA F T CY QMRQD - - - - - - - - L I H L T TG SRE I DR I L K - GG I E TGS I T E L FGE F R TGK T Q I CH T L CV T C QL S I DQGGGE GR - V L Y I D T E GT F PA T - T AAS L L RSK L T NHSGT AQQNRQ I V S F SQS L DV L LG - GGVA L AE L T E I VGRPGSGK T QL TMQL CVDA R L P SKYGGV E GS - V V V I DAE GSW P V T K T AAA L L E ENV EGQGC - - - - - - - I I T F CRHVD T L LG - GG I AMGE L T E I AGP PGVGK T QWGMQL AVDA R L PN T FGGVA GE - T V Y VD T E GS F PAV - - SAR L L F EAERAQV - - - - - - - R I I T F S S E L DR L LG - GG I ARGQV T E F CGAPGLGK T Q I GMQL A I DV Q I PAV FGGL E GE - AV Y I - - E GS F PGCR T VREMH AE FQARQA - - QGF S T HV T T F SGE L DGV LG - GGV P VGGV T E I SGP PGVGK T QL LMQL AV SC AMP V E FGGMG GA - C L F VD T E GS F - - - - - - - - - - - - - - - - - - - - - - - V E T I S TG S L S L D I A LGA GGL PMGR I V E I YGP E S SGK T T L - - T LQV - - - - - I AAAQRE GK T CA F I DAE HA L 3 LGP I ANR F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - GL SHAECVA N I V Y VK - - - - V SN - - - - - - - L L L L Y T K I EN KGR T AA F TMK RP I T P EMSMG MNRNAPNNQE E F NKV L H F A I P L YW - - - - - - - - - - - - - - - - - - LMPAP S I E EA I YA F HP T F AYNE PKEGT N I I EHKRK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - SKNSR F KG AP LGPRE I AK H L R I YRP S E P T L - - - - - - - - - I L NQER F L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Y L R L F DHE E L D - I A L S E F YDA L V - - - - - - - - V VA I AKR F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - GL S PASC L E N I AYAK - 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- - - - - - I V S I AKR F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - GL NANDC L D NVAYAR - - - - AYN - - - - - - - I VA I AKRY - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - GL HP T DC L N N I AYAK - - - - AYN - - - - - - - I V S I AKR F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - GL S P SDC L D NVAYAR - - - - AYN - - - - - - - I V S I AKR F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - GL S P SDC L D NVAYAR - - - - AYN - - - - - - - I V S I AKR F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - GL S P SDC L D NVAYAR - - - - AYN - - - - - - - V V S I AKR F - - - - - - - - - - - - - - - - - - - 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- - - - - - - - - T HN L T RM AA F L T DMANE F D L AV VA - I N H L T T - R I DKD SNSN - GG - - - - - - - - SN T K - - - - - - - - - - - - - - - L V PA LG E SWAHS V T SR VGR - - - - - - - - - - T RS L T SQ AA F L T N L AAQ SG I AV VA - I N QMT T - KMT T S EAS - - - - - - - - - - - - - - - K - - - - - - - - - - - - - - - QV PA LG E SWAHAV T T R AP T PGT VAPA P PRSRQL AQL ASA L S S L A T R HHVAV V L - T N QL T T - R L T SA T E T P - LGGP S L PAPDAGAR - - - - - - - - - - - - - - - L V PA LG EGWL HAC T SR PNR L L SKHGL WRRSR L L FQC S T L L EGL AA T FQL A I V V - T N HMT T - KV L HG T AAN - GT ANG S SAE E SGCSA PNS E ENAGQR RQS V L V PA LG DAWGQGL S T R - - - - - - - - HM GL AARMMSQA MRK L AGN L KQ SN T L L I F - I N Q I RM - K I GVM FGNP E T T TGG NA L K F YAS VR L D I RR I GAVK EGENV - - VGS E T RVKV VKNK 6 L F L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KAR GNSRVCK - V Y NS P S L - - - - - - - - - - - - - - - P EGEAV F A I A EG - G I VDYDD AMY - 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I Y DS P S L - - - - - - - - - - - - - - - P EGEA I F C I S QG - G I KDCDD L Y L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GE T R I CK - I Y DS PC L - - - - - - - - - - - - - - - P EAEAMF A I N AD - GVGDAKD LGF K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGK GCQR L CK - V V DS PC L - - - - - - - - - - - - - - - P EAECV F A I Y ED - GVGDPRE L Y L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GE SR I CK - I Y DS P V L - - - - - - - - - - - - - - - P EGEAV F A I T EG - G I ADY E E L Y L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GE SR I CK - I Y DS P S L - - - - - - - - - - - - - - - AEGEAV F A I G EG - G I GDY ED L S L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GEQR I I K - V Y DS PC L - - - - - - - - - - - - - - - AE S EA I FG I Y EN - GVGDVRD L Y L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GE SR I CK - I Y DS P V L - - - - - - - - - - - - - - - P EGEAV F A I T EG - G I ADY E E L Y L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GE SR I CK - I Y DS P V L - - - - - - - - - - - - - - - P EGEAV F A I T EG - G I ADY E E L Y L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GE SR I CK - I Y DS P V L - - - - - - - - - - - - - - - P EGEAV F A I T EG - G I ADY E E L Y L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GE SR I CK - I Y DS P V L - - - - - - - - - - - - - - - P EGEAV F A I T EG - G I ADY E E L Y L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GE SR I CK - I Y DS P V L - - - - - - - - - - - - - - - P EGEAV F A I T EG - G I ADY E E L Y L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GE SR I CK - I Y DS P V L - - - - - - - - - - - - - - - P EGEAV F A I T EG - G I ADY E E L Y L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GE SR I CK - I Y DS P V L - - - - - - - - - - - - - - - P EGEAV F A I T EG - G I ADY E E L Y L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KSR GE SR I CK - I Y DS P V L - - - - - - - - - - - - - - - P EAEAV F A I T EG - G I ADY E E L Y L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GE SR I CK - I Y DS P S L - - - - - - - - - - - - - - - AEGEAV F A I G EG - G I GDY ED L Y L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GE SR I CK - I Y DS P S L - - - - - - - - - - - - - - - AEGEAV F A I G EG - G I GDY ED - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L F L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - QSK GE SR I CK - V Y DS P V L - - - - - - - - - - - - - - - P EGEAV F A I T DG - G I NDYHD L F L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - QSK GE SR I CK - I Y DS P V L - - - - - - - - - - - - - - - P EGEAV F A I T DG - G I NDYHD L F L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - QSK GE SR I CK - V Y DS P V L - - - - - - - - - - - - - - - P EGEAV F A I T DG - G I SDYQD L F L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - QSK GE SR I CK - I Y DS P V L - - - - - - - - - - - - - - - P EGEAV F S I T DG - G I SDYND L F L K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GE T R I CK - I Y DS PN L - - - - - - - - - - - - - - - P EGDA T F S I T EG - G I NDPKD L F L K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GE T R I CK - I Y DS PN L - - - - - - - - - - - - - - - P EGDA T F S I T EG - G I NDPKD L Y L K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR AE T R I CK - I Y DS PN L - - - - - - - - - - - - - - - P EGD T A F A I T EG - G I NDPND L YMK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGK GQNRVCK - I V DS PN L - - - - - - - - - - - - - - - P EAEAQF S I K EG - G I DKADN L Y L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GE SR I CK - I Y DS PC L - - - - - - - - - - - - - - - P E S EA I YA I G KG - G I ED F NE L Y L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GENR I VK - I Y DS PC L - - - - - - - - - - - - - - - P E S E EQY T I S PG - G I DDCAD L F L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR AE ER I CK - V V S S PC L - - - - - - - - - - - - - - - AEAEAR FQ I S P E - GV T DVKD L S VR - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KGR GENRV VK - I I AS P S L - - - - - - - - - - - - - - - P EREAN F A I G QE - GV T DAKD L Y L K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - KNK GNNR T CK - I Y DS PN L - - - - - - - - - - - - - - - P SNECV F S I S E L - G I GDP I D LM I DHYRH - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - PQF DMNE V - - RS V S - L L KS PHK - - - - - - - - - - - - - - - P PGT A L L L I T EK - G I RGV P S I L L SR - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P I SD T NG V - - R T C T - L V KS PR L - - - - - - - - - - - - - - - ASGSADYQ I L QC - G I RGVDA VR L EW - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - REG A - - RVAR - L E KS P S L - - - - - - - - - - - - - - - PGGCAA F DV T AE - GVRGVRS L L L S F HHYDV PACS F ADRP T S P SAA T EC T E DV V Y S L S SQQ QQL P T S L RRV VQHRV VR - V L KCSGQ - - - - - - - - - - - - - - - PRRE T C F AV T SK - G I RDARR I AAP F KQAE F Q I L YGEG I N F YGE L VD LGVK EK L I EKAGAW Y S YKGEK I GQ GKANA T AWL K DNP E T AKE I E KKVRE L L L SN PNS T PD F S VD DS EGVAE T NE 7 S S V - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - F - - - - - - - - - - - - V T H I DG - - - - - - - - - - - - - - - - - - KS L * - - - - - - - - - - - - - - KS L * - - - - - - - - - - - - - - KS L HS - - - - - - - - - - - - S - - - - - - - - - - - - - - - - - - ED - - - - - - - - - - - - - - - E K - - - - - - - - - - - - - - - - - N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K - - - - - - - - - - - - - - - - - K - - - - - - - - - - - - - - - - - K - - - - - - - - - - - - - - - - - K - - - - - - - - - - - - - - - - - K - - - - - - - - - - - - - - - - - K - - - - - - - - - - - - - - - - - K - - - - - - - - - - - - - - - - - K - - - - - - - - - - - - - - - - - N - - - - - - - - - - - - - - - - - N - - - - - - - - - - - - - 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https://openalex.org/W2918036813	https://cdr.lib.unc.edu/downloads/9019s476s	English	null	Journal of Neurodevelopmental Disorders reviewer acknowledgement 2015	Journal of neurodevelopmental disorders	2,016	cc-by	786	REVIEWER ACKNOWLEDGEMENT Open Access REVIEWER ACKNOWLEDGEMENT Journal of Neurodevelopmental Disorders reviewer acknowledgement 2012 Joseph Piven © 2013 Piven; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Contributing reviewers The editors of Journal of Neurodevelopmental Disorders would like to thank all of our reviewers who have contributed to the journal in volume 4 (2012). High quality and timely reviews are critical to the overall quality of the journal. We are committed to providing a unique and important outlet for scholarship regarding neurodevelopmental disorders and are indebted to the outstanding reviewers who have contributed their time over the last year in helping us to reach this goal. Piven Journal of Neurodevelopmental Disorders 2013, 5:5 http://www.jneurodevdisorders.com/content/5/1/5 Piven Journal of Neurodevelopmental Disorders 2013, 5:5 http://www.jneurodevdisorders.com/content/5/1/5 Contributing reviewers Joost Janssen Netherlands Warren Jones United States of America Emily Jones United States of America Annette Karmiloff-Smith United Kingdom Cary Kogan Canada Gregor Kohls United States of America Meng-Chuan Lai United Kingdom Amir Levine United States of America Alice Lin United States of America Rhiannon Luyster United States of America Carla Mazefsky United States of America James McPartland United States of America Joost Janssen Netherlands Warren Jones United States of America Emily Jones United States of America Annette Karmiloff-Smith United Kingdom Cary Kogan Canada Gregor Kohls United States of America Meng-Chuan Lai United Kingdom Amir Levine United States of America Alice Lin United States of America Rhiannon Luyster United States of America Carla Mazefsky United States of America James McPartland United States of America Melissa Allen United Kingdom Elizabeth Aylward United States of America Margaret Bauman United States of America Raphael Bernier United States of America Elina Birmingham Canada Dorothy Bishop United Kingdom Susan Bookheimer United States of America Marc Brysbaert Belgium Linda Campbell Australia Neva Corrigan United States of America Ana Cubillo United Kingdom Geraldine Dawson United States of America Saumitra Deb United Kingdom Manny DiCicco-Bloom United States of America Maria Luz Dizon United States of America Jed Elison United States of America Deborah Fidler United States of America Carsten Finke Germany Chris Frith United Kingdom Susanna Fryer United States of America Dennis Grayson United States of America Paul Hagerman United States of America Francesca Happe United Kingdom Todd Hare Switzerland Correspondence: jpiven@med.unc.edu Carolina Institute for Developmental Disabilities, University of North Carolina at Chapel Hill, 100 Renee Lynne Court, Carrboro, NC 27510, USA Joost Janssen Netherlands Warren Jones United States of America Emily Jones United States of America Annette Karmiloff-Smith United Kingdom Cary Kogan Canada Gregor Kohls United States of America Meng-Chuan Lai United Kingdom Amir Levine United States of America Alice Lin United States of America Rhiannon Luyster United States of America Carla Mazefsky United States of America James McPartland United States of America Melissa Allen United Kingdom Geraldine Dawson United States of America © 2013 Piven; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Page 2 of 2 Piven Journal of Neurodevelopmental Disorders 2013, 5:5 http://www.jneurodevdisorders.com/content/5/1/5 Catherine Stoodley United States of America Bruce Tomblin United States of America Anna Tury United States of America Jaap van Pelt Netherlands Katarina Varnäs Sweden David Walker Australia Karli Watson United States of America Zhengang Yang China doi:10.1186/1866-1955-5-5 Cite this article as: Piven: Journal of Neurodevelopmental Disorders reviewer acknowledgement 2012. Journal of Neurodevelopmental Disorders 2013 5:5. Catherine Stoodley United States of America Bruce Tomblin United States of America Anna Tury United States of America Jaap van Pelt Netherlands Katarina Varnäs Sweden David Walker Australia Karli Watson United States of America Zhengang Yang China Nancy Minshew United States of America Sandra Mooney United States of America Jeff Munson United States of America Charles Nelson United States of America Joshua New United States of America Richard Nowakowski United States of America John O'Doherty United States of America Noriko Osumi Japan Daniela Plesa Skwerer United States of America Armin Raznahan United States of America Allan Reiss United States of America Lawrence Reynolds United States of America Todd Richards United States of America Cary Savage United States of America Gaia Scerif United Kingdom Stephen Scherer Canada Emily Simonoff United Kingdom Mikle South United States of America Nancy Minshew United States of America Sandra Mooney United States of America Jeff Munson United States of America Charles Nelson United States of America Joshua New United States of America Richard Nowakowski United States of America John O'Doherty United States of America Noriko Osumi Japan Daniela Plesa Skwerer United States of America
https://openalex.org/W3196565435	http://izvestia-soigsi.ru/izvestia/2019/33/narbut-krashennikov.pdf	Russian	null	DONOR TEXTS CORPUS IN THE TRANSLATION RECEPTION OF KOSTA KHETAGUROV'S CREATIVITY	Izvestiâ SOIGSI	2,019	cc-by-sa	2,540	КОРПУС ТЕКСТОВ-ДОНОРОВ В ПЕРЕВОДЧЕСКОЙ РЕЦЕПЦИИ ТВОРЧЕСТВА КОСТА ХЕТАГУРОВА Е. В. Нарбут А. Е. Крашенинников В статье предлагается новый подход для теоретического осмысления опыта художе- ственного перевода поэзии Коста Хетагурова, а также для практического применения в дальнейшей переводческой практике. Категория текстов-доноров рассматривается как база для создания новых текстов. Разработчики данного подхода исходят из идеи принципиальной интертекстуальности художественного перевода и учета образцов ху- дожественной речи в принимающей культуре. Текст-донор определятся как текст или совокупность текстов близкой тематики (гипертекст), которые могут послужить для переводчика источником решения задач, возникающих при работе с соответствующим оригиналом. Ключевые слова: осетинская литература, глобализация, национальная литература, художественный перевод, текст-донор, интертекстуальность, адекватность, эквива- лентность. Сохранение и развитие национальных литератур – это совсем не праздный во- прос. Он напрямую связан с теми вызова- ми, с которыми сталкиваются народы и их культуры в процессе глобализации. Для на- шей страны он имеет бóльшую важность, чем для многих иных стран, так как Россия – многонациональное государство, в кото- ром явлены признаки не только современ- ной культуры, но и традиционных культур народов нашей страны. Поэтому в культурной палитре России осетинская литература является неотъем- лемой частью, которая развивается, взаи- модействует с другими культурами наро- дов России и представляет интерес не толь- ко для широкого круга читателей, но и для научного, в частности филологического, анализа. Об этом свидетельствуют много- численные статьи, монографии, диссерта- ционные исследования, имеющие продол- жительную историю. В этой связи можно вспомнить «Очерк развития осетинской литературы» Х. Н. Ардасенова, вышедший в далеком 1959 г. [2], а из современных – диссертацию «Осетинская поэзия и рус- ская литература 30-40-х гг. XX в.» Л. Л. Са- накоевой [3], а также сборник материалов международной конференции «Коста и мировой историко-культурный процесс», посвященной 155-летию со дня рождения Коста Хетагурова [4]. Сохранение и развитие национальных литератур – это совсем не праздный во- прос. Он напрямую связан с теми вызова- ми, с которыми сталкиваются народы и их культуры в процессе глобализации. Для на- шей страны он имеет бóльшую важность, чем для многих иных стран, так как Россия – многонациональное государство, в кото- ром явлены признаки не только современ- ной культуры, но и традиционных культур народов нашей страны. Так, в своих размышлениях об исто- рическом дискурсе России в рамках дихо- томии многонациональная империя – на- циональное государство Х. Х. Соблиров приходит к выводу, что на территории страны продолжается процесс националь- ного строительства государства на основе россиян, т.е. на основе всех народов Рос- сии [1]. СОИГСИ СОИГСИ Языкознание. Литературоведение. Фольклористика DOI: 10.23671/VNC.2019.72.35262 КОРПУС ТЕКСТОВ-ДОНОРОВ В ПЕРЕВОДЧЕСКОЙ РЕЦЕПЦИИ ТВОРЧЕСТВА КОСТА ХЕТАГУРОВА В этой связи безусловно возрастает роль межкультурного диалога, без которо- го невозможно крепкое объединение наро- дов хоть под крышей многонациональной империи, хоть под крышей, разумеется, не просто национального, а многонациональ- ного государства. Л. Л. Санакоева отмечает, что история становления и развития осетинской ли- тературы, а также творческие биографии авторов весьма характерны для современ- ного литературного процесса. Данный про- 73 ИЗВЕСТИЯ СОИГСИ 33 (72) 2019 Языкознание. Литературоведение. Фольклористика СОИГСИ цесс характеризуется несколькими тенден- циями. Это, с одной стороны, тесная связь с традициями собственной литературы, с художественными произведениями, ко- торые созданы на родном языке. С другой стороны, опора на традиции не исключает ориентированность на мировой литератур- ный опыт и усвоение, а затем и творческое воплощение традиций более развитых ли- тератур. Санакоева подчеркивает, что осе- тинская литература, видные ее представи- тели тесно связаны с классической русской литературой и культурой, а Коста Хетагу- ров, родоначальник осетинской литерату- ры, создавал свои произведения как на осе- тинском, так и на русском языках. тагурова на русский язык может, как нам представляется, корпус текстов-доноров. Категория текстов-доноров рассматри- вается нами в аспекте «текст – база для соз- дания новых текстов». Несмотря на то, что за многовековую историю художественного перевода возникло и развивается разно- образное количество приемов и методов, нацеленных на достижение адекватности и эквивалентности перевода в самых раз- нообразных комбинациях языков, метод использования речевых ресурсов корпуса текстов-доноров может помочь переводчи- ку обеспечить соответствие переводного текста оригиналу на уровне содержания и формы. Исходя из идеи принципиальной интертекстуальности художественного пе- ревода и учета образцов художественной речи в принимающей культуре, высказан- ной Л. С. Макаровой [7, 11], мы определя- ем текст-донор как текст или совокупность текстов близкой тематики (гипертекст), ко- торые могут послужить для переводчика источником решения задач, возникающих при работе с соответствующим оригиналом. Значение Коста Хетагурова для осетин- ской культуры и культуры России трудно переоценить. Поэтому его творчество на осетинском языке представляет большой интерес в аспекте художественного пере- вода с осетинского языка на русский. При этом перед переводчиком стоит сложная задача, связанная с самобытностью его творчества: при попытке найти подходя- щие переводческие решения нужны, по сути дела, глубокие знания осетинского языка и особое поэтическое чутье [5]. Как отмечал Н. Языкознание. Литературоведение. Фольклористика – Ушаков Николай Николаевич: «Кому что…», «Синица»; – Ушаков Николай Николаевич: «Кому что…», «Синица»; – Ушаков Николай Николаевич: «Кому что…», «Синица»; – Иринин Борис: « В пастухах», «Горе», «Желание», «Мать сирот», «Надежда», «Тревога»; – Фроман Михаил Александрович: «Без доли» «В разлуке»; – Исаковский Михаил Васильевич: «Знаю», «Тревога», «У могилы» («У гроба»); – Исаковский Михаил Васильевич: «Знаю», «Тревога», «У могилы» («У гроба»); – Шпирт Александр Исаакович: «Будь мужчиной», «В новогоднюю ночь», «Вса- ти», «Дума», «Зима», «Киска», «Олень и еж», «Осень», «Раздумье», «Редька и мед», «Хе- таг» (совместно с П. Панченко), «Шалун», «Школьник». – Шпирт Александр Исаакович: «Будь мужчиной», «В новогоднюю ночь», «Вса- ти», «Дума», «Зима», «Киска», «Олень и еж», «Осень», «Раздумье», «Редька и мед», «Хе- таг» (совместно с П. Панченко), «Шалун», «Школьник». – Кедрин Дмирий Борисович: «Мужчи- на или женщина?», «Прощай»; – Корчагин Александр Иванович: «О, если бы!»; – Липкин Семен Израилевич: «Безум- ный пастух», «О, если бы!»; Необходимо отметить, что огромную помощь при каталогизации ядра корпуса текстов-доноров оказал ресурс «Речь и речь нерасторжимы…» (I часть. Осетинская по- эзия в переводах русских поэтов) [9]. Ав- тор проекта и составитель И. А. Хайманова проделала удивительную, кропотливую ра- боту, которая стала не только подспорьем для исследователей, но и познавательным чтением для любого ценителя поэзии и ху- дожественного перевода. – Озеров Лев Адольфович: «А-ло-лай», «Без пастуха», «Будь мужчиной», «В но- вогоднюю ночь», «Дума жениха», «Если б запеть мне…», «Зима», «Кто ты?», «Лето», «Песня бедняка», «Привет», «Прощай», «Синица», «Шалун»; – Олендер Семен: «А-лол-лай», «Безум- ный пастух», «Волк и журавль», «Ворона и лисица», «Гуси», «Дума жениха», «Если бы я пел, как нарт вдохновенный…», «Желание», «Лиса и барсук», «О чем?», «Походная песня», «Привет», «Солдат», «У могилы», «Упрек»; Со своей стороны мы можем добавить в ядро корпуса текстов-доноров Елену Александровну Благинину, П. Сосиева (к сожалению, нам не удалось уточнить его имя и отчество) и их переводы поэмы «Кто ты?». – Панченко Павел Михайлович: «Вес- на», «Взгляни!..», «Завещание», «Кубады», «Ласточка», «Новогодняя песня», «Посла- ние», «Песня бедняка», «Постник», «Про- пади!..», «Тоска влюбленного», «Хетаг» (со- вместно с А. Шпиртом); – Панченко Павел Михайлович: «Вес- на», «Взгляни!..», «Завещание», «Кубады», «Ласточка», «Новогодняя песня», «Посла- ние», «Песня бедняка», «Постник», «Про- пади!..», «Тоска влюбленного», «Хетаг» (со- вместно с А. Шпиртом); К периферии корпуса текстов-доноров для переводчика поэзии Коста Хетагурова можно отнести различные качественные переводы других авторов осетинской поэ- зии. СОИГСИ Языкознание. Литературоведение. Фольклористика КОРПУС ТЕКСТОВ-ДОНОРОВ В ПЕРЕВОДЧЕСКОЙ РЕЦЕПЦИИ ТВОРЧЕСТВА КОСТА ХЕТАГУРОВА Джусойты – осетинский поэт, драматург, публицист, литературовед, переводчик, народный писатель Осетии – при художественном переводе необхо- димо умение адекватно и художественно передавать форму и содержание подлин- ника средствами переводящего языка, не говоря уже о трудностях, связанных с ритмикой и силлабо-тонической струк- турой осетинского стихосложения, чья традиция была создана и прочно утвер- ждена именно поэтическим творчеством Коста Хетагурова. А это в случае с Коста Хетагуровым очень сложно: «Коста – поэт, теряющий в переводе независимо от ран- га переводчика столько, что он начинает походить на Антея, оторванного от мате- ри-земли» [6, 47]. В данной работе мы предприняли первые шаги, связанные с применением текста-донора, в изучении опыта художе- ственного перевода лирики Коста Хетагу- рова на русский язык. Ядром корпуса текстов-доноров, кото- рые могут помочь потенциальному пере- водчику, являются уже созданные перево- ды поэзии Коста Хетагурова. И в первую очередь необходимо назвать переводы, сде- ланные Анной Ахматовой. Хотя сама поэ- тесса относилась к переводческой деятель- ности скептически, ее переводы обладают непреходящей ценностью, о чем свидетель- ствует тот факт, что не так давно во Влади- кавказе вышел в свет сборник ее переводов из осетинской поэзии [8]. К ядру корпуса также необходимо отне- сти тексты следующих переводчиков: – Брик Борис Ильич: «Зима», «Лето», «Привычка», «Сердце бедняка», «Солдат»; – Заболоцкий (Заболотский) Николай Алексеевич: «На кладбище»; Значительно помочь переводчику на сложном пути перевода лирики Коста Хе- ИЗВЕСТИЯ СОИГСИ 33 (72) 2019 74 ИЗВЕСТИЯ СОИГСИ 33 (72) 2019 74 Языкознание. Литературоведение. Фольклористика Напрямую не связанные с творчеством Коста Хетагурова, они позволят перевод- чику впитать колорит осетинского поэти- ческого языка, а также помогут в сложном случае определиться в собственном выборе того или иного эквивалента. – Потапова Вера Аркадьевна: «Сердце бедняка»; – Рождественский Всеволод Алексан- дрович: «Горе»; – Семынин Петр Андреевич: «Ласточ- ка», «Песня бедняка»; – Серебряков Борис Яковлевич: «Без пастуха», «Песня бедняка»; – Синельников Михаил Исаакович: «Безумный пастух», «Знаю», «Сердце бед- няка»; Дальнейшее изучение корпуса тек- стов-доноров в области художественного перевода позволит, на наш взгляд, не толь- ко расширить наше представление о фе- номене текстов-доноров, но и сформули- ровать более определенные рекомендации для переводчиков. – Тихонов Николай Семенович: «Вес- на», «Зима», «Лето», «На кладбище», «Олень и еж», «Осень», «Походная песня», «Прислужник»; ИЗВЕСТИЯ СОИГСИ 33 (72) 2019 75 ИЗВЕСТИЯ СОИГСИ 33 (72) 2019 75 Языкознание. Литературоведение. Фольклористика СОИГСИ 1. Соблиров Х.Х. Исторический дискурс о России: «Многонациональная империя» или «Национальное государство» // Научные проблемы гуманитарных исследований. 2009. №10-2. С. 104. 1. Соблиров Х.Х. Исторический дискурс о России: «Многонациональная империя» или «Национальное государство» // Научные проблемы гуманитарных исследований. 2009. №10-2. С. 104. 2. Ардасенов Х.Н. Очерк развития осетинской литературы. Орджоникидзе, 1959. 3. Санакоева Л.Л. Осетинская поэзия и русская литература 30–40-х гг. XX в.: Вопросы перевода и литературных взаимосвязей: Дис... канд. филол. наук. Владикавказ, 2002. 3. Санакоева Л.Л. Осетинская поэзия и русская литература 30–40-х гг. XX в.: Вопросы перевода и литературных взаимосвязей: Дис... канд. филол. наук. Владикавказ, 2002. 4. Коста и мировой историко-культурный процесс: Сборник материалов Междуна- родной конференции, посвященной 155-летию со дня рождения К.Л. Хетагурова. Влади- кавказ, 2014. 5. Томеллери В.С., Сальватори М. Несколько соображений о переводе «Осетинской лиры» Коста на итальянский язык // Известия СОИГСИ. 2013. Вып. 10(40). С. 10-19. 5. Томеллери В.С., Сальватори М. Несколько соображений о переводе «Осетинской лиры» Коста на итальянский язык // Известия СОИГСИ. 2013. Вып. 10(40). С. 10-19. 6. Джусойты Н. Коста Хетагуров // Коста Хетагуров. Стихотворения и поэмы. Вступ. статья, составление, подготовка текста и примечания Н. Джусойты. Л., 1976. С. 5-48. 6. Джусойты Н. Коста Хетагуров // Коста Хетагуров. Стихотворения и поэмы. Вступ. статья, составление, подготовка текста и примечания Н. Джусойты. Л., 1976. С. 5-48. 7. Макарова Л.С. Коммуникативно-прагматические основы художественного перево- да. М., 2004. 7. Макарова Л.С. Коммуникативно-прагматические основы художественного перево- да. М., 2004. 8. Ахматова А. Переводы из осетинской поэзии. Владикавказ, 2015. 8. Ахматова А. Переводы из осетинской поэзии. Владикавказ, 2015. 9. «Речь и речь нерасторжимы…» I часть. Осетинская поэзия в переводах русских по- этов [электронный ресурс]. DONOR TEXTS CORPUS IN THE TRANSLATION RECEPTION OF KOSTA KHETAGUROV’S CREATIVITY DONOR TEXTS CORPUS IN THE TRANSLATION RECEPTION OF KOSTA KHETAGUROV’S CREATIVITY DONOR TEXTS CORPUS IN THE TRANSLATION RECEPTION OF KOSTA KHETAGUROV’S CREATIVITY Keywords: Ossetian literature, globalization, national literature, literary translation, donor text, intertextuality, adequacy, equivalence. The article offers a new approach to theoretical understanding of the experienceof literary translation of KostaKhetagurov’s poetry as well as to practical application in further translation practice. The category of donor texts is considered to be the basis for creating new texts. The developers of this approach proceed from the idea of the fundamental intertextuality of literary translation and considering the patterns of artistic speech in the receiving culture. A donor text is defined as a text or a set of texts of a similar subject (hypertext) which can serve as a source for the translator in solving problems arising while working with the corresponding original. Языкознание. Литературоведение. Фольклористика URL: http://nslib.tmweb.ru/perevodi/index.html 8. Ахматова А. Переводы из осетинской поэзии. Владикавказ, 2015. 9. «Речь и речь нерасторжимы…» I часть. Осетинская поэзия в переводах русских по- этов [электронный ресурс]. URL: http://nslib.tmweb.ru/perevodi/index.html Narbut, Elena V. – Northeastern State University; narbut@rambler.ru Narbut, Elena V. – Northeastern State University; narbut@rambler.ru Krasheninnikov, Andrey E. – Northeastern State University; narbut@rambler.ru Krasheninnikov, Andrey E. – Northeastern State University; narbut@rambler.ru REFERENCES 1. Soblirov Kh.Kh. 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Poems. Entry article, compilation, preparation of the text and notes by N. Dzhusoity]. Leningrad, Sovetskiy pisatel’, Leningradskoye otdeleniye, 1976, pp. 5-48. 7. Makarova L.S. Kommunikativno-pragmaticheskiye osnovy khudozhestvennogo perevoda [Communicative-pragmatic foundations of literary translation]. Moscow, Moscow State Regional University, 2004. 256 p. 7. Makarova L.S. Kommunikativno-pragmaticheskiye osnovy khudozhestvennogo perevoda [Communicative-pragmatic foundations of literary translation]. Moscow, Moscow State Regional University, 2004. 256 p. y 8. Akhmatova A. Perevody iz osetinskoy poezii [Translations from Ossetian poetry]. Vladikavkaz, Ir, 2015. 51 p. 8. Akhmatova A. Perevody iz osetinskoy poezii [Translations from Ossetian poetry]. Vladikavkaz, Ir, 2015. 51 p. 9. “Rech’ i rech’ nerastorzhimy…» I chast’. Osetinskaya poeziya v perevodakh russkikh poetov [“Speech and speech are indissoluble...” Part I. Ossetian poetry in translations of Russian poets]. [electronic resource]. URL: http://nslib.tmweb.ru/perevodi/index.html 9. “Rech’ i rech’ nerastorzhimy…» I chast’. Osetinskaya poeziya v perevodakh russkikh poetov [“Speech and speech are indissoluble...” Part I. Ossetian poetry in translations of Russian poets]. [electronic resource]. URL: http://nslib.tmweb.ru/perevodi/index.html ИЗВЕСТИЯ СОИГСИ 33 (72) 2019 77 ИЗВЕСТИЯ СОИГСИ 33 (72) 2019 77
W3011934113.txt	https://content.sciendo.com/downloadpdf/journals/rtuect/24/1/article-p162.pdf	en		Calculation of Kinetic Parameters of Thermal Decomposition of Forest Waste using the Monte Carlo Technique	Environmental and Climate Technologies	2,020	cc-by	3,718	Environmental and Climate Technologies 2020, vol. 24, no. 1, pp. 162–170 https://doi.org/10.2478/rtuect-2020-0010 https://content.sciendo.com Calculation of Kinetic Parameters of Thermal Decomposition of Forest Waste using the Monte Carlo Technique Alok DHAUNDIYAL1, Laszlo TOTH2 1,2Institute of Process Engineering, Doctoral School of Mechanical Engineering, Szent István University, Gödöllő 2100, Hungary Abstract – This paper deals with the pyrolysis of forest waste in the presence of an inert atmosphere. Experiments are carried out at different heating rates (5 °C, 10 °C and 15 °C) to determine derivative thermogravimetric behaviour of the material. Unlike the conventional scheme, the Monte Carlo technique is implemented to solve the distributed activation energy model (DAEM). DAEM is transformed into the inverse pyrolysis problem to determine the kinetic parameters of thermal degradation of forest waste. Activation energy, the preexponential factor and the distribution parameters are estimated by introducing the Monte Carlo Technique in the thermal conversion process. Keywords – Biomass; distributed activation energy model; Monte Carlo technique; pyrolysis. 1. INTRODUCTION Modelling scheme of biomass pyrolysis can be performed on the basis of single reaction and multi-reaction models. Comprehensive details of these models are reviewed in previous research [1]–[4]. The latest and up-to-date approach to describe biomass pyrolysis pivots around the distributed activation energy model [5]–[7]. The numerical solution of DAEM is used to estimate kinetic parameters. The DAEM is converted into an inverse problem, so that realistic results can be derived. As the trade-off relationship between material decomposition and mathematical model is fail to impart the robustness in the results, therefore the Monte Carlo scheme is adopted to make the solution more pragmatic. Some of the latest developments and the effect of various parameters on the single-reaction model are investigated by Brown [8]. Gune and Gune [9] reported the effect of miscellaneous parameters on the behaviour of first Order DAEM. Dhaundiyal et al. [10], [11] performed a comprehensive analysis of various density functions to predict the nature of thermal decomposition of biomass through DAEM. In this study, kinetic parameters are determined by using the Monte Carlo scheme. The primary objective of this paper is to introduce a new method of solving in -exact integrals encountered while solving the solid-state kinetic models. It is assumed that the approximated solution forms an injective set. The Gaussian function is considered to be the initial distribution function. Only with the help of derivative thermogravimetric (DTG) analysis, the kinetic parameters of thermal degradation of forest waste is obtained. Corresponding author. E-mail address: alok.dext@hotmail.com ©2020 Alok Dhaundiyal, Laszlo Toth. This is an open access article licensed under the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0), in the manner agreed with Sciendo. 162 Environmental and Climate Technologies ____________________________________________________________________________ 2020 / 24 2. NON-ISOTHERMAL DAEM AND ITS NUMERICAL SOLUTION DAEM is one of the multi-reaction models. The postulations, restriction and derivation of the non-isothermal n th DAEM equations can be found in literature [12], [13]. Non-isothermal equations for the first order as well as n th DAEM are given below:   t 0  0 (1 −  ) = exp  −  A  −E exp    RT    dt  f ( E ) dE ,   (1) 1  t A   1−n −E (1 −  )n1 = 1 − (1 − n )  exp   dt  f ( E ) dE ,    RT    0 0    (2) where E activation energy; α conversion of biomass; A frequency factor; R gas constant; t time; T temperature; N reaction order; f(E) density function of the activation energy of the volatile release. In this case, the function f(E) represents density function of the Gaussian distribution. The approximated solution of equations (1) and (2) can be done using the asymptotic technique. Therefore, the whole integral comprises two different kinds of density functions. The first term of DAEM is density function of temperature distribution, or temperature integral, whereas the density function of activation energy forms the second part of computational process. On the basis of these demarcations, the approximation is performed. The asymptotic form of the mean (Es) and   the step width (Ew) of the temperature integral exp  A exp  − E   is derived for the linear ramp    RT   t  0  rate of temperature as mentioned in the study [13], thus, Es = RTY (  ) and Ew = Es , applying 1 + Y ( ) the energy scaling and introducing the energy correction factor, we get ys = E Es and yw = w . E0 E0 (3) Therefore, the approximated form of temperature integral is:    ys − y    t A  −E   exp   exp     ,   ~ exp  −exp    RT y   w   0     where E₀ Y(τ) τ = A.t (4) mean value of activation energy; Lambert W function; time-scale factor. 163 Environmental and Climate Technologies ____________________________________________________________________________ 2020 / 24 Equations (1) and (2) are simplified by incorporating functional expressions of the temperature integral and the distribution of activation energies.    0     ys − y     2     exp − ( y − 1) dy  y w     ) ( (1 −  ) ~ exp  −exp  −exp   (5) Similarly, for nth order reactions apply the binomial expansion on the equation (2): (1 −  )n1 ~        ys − y    ys − y   n  2  +   exp  2    + exp − ( y − 1) dy , yw   2  y   w   (  1 − exp  0 ) (6) E0 2 is a constant and σ is standard deviation of Gaussian distribution function. 2 2 Using the Laplace asymptotic scheme for the interior points of the integral ‘dy’ (Equation (5) and Equation (6)), we get a leading behaviour of the remaining mass fraction of the volatile (1-α) for the first and n th order reactions. where  = (1 −  ) ~ (1 −  )n1 ~1 − 1 2 exp ( − ( ye − 1)( ye + 2 yw − 1) ) (7)  y −1  1+  e   yw  n   exp −  ( ye,n − 1)( 2 ys − ye,n − 1) + 2 exp −2 ( ye,n − 1)( ys − ye,n )    ( ) ( ) (8) Equations (7) and (8) are the desired expressions for the first and nth order reactions. The values of ye, and ye,n and yʹe,n represent the points where the exponents of the exponential term in equations (5) and (6) attain maximum values, respectively.  1  y −1   ye = 1 + ywY  exp  s    2y 2   yw   w  (9) 1 2yw (10) ye ,n = 2 ye,n −1 (11) ye,n = 1 − 3. MONTE-CARLO SCHEME The Monte Carlo method is a computational technique used to obtain an unknown parameter. It is a fundamental tool to calculate and simulate a set of maps which are independent and identically distributed with the same distribution as the set of all maps [14]. Guo et al. [15] reported that the evolution of bonds in pyrolysis could be simulated by combining the Boltzmann-Monte-Carlo algorithm with the percolation theory. In pyrolysis problem of coal, the Monte Carlo algorithm delineates the bond formation by using coupling 164 Environmental and Climate Technologies ____________________________________________________________________________ 2020 / 24 or collision of radical fragment [15]. The concept of Monte-Carlo analysis is to understand the uncertainty in the system through random sampling. Random number generator assigns a random value to each variable within the demarcated limit. Thus, it helps to develop a robust scheme to solve the inverse problem. Mainly, the performance of any given model is carried out by the deterministic value of parameters, but Monte Carlo is very effective for those models that have no analytical solutions. However, sometimes deterministic approach ma y not be able to delineate the actual process, therefore it is necessary to enhance the decision ability of the model and the gap between numerical approximations and analytical solutions can be abridged. In this study, the area of the DTG curve is calculated by using the MonteCarlo scheme. The MATLAB software is used to generate random particles of 17 to 20 million, so that the approximated area is nearly equal to the area bounded by the DTG plane. The area under the DTG curve is estimated by using binary logic matrices, which helps to determine the number of particles enclosed by the DTG curves, or the number of particles bombarded on the DTG plane. Programming for computing the area under the curve is performed on the MATLAB interface. m   r  dm  1      dT  ( m0 − mr )  mt  dT    p  n .A   (12) Here: p inbound particles; n total number of particles bombarded on the DTG plane; A area under the DTG plane; m0, mt and mr are the initial, instantaneous and residual mass of biomass respectively. 4. APPLICATION OF BIOMASS Thermal degradation of pine waste is conducted in the presence of inert gas (Nitrogen) by using the thermogravimetry technique. The volumetric flow rate of t he gas is 200 mL/min. The crucible holder of Alumina (Al 2O3) is used to hold the sample inside the heating chamber. The thermocouple type ‘R’ is used to measure the temperature of the sample. The derivative thermogravimetric data is used to explore the kinetic parameters of thermal degradation of pine samples. The test is conducted at the heating rates of 5 °C/min, 10 °C/min and 15 °C/min. The chemical composition of the pine samples is shown in Table 1. TABLE 1. CHEMICAL COMPOSITION OF PINE NEEDLES WITH HIGH HEATING VALUE (H.H.V) C, % 54.28 H, % 6.55 N, % 0.61 O, % 35.50 Ash content, % 2.92 S, % 0.11 H.H.V, MJ/kg 20.65 5. RESULTS AND DISCUSSION Mathematical computation of the kinetic model is performed by using the Monte Carlo simulation method. The given pyrolysis problem is converted into the inverse form assuming the non-surjective nature of the co-domain of kinetic parameters. The estimated range of variation of activation energy is distinguished by its integral limit. The Brownian movements of bombarded particles on the DTG plane at different heating r ates are illustrated in Fig. 1, 165 Environmental and Climate Technologies ____________________________________________________________________________ 2020 / 24 Fig. 2, and Fig. 3. The number of inbound particles (p) is used to determine the area (Equation (12)) enclosed by DTG curves at the heating rates of 5 °C, 10 °C and 15 °C. The estimated areas under the DTG curve at different heating rates are shown in Table 2. It is assumed that the significant behaviour of total integrand (Equation (1) and (2)) is about the central value (Es) of temperature integral, thus it reflects the activation energy required to initiate the thermal decomposition of the pine waste. The upper limits of activation energies are estimated to be 154.47 kJ/mol, 144.62 kJ/mol and 147.64 kJ/mol at the heating rates of 5 °C/min, 10 °C/min and 15 °C/min for first-order reactions, respectively, whereas the upper range of activation energies for nth order reactions lies in the domain of 66.20 kJ/mol to 69.56 kJ/mol (Table 3). Frequency factors calculated using the proposed scheme lie in between 10 4 and 10 7 min−1. The activation energy distribution (on the basis of mean value) among the different stages of decomposition of pine needles is illustrated in Fig. 4. It is indicated that the energy required during the dehydration phase of pine needles vary from 25 kJ/mol to 50 kJ/mol, whereas the activation energy rises steadily until the char formation. The activation energy increases by 40 % till the end of 58 % conversion of pine waste. The activation energies of sigmoidal reactions get changed due to the variation of the heat of reactions (exothermic to endothermic). Unlike the drift in the activation energy at the end of dehydration of the pine needle sample, it is marginally very low at the onset of the char formation stage. The activation energies of pine needles during char formation gets increased by 9 %. The results are compared with different model-free methods and multi-component devolatisation mechanisms, and it has been found that the derived solution provided the same solution with comprehensive details of the variation of activation energies caused by temperature integral , as well as the density function. The average variation of activation energy due to the density function varies from 66 kJ/mol to 76 kJ/mol, whereas the approximated solution of temperature integral has a significant role in deciding the overall activation energy of the pine-needles, as both the functions are intertwined in the distributed activation energy model, therefore it is necessary to demarcate the individual merit of them. Fig. 1. Brownian motion of inbound and out bound particles on the DTG plane at 5 °C/min. The significant contribution of the double exponential term to decide the shape and amplitude of the resultant curve is relatively high to the density function (the Gaussian distribution). Thus, the solution converges at the energy scaling factor ( y) from 1.3 to 1.4. 166 Environmental and Climate Technologies ____________________________________________________________________________ 2020 / 24 The energy spacing of the temperature integral (or double exponential term), the kinetic parameters of n th order and the variance of the Gaussian energy distribution are estimated in Table 4. It can be concluded that the proposed scheme has good agreement with other statistical methods, where the solution is predicted by the trade-off relationship between experimental and mathematical models. However, Monte Carlo simulation is also an approximated methodology, yet it reduces the scope of multiple iterations through the direct correlation of experimental data with a mathematical solution. Fig. 2. Brownian motion of inbound and out bound particles on the DTG plane at 10 °C/min. Fig. 3. Brownian motion of inbound and out bound particles on the DTG plane at 15°C/min. 167 Environmental and Climate Technologies ____________________________________________________________________________ 2020 / 24 Conversion, α Fig. 4. Energy distribution at different stages of decomposition of pine needles. TABLE 2. CALCULATED AREA OF THE DTG CURVES FOR DIFFERENT HEATING RATES Heating rate Area under curve (sq. Unit)   1    ( m0 − mr )    mr  mt  dm    dT = (1 −  )  dT  5 °C/min 10 °C/min 15 °C/min 52.4866 104.3758 153.6810 TABLE 3. PARAMETRIC INFORMATION OF THERMAL DECOMPOSITION OF PINE NEEDLES Parameters First order reactions 5 °C/min 10 °C/min 15 °C/min nth order reactions (n = 0.002) 5 °C/min 10 °C/min 15 °C/min E0, kJ/mol 76 65.94 20 26 −1 66.72 A, min 6.39·10 3.10·10 3.96·10 5.44·10 6.56·10 7·102 σ , kJ/mol 89.3 75.49 81.76 2 2.0670 2.29 Es, kJ/mol 99.50 92.08 92.78 43.022 40.55 38 Ew, kJ/mol 4.95 4.91 4.98 4.64 4.59 4.64 Integral limit 26.79 ≤ E∞ ≤ 154.45 25.15 ≤ E∞ ≤ 144.62 25.80 ≤ E∞ ≤ 147.64 3.15 ≤ E∞ ≤ 69.56 11.44 ≤ E∞ ≤ 66.38 3.53 ≤ E∞ ≤ 66.20 2 7 7 4 27 2 2 TABLE 4. VALIDATION OF THE PROPOSED SCHEME WITH THE OTHER METHODOLOGIES Activation Energy Monte Carlo Technique E, kJ/mol 92–100 Model- free methods [16] FWO 82.38 KAS 74.83 Kissinger 132.77 Multi-component devolatilization mechanism [17] 90–132 6. CONCLUSION The kinetic parameters of thermal decomposition of pine needles have been derived using the Monte Carlo technique. The variation of activation energy is demarcated on the basis of temperature integral, as the significant contribution of temperature integral governs the 168 Environmental and Climate Technologies ____________________________________________________________________________ 2020 / 24 integral problem of DAEM. The estimated value of activation energy varies between 92 kJ/mol and 100 kJ/mol, whereas the lower limit of activation energy changes from 25 kJ/mol to 26 kJ/mol. Similarly, the activation energy for the nth reactions varies from 38 to 43 kJ/mol at the reaction order of 0.002. The obtained values are within the permissible limit of variation as mentioned in literature [16]–[19]. The proposed scheme is relatively robust and precise to other methodologies. By applying the inverse function to calculate the kinetic parameters, the computational burden gets lessened. Secondly, it demarcates the activation energy variation caused by temperature integral from the energy distribution introduced by the density function of the distribution function. However, the nature of the inverse function for the proposed scheme must be non-surjective in its validation. It implies that the conversion at a particular time cannot have two different activation energies at the given heating rate. So, the solution must be non-trivial in nature. Apart from precise calculation of kinetic parameters, the mathematically formulated solution demands one to one mapping of variables, thus the solution must be injective, and the kinetic parameters must have a single value at the given conversion (α). Furthermore, should the bombarded particles cover the DTG plane completely, the technique would be more effective. ACKNOWLEDGEMENT This work was supported by the stipendium Hungaricum Programme and by the Institute of Process Engineering, Doctoral School of Mechanical Engineering, Szent Istvan University, Godollo, Hungary. REFERENCES [1] Capart R., Khezami L., Burnham A. K. Assessment of various kinetic models for the pyrolysis of a microgranular cellulose. Thermochimica Acta 2004:417:79–89. https://doi.org/10.1016/j.tca.2004.01.029 [2] Conesa J. A., Caballero J., Marcilla A., Font R. Analysis of different kinetic models in the dynamic pyrolysis of cellulose. Thermochimica Acta 1995:254:175–192. https://doi.org/10.1016/0040-6031(94)02102-T [3] Dhaundiyal A., Singh S. B., Hanon M. M., Rawat R. Determination of Kinetic Parameters for the Thermal Decomposition of Parthenium hysterophorus. Environmental and Climate Technologies 2018:22(1):5–21. https://doi.org/10.1515/rtuect-2018-0001 [4] Yaroshenko A. P. Theoretical model and experimental study of pore growth during thermal expansion of graphite intercalation compounds. Journal of Thermal Analysis and Calorimetry 2005:79:515–519. https://doi.org/10.1007/s10973-005-0571-3 [5] Dhaundiyal A., Tewari P. Kinetic Parameters for the Thermal Decomposition of Forest Waste Using Distributed Activation Energy Model (DAEM). Environmental and Climate Technologies 2017:19(1):15–32. https://doi.org/10.1515/rtuect-2017-0002 [6] Dhaundiyal A., Singh S. B., Hanon M. M. Study of Distributed Activation Energy Model Using Bivariate Distribution Function, f(E1, E2). Thermal Science and Engineering Progress 2018:5:388–404. https://doi.org/10.1016/j.tsep.2018.01.009 [7] Galgano A., Blasi C. Di. Modeling Wood Degradation by the Unreacted-Core-Shrinking Approximation. Industrial & Engineering Chemistry Research 2003:42:2101–2111. https://doi.org/10.1021/ie020939o [8] Morgan D. J., Brown M. A. Introduction to Thermal Analysis: Techniques and Applications. London and New York: Chapman and Hall, 1988. [9] Güneş M., Güneş S. The influences of various parameters on the numerical solution of non-isothermal DAEM equation. Thermochimica Acta 1999:336(1–2):93–96. https://doi.org/10.1016/S0040-6031(99)00207-5 [10] Dhaundiyal A., Singh S. B., Hanon M. M. Application of Archimedean copula in the non-isothermal nth order distributed activation energy model. Biofuels 2019:10:1–12. https://doi.org/10.1080/17597269.2018.1442662 [11] Dhaundiyal A., Singh S. B. Mathematical insight to non-isothermal pyrolysis of pine needles for different probability distribution functions. Biofuels 2018:9(5):647–658. https://doi.org/10.1080/17597269.2017.1329495 [12] Burnham A. K. Introduction to Chemical Kinetics. Global Chemical Kinetics of Fossil Fuels 2017:25–74. https://doi.org/10.1007/978-3-319-49634-4_2 169 Environmental and Climate Technologies ____________________________________________________________________________ 2020 / 24 [13] Dhaundiyal A., Singh S. B. Distributed activation energy modelling for pyrolysis of forest waste using Gaussian distribution. Proceedings of the Latvian Academy of Sciences. Section B. Natural, Exact, and Applied Sciences 2016:70(2):64–70. https://doi.org/10.1515/prolas-2016-0011 [14] Cho W. K. T., Liu Y. Y. Sampling from complicated and unknown distributions: Monte Carlo and Markov Chain Monte Carlo methods for redistricting. Physica A: Statistical Mechanics and its Applications 2018:506:170–178. https://doi.org/10.1016/j.physa.2018.03.096 [15] Guo X., Liu Z., Xiao Y., Xu X., Xue X., Liu Q. The Boltzmann-Monte-Carlo-Percolation (BMCP) model on pyrolysis of coal: The volatiles’ reactions. Fuel 2018:230:18–26. https://doi.org/10.1016/j.fuel.2018.05.016 [16] Dhaundiyal A., Abdulrahman T. M., Laszlo T. Thermo-kinetics of Forest Waste Using Model-Free Methods. Multidisciplinary Sciences 2019:24(1):465–495. https://doi.org/10.11144/javeriana.sc24-1.tofw [17] Korobeinichev O. P., Paletsky A. A., Gonchikzhapov M. B., Shundrina I. K., Chen H., Liu. N. Combustion Chemistry and Decomposition Kinetics of Forest Fuels. Procedia Engineering 2013:62:182–193. https://doi.org/10.1016/j.proeng.2013.08.054 [18] Dhaundiyal, A., Toth, L. Modeling of Hardwood Pyrolysis Using the Convex Combination of the Mass Conversion Points. Journal of Energy Resources Technology, Transactions of the ASME 2019:142(6):061901. https://doi.org/10.1115/1.4045458 [19] Dhaundiyal, A. et al. Analysis of pyrolysis reactor for hardwood (Acacia) chips. Renewable Energy 2020:147(Part 1):1979–1989. https://doi.org/10.1016/j.renene.2019.09.095 Alok Dhaundiyal had been awarded the master’s degree in mechanical engineering from GBPUAT, Uttarakhand, India in 2014. From 2015 to 2017, he worked as an Assistant Professor in Himgiri Zee University. He has also worked on Deutsche Gesellschaft für Internationale Zusammenarbeit (GIZ) projects, which are the part of Indo-German Govt. Programme to promote renewable energy sector of India under the patronage of state government agency of Uttrakhand, India, UREDA; and Central ministry, Ministry of New Renewable Energy. The author’s area of expertise is the energy generation through the biomass waste utilization. In 2012, he has been awarded the Graduate Aptitude Test Engineering (GATE) Scholarship for Master of Engineering by the govt. of India. The Faculty of Engineering Excellence award has been conferred on him by the University of Strathclyde, Scotland, United Kingdom. He has also received University Grant Commission (2017 to 2022) scholarship. In 2017, the Stipendium Hungaricum Scholarship has been bestowed upon him by the government of Hungary. Currently, he has been pursuing his doctoral studies (Mechanical) at Szent István Egyetem (St. Stephen University), Hungary. ORCID iD: https://orcid.org/0000-0002-3390-0860 Prof. (Emeritus) Laszlo Toth (DSc.) had received his PhD Degree from University of Agricultural Sciences, Gödöllő in 1967. In 1986, he had been awarded the Doctor of Science by the Hungarian Academy of Science, Budapest, Hungary. He has been working as Professor in the Department of Energetics, Szent István University, Hungary, since 1995. He has research experience of 34 years and teaching experience of 20 years. Currently he is the Editor-in-chief of ‘Journal of Agricultural Mechanisation’, Hungary. He is also the President of the Hungarian Scientific Wind Energy Association, and a member of the World Wind Energy Association. He is also a member of the American Chemical Society and the American Society of Mechanical Engineers. ORCID iD: https://orcid.org/0000-0003-0161-1375 170
https://openalex.org/W4232299740	https://www.scienceopen.com/document_file/87c7c4ab-78d0-4af4-a13f-2ba3e5887ad1/ScienceOpen/299_Davies.pdf	English	null	Managing Gravity Infusion using a Mobile Application	Electronic workshops in computing	2,014	cc-by	4,455	Managing Gravity Infusion using a Mobile Application Paul Lee Singleton Hospital Swansea SA2 8QA paul.lee@wales.nhs.uk Harold Thimbleby University of Swansea Swansea SA2 8PP harold@thimbleby.net Mark Davies, Alan Chamberlain University of Nottingham Nottingham NG8 1BB mark.davies, alan.chamberlain @nottingham.ac.uk Mark Davies, Alan Chamberlain University of Nottingham Nottingham NG8 1BB mark.davies, alan.chamberlain @nottingham.ac.uk Gravity infusion, also known as “the drip,” is a common and basic method for delivering ﬂuids to a patient, without the use of any complex medical devices, such as an infusion pump or a syringe driver. Nevertheless there are many quite complex and error-prone steps involved in setting up a gravity infusion for the correct dose, and since there is no computer or similar technology involved to assist with the procedure, it can be difﬁcult to guarantee the accuracy and consistency of the ﬂuid delivery. This paper presents a new method for accurately setting gravity infusion drug delivery, based on a handheld mobile application that includes a novel approach to help estimate ﬂow rate and double-check the steps involved in setting it up. We demonstrate how simple visual interfaces can play an important role in the healthcare setting, and we explain safety features that have been implemented to catch common errors and slips that can occur. Human Computer Interaction, Mobile Medical Applications, Gravity Infusion, Intravenous Therapy levels of nurses identiﬁed the lack of conﬁdence when carrying out basic infusion rate calculations. In their day-to-day work, nurses regularly need to carry out different types of mathematical calculations when administering ﬂuids and drugs to patients. Calculators can be used as a way of double-checking their calculations, however calculators have been criticized for their design in regard to human error (Thimbleby 1997, 1998, 2000). Another issue is that similar calculators (sometimes even from the same manufacturer) can work differently to each other, and can produce different answers when using the same key sequences (Thimbleby 2000). Calculators also have a tendency to display math equations differently to how we would usually write them out and this can sometimes lead to confusion, especially when performing a long calculation (Thimbleby 2000). Calculators cannot be depended on for correct calculations. 2.1. Mathematical Calculation The ﬁrst step in carrying out a gravity infusion is to calculate the correct dosage based on the patient’s prescription. Each patient’s prescription is delivered from a ﬂuid bag that can vary in size from 50 mL to 2 litres and is delivered through an IV “administration set” (ﬂexible tubing to deliver the drug). Each administration set will have a “drop factor” size. These sizes can be 10, 15, 20 or 60 drops per mL. Figure 1: A mobile application to help practitioners carry out intravenous drip-rate infusions when medical pumps are not available. inappropriate doses that they may try to administer; typically smart pumps have “hard” and “soft” limits, and while they avoid some types of error, they are complex to use and are often circumvented — it is easier to set a smart pump to a harmless drug like “saline” so it does not interfere with what the practitioner does. However, whether smart pumps are used or not this still does not guarantee the calculation or setting-up is right. (Throughout this paper we use the term “practitioner” to mean any suitable qualiﬁed clinician, such as a nurse.) To calculate the intended drip-rate of a gravity infusion you need to divide the total volume of ﬂuid (in milliliters) by the total time required for the delivery (in hours) and then multiply by the drop factor (number of drops per mL). This gives you the total number of drops required per hour. To convert this to drops per minute, you need to divide by 60. To calculate the intended drip-rate of a gravity infusion you need to divide the total volume of ﬂuid (in milliliters) by the total time required for the delivery (in hours) and then multiply by the drop factor (number of drops per mL). This gives you the total number of drops required per hour. To convert this to drops per minute, you need to divide by 60. The full formula is: Drops per minute = Total volume, mL Total time, hr × Drip factor 60 60 Modern electronic infusion pumps provide many features and are generally recommended to be used for delivering drugs to patients, but there are situations where practitioners are faced with having to carry out infusions by gravity drips. Gravity drips are simpler and do not suffer from battery failure: Lee et al (2012) review some of the problems. 2.1. Mathematical Calculation 60 Although this type of calculation is not particularly difﬁcult to calculate, there are several steps involved before the ﬁnal answer can be obtained, also each variable in the calculation needs to be converted to the correct unit of measurement. Practitioners who do not regularly carry out this calculation can easily forget some of the steps involved. One should remember that remembering and correctly performing the calculation under clinical conditions is very much harder than reading the explicit calculation above while reading a research paper! In this paper we present a novel mobile application for the training and checking of gravity infusions. Our aim is to explore new ways of supporting healthcare tasks through the use of our everyday mobile devices. The application includes features for assisting healthcare staff through each of the tasks involved when carrying out gravity infusions — the initial medication calculation, setting up the drip and checking and monitoring the drip. Another calculation is also required. The practitioner has to adjust the actual drip rate to be equal to the calculated drip rate. This involves counting the drips in one minute and making appropriate adjustments. This is a lengthy process, and fortunately it sufﬁces to count drips in 15 seconds and then multiply by 4. The advantage of having a shorter time and counting fewer drops (and therefore missing fewer drops and having less time to be interrupted by other tasks) is ﬁnely balanced against the additional step of multiplying in by 4. Another calculation is also required. The practitioner has to adjust the actual drip rate to be equal to the calculated drip rate. This involves counting the drips in one minute and making appropriate adjustments. 1. INTRODUCTION Intravenous infusion therapy, administering medica- tion ﬂuids to a patient directly into their veins, is a frequently used practice throughout healthcare. In the UK National Health Service (the NHS) around 15 million infusions are carried out every year. Unfortunately, there are approximately 700 unsafe incidents reported annually in the UK and many more going unreported (NPSA 2004); between 44,000–98,000 people die each year as a result of preventable medical errors in the USA (Kohn, et al 1999). These rates are higher than those of deaths from motor-vehicle accidents, breast cancer and AIDS per year in the USA (Kohn, et al 1999) and no doubt in any other country with western healthcare standards (or roads). The problem of drug dose calculation error has been identiﬁed in the literature (Wright 2012; Warburton 2010); there have been numerous incidents where calculating or entering the wrong dose — and administering it — has led to patient harm or death (e.g., ISMP 2007; American Medical News 2011 etc). The use of a modern pump such as an infusion pump assists the safe delivery of IV (intravenous) medication. Around 90% of hospitalized patients receive IV medication through an infusion pump. Some “smart pumps” have a pre-installed drug libraries with ﬁxed thresholds set for each drug. This, in effect, can warn the practitioner of potentially A case study by Lee (2008) carried out in a large NHS hospital on the mathematical conﬁdence © The Authors. Published by BCS Learning and Development Ltd. Proceedings of HCI 2014, Southport, UK 299 Managing Gravity Infusion using a Mobile Application Davies • Chamberlain • Thimbleby • Lee Managing Gravity Infusion using a Mobile Application Davies • Chamberlain • Thimbleby • Lee infusion is a popular method of infusion due to its low cost. However as there is no assistance from a device, there are more steps involved with the calculation relating to the delivery of any drugs to be administered in this fashion. Getting each of the steps right in calculating and delivering the drug is important to guarantee the safety and well being of a patient. Figure 1: A mobile application to help practitioners carry out intravenous drip-rate infusions when medical pumps are not available. 3.1. Calculating the Right Dose Infusion calculations are carried out in a hospital on a daily basis. It is crucial that the dose is calculated correctly to ensure the safety and well-being of a patient. Figure 2 shows the design of the interface for calculating drip-rates. There are three main steps to the calculation: (i) Total volume of the drug to be infused (in milliliters), (ii) Total time of infusion (hours/minutes), and (iii) The drop factor (which is written on the administration set). 2.2. Setting the Drip-Rate Once the drip-rate has been calculated, the practitioner can then begin to set up the gravity infusion. This involves hanging the bag of ﬂuid on a steady drip stand and connecting the correct administration set from the bag to the patient. The administration set has a “drip chamber” (shown in ﬁgure 3) built on to it, which is used by the practitioner to measure the ﬂow rate of the ﬂuid (that is, watch the drips and time them against their watch) and adjust a roller clamp to make the ﬂow rate correct. The ﬂow rate is measured in drops per minute. One of the problems with this is that it can be time consuming; adjusting the roller clamp, looking at your pocket watch, counting the drops, and trying to adjust the clamp to achieve the precise drip-rate. Gravity infusion rates are often therefore approximated. Figure 2: The user interface for calculating a drip-rate for an infusion. The practitioner must enter the total volume (in millilitres, mL), total time (in hours and/or minutes) and a drop-factor (which can be 10, 15, 20 or 60 per mL). When a value has been set in each of the steps, the drip-rate will be calculated and displayed. “checker” for practitioners when carrying out gravity infusions. The mobile application has features for calculating, setting and checking infusions. 2. GRAVITY INFUSION Gravity infusion is the most basic form of delivering ﬂuids to a patient. It is delivered intravenously (into the vein) and can be used in cases where drug delivery pumps are not available. Since there is no device to control the delivery, the infusion rate depends entirely on gravity. The ﬂow rate is measured by counting the drops per minute. Gravity Practitioners often write down calculations on anything handy, such as wooden tongue depressors. 300 Managing Gravity Infusion using a Mobile Application Davies • Chamberlain • Thimbleby • Lee Figure 2: The user interface for calculating a drip-rate for an infusion. The practitioner must enter the total volume (in millilitres, mL), total time (in hours and/or minutes) and a drop-factor (which can be 10, 15, 20 or 60 per mL). When a value has been set in each of the steps, the drip-rate will be calculated and displayed. This workaround indicates the difﬁculty of the task for practitioners in real clinical environments. 2.3. Checking and Monitoring the Drip-Rate Even after completing the process you still need to check that the rate has not changed of a period of time. Many factors can inﬂuence the drip-rate of a gravity infusion, from the patient moving their position, changes in temperature, through to change in the position/height of the ﬂuid bag. As this is gravity infusion there are no devices used to monitor a drip’s activity or alarms to notify staff of changes. It is up to the practitioner to do regular checks by manually counting the falling drops against the second hand on their watch. They will usually count the drops and estimate the drip-rate; that is, they count drops for approximately 15 seconds on their watch and then multiply by 4 to get an estimated number of drops per minutes. We will now discuss how each of the implemented features in the mobile solution can be used to carry out gravity infusions safer and brieﬂy describe the usability techniques that were used in the development of the system. 4.6. Recognition Rather Than Recall Each step in the calculation is visible and nothing is hidden. The user is not required to remember any information from one step to the next. 4.2. Aesthetic and Minimal Design Calculations are very clear to read by breaking down each calculation step and using a larger font for answers. The user interface contains only what is needed for the calculation. on the administration set. For this we used graphical icons to represent the values, which matches what is printed on the administration set. Only one value can be selected at a time and by default, no value is selected. When a value becomes selected, its button will change its state and display as highlighted. This eliminated the chances of a user entering an invalid value and saves time key pressing. 3. A MOBILE APPLICATION FOR TRAINING AND CHECKING GRAVITY INFUSIONS We worked directly with experts in the NHS and carried out user-centred design approaches with agile methodologies to allow users to feed into the design process to develop a system that would suit their needs. This included rapid prototyping and evaluating features through one-on- one meetings and focus group sessions. We followed the recommendations of ISO Standard 14971, Medical devices – Application of risk management to medical devices and ISO Standard 62366 Medical devices – Application of usability engineering to medical devices. One of the key problems with many software systems is that they do not always check for human error. As humans, we make mistakes, which is why it is critical that we design safety mechanisms into healthcare systems to detect input errors from the user. Some of the problems with modern calculators and how they ignore human error are raised by Thimbleby (2000), and there are many reports — both individual cases in the media and in research — of calculation errors being a leading cause of medication errors (e.g., ISMP 2007; American Medical News 2011; BBC 2011). We developed a mobile solution (see ﬁgure 1), which can be used for training purposes and also as a 301 ng a Mobile Application Thimbleby • Lee Figure 3: The user interface to help with setting a drip-rate accurately. The practitioner adjusts the roller clamp on the infusion and tries to synchronize the real drip-rate (shown on the left) with the accurate on-screen simulation in the app. Managing Gravity Infusion using a Mobile Application Davies • Chamberlain • Thimbleby • Lee 4. USABILITY ENGINEERING We followed the classic Nielsen (1993) for usability heuristics for user interface design. We sketched user interfaces, prototyping them, and ran focus group with nurses, which clearly established the beneﬁt of the animation. We now summarize some of the error-prevention methods we engineered into the design to help carry out safer calculations for infusion drip-rates: 4.3. Help Guides Located in the corner of the interface, the “information” icon opens the help features, which explain each calculation step to the user in case they get stuck or confused. It also includes the formula used by the app for the drip-rate calculation. Decimal points have been removed from the number pad, as they are not needed in this type of calculation, eliminating the chances of decimal point errors, which Thimbleby & Cairns (2010) discuss the risks of. 4.1. Minimize the User’s Memory Load All steps of the calculation appear on the same screen. This reduces the memory load and allows users to see previous steps in the calculation, which lead to the answer. Figure 3: The user interface to help with setting a drip-rate accurately. The practitioner adjusts the roller clamp on the infusion and tries to synchronize the real drip-rate (shown on the left) with the accurate on-screen simulation in the app. 4.5. Visibility of System Status The interface keeps the user informed about the state of the calculation by revealing an answer when all three parts of the calculation have been correctly entered. Also, whenever a user inputs a number, if the value is acceptable after a validation check the number will turn blue to indicate that it has been accepted and allow the user to progress to the next step in the calculation. 4.4. Error Prevention Units and conversions are automatically calculated. For example, if a practitioner needs to enter ’75 minutes’ as the total time, there is a feature to allow them to enter ’75’ as an option directly into the minutes input ﬁeld. This also removes the need to convert 75 minutes into 1 hour and 15 minutes (which is also an acceptable input). Correct symbols and abbreviations have been used, and adhere to the Institute for Safe Medication Practices guidelines (ISMP 2006). For example “100mL” is correctly represented as “100 mL” with a space between the dose and unit of measure, and a capital L is used for litres, since l (the letter) is too easily confused with 1 (the digit). The space helps prevent the “m” from being mistaken as a zero or two zeros (e.g., when written badly by hand), risking a 10- to 100- fold overdose. 4.7. Visual, Audio and Vibration Output for Better Guidance This visualization feature is further supported by the use of sound (“drip falling” sound) and vibration feedback when a drip falls into the chamber, to guide the practitioner if they are unable to view the screen continually — which is likely, as they are also watching the actual drip chamber. Figure 4: The user interface for checking a drip-rate. The practitioner taps the icon on the screen every time a drop falls in the drip chamber. The application will analyse the taps and time, and calculate the rate, which will then be displayed on the screen to the practitioner. Figure 4 shows a feature that we developed for checking the drip-rate of a set infusion. The approach should be compared to current methods for counting the rate of drips, which involves a practitioner holding out a pocket watch, simultaneously counting the number of drops that fall against the time elapsed, and performing the appropriate calculations. The idea behind this feature was to guide practitioners better when setting up a drip. A practitioner can hold the visualization up against the real drip chamber and try to synchronize the accurate on-screen representation against the real one, which they try to achieve by increasing/decreasing the wheel of the roller clamp on the administration set. We suggest that it can reduce practitioner workload by not having to count the drops and time manually, instead matching the rate of the app’s visualization. It may also be quicker and could even help achieve the precise rate that needs to be set. See Future Work, below, in section 5. As we earlier said, counting how many drops that fall in 15 seconds, then multiplying this value by 4 to get an average total number of drops per minute (of course 4 × 15 s = 1 m). The feature that we have developed works by touching the large “drop” icon displayed on the interface, each time a drop falls. When the practitioner ﬁrst touches the screen, it will begin a timer and count the number of drops against the time. The practitioner continues to tap the interface whenever a drip falls in the chamber. The software measures the intervals between each screen press and looks for a consistent, steady pattern in the rate. When a steady pattern has been made, the software prints a screen alert, informing the practitioner of the drip-rate value. 4.7. Visual, Audio and Vibration Output for Better Guidance Each input ﬁeld has been capped with a threshold to prevent any accidental lethal doses. “Total volume” cannot be bigger than 4 digits and the “hours” and “minutes” ﬁelds cannot be greater than 2 digits each. The “drop factor” can only be a value of 10 mL, 15 mL, 20 mL or 60 mL corresponding to the value Once the drip-rate to an infusion has been calculated, the next step for the practitioner to do is set this rate. This involves adjusting a roller clamp and counting the drops in the drip chamber against 302 Managing Gravity Infusion using a Mobile Application Davies • Chamberlain • Thimbleby • Lee Managing Gravity Infusion using a Mobile Application Davies • Chamberlain • Thimbleby • Lee Figure 4: The user interface for checking a drip-rate. The practitioner taps the icon on the screen every time a drop falls in the drip chamber. The application will analyse the taps and time, and calculate the rate, which will then be displayed on the screen to the practitioner. the seconds-hand on their watch. Carrying out this process and getting the right rate can be tricky and therefore the rate is usually approximated. One of the ways that we thought could help this procedure was through using some of the affordances that a mobile device offers — namely visual, audio and vibrations. Figure 3 shows the user interface for supporting the setting of a drip-rate. The practitioner must ﬁrst enter the drip-rate value, which may or may not have been calculated using the calculate feature. Once a drip-rate value has been entered, checks are made to ensure the value is not over the threshold limit. Once the value has been validated and approved, the visualization can start. The visualization mimics the design and characteristics of a real drip chamber. Drops will fall from the top and down into the chamber, repeatedly at the speed of the dripping rate that was entered by the practitioner. When this visualization begins, a timer and counter also begins. The timer starts from zero and counts the time of the drips. The counter counts the number of drops that have fallen for each second. 4.7. Visual, Audio and Vibration Output for Better Guidance Instructions and previous recordings are displayed on the interface to avoid confusion and reduce memory load. 4.8. Touch Input for Counting Drops Infusions that are administered by a medical pump (such as an infusion or syringe pump) have the advantage of monitoring the infusion over the infusion’s total time set. With gravity infusion there is no pumping mechanism to deliver the medication or any tools to monitor the infusion. The practitioner is required to regularly check that the drip-rate has not changed and that the patient receives the total speciﬁed amount of medication over the prescribed amount of time. The idea behind this feature is that practitioners might be able to perform these regular infusion-rate checks quicker and also remember what previous recorded rates were. As this feature relies on the practitioner’s reactions as input, it is only safe to say that the ﬁnal ﬁgure is an approximation. Some initial testing, which has been carried out on a fake infusion, using a bag of saline showed that this feature achieved drip-rate values that were within 303 Managing Gravity Infusion using a Mobile Application Davies • Chamberlain • Thimbleby • Lee profession/308159942/4 [last accessed: March 2014]. a ±3 range. Although it might not be possible to achieve the exact ﬁgure every time, it has shown new ways of carrying out infusion checks, which can act as a useful training tool for trainee staff. We aim to carry out further in-depth studies and focus groups on the evaluation of this feature with qualiﬁed staff practitioners. BBC News, 2011. Fatal insulin overdose nurse suspended for year. Available from: www.bbc.co.uk/news/uk-wales-south-east-wales- 13625168 [last accessed: March 2014]. Wright, K., 2012. How to ensure patient safety in drug dose calculation. Nursing Times, 108(42), pp.12–13. 5. FUTURE WORK When we designed this system we used focus groups and expert users to inform the design, which allowed us to ﬁne-tune the design. We are now carrying out user studies with nurses and related healthcare professionals to evaluate the differences of carrying out gravity infusion with and without the app. We are also interested in knowing how practitioners would use the app and how we can build on from its current features. Technically, we are examining the use of augmented reality interfaces to allow the user to capture drip-rates using the phone’s camera. We are also examining the use of the system in a training environment and the way that as part of training the system could be adapted to have more social elements. Warburton, P., 2010. Numeracy and patient safety: the need for regular staff assessment. Nursing Standard, 24(27), pp.42–44. Kohn, L., Corrigan, J., and Donaldson, M., 1999. To Err Is Human: Building a Safer Health System. Washington DC: The National Academy Press. Institute for Safe Medication Practices, 2006. List of Error Prone Abbreviations, Symbols and Dose Designations. Available from: ismp.org/tools/errorproneabbreviations.pdf [last accessed: March 2014]. Institute for Safe Medication Practices, 2007. Fluorouracil Incident Root Cause Analysis. Available from: www.ismp-canada.org [last accessed: March 2014]. 6. CONCLUSIONS This paper has addressed some of the issues and difﬁculties with carrying out gravity infusions. We have presented a mobile solution and discussed its design features, which aim to help nurses and practitioners carry out drug infusions safely and accurately. Although we still need to carry out user studies to evaluate the efﬁciency and usefulness of these features, our development has shown new ways of thinking about how we can develop simple and available mobile solutions that may improve the quality of safety when carrying out important drug infusions. We have also demonstrated how we can apply sound usability engineering techniques and interactions for future mobile medical software. Lee, P., 2008. Risk-score system for mathematical calculations in intravenous therapy. Nursing Standard. 22(33), pp.35–42. Lee, P., Thompson, F., and Thimbleby, H. 2012. Analysis of Infusion Pump Error Logs and Their Signiﬁcance for Healthcare. British Journal of Nursing. 21(8), pp.S12–S22. National Patient Safety Agency, 2004. Safer Practice Notice 1, Improving Infusion Device Safety. London. Nielsen, J., 1993. Usability Engineering. Burlington, MA: Morgan Kaufmann. Acknowledgements Thimbleby, H., 1997. A true calculator. Engineering Science and Education Journal, 6(3), pp.128–136. The research on which this article is based was funded by RCUK research grants [EP/J000604/1, EP/J000604/2, and EP/G065802/1] and partly funded by EPSRC grants [EP/G059063/1, EP/L019272/1]. Thimbleby, H., 1995. A new calculator and why it is necessary. Computer Journal, 38(6), pp.418–433. Thimbleby, H., 2000. Calculators are needlessly bad. International Journal of Human-Computer Studies, 52(6), pp.1031–1069. REFERENCES American Medical News, 2011. Revealing their medical errors: Why three doctors went public. Available from: Thimbleby, H. and Cairns, P., 2010. Reducing Number Entry Errors: Solving a Widespread, Serious Problem. Journal Royal Society Interface, 7(51), pp.1429–1439. www.amednews.com/article/20110815/ 304
https://openalex.org/W4368347764	https://www.researchsquare.com/article/rs-2876848/latest.pdf	English	null	NME4 promotes cell malignant process by targeting SMAD2 in colorectal cancer	Research Square (Research Square)	2,023	cc-by	6,514	NME4 promotes cell malignant process by targeting SMAD2 in colorectal cancer Zhengyu FANG The First Hospital of Zhejiang University of Traditional Chinese Medicine Qiongxiang GE Zhengyu FANG The First Hospital of Zhejiang University of Traditional Chinese Medicine Qiongxiang GE The First Hospital of Zhejiang University of Traditional Chinese Medicine Zhi CHEN The First Hospital of Zhejiang University of Traditional Chinese Medicine Haixin QI The First Hospital of Zhejiang University of Traditional Chinese Medicine Boan ZHENG (  13858180116@163.com ) Zhejiang Provincial People's Hospital Abstract Nucleoside diphosphate kinase 4 (NME4) is aberrantly expressed in several cancer types. However, the function of NME4 in colorectal cancer (CRC) remains to be elucidated. Bioinformatic analysis and clinical sample collection revealed that NME4 was highly expressed in CRC tissues and positively correlated with stage and tumor size. Knockdown of NME4 expression inhibited the proliferation and migration of CRC cells and promoted apoptosis. Moreover, NME4 inhibition in vivo markedly suppressed the size and weight of tumors. Then, cDNA microarray and Ingenuity Pathway Analysis (IPA) analysis were applied to identify SMAD2 and mTOR signaling pathways as downstream targets of NME4. And it was confirmed that SMAD2 was significantly reduced in CRC tissues and significantly correlated with AJCC staging and positive numbers. Furthermore, overexpression of NME4 resulted in a substantial increase in SMAD2 expression. NME4 overexpression promoted cell proliferation and migration, whereas knockdown of SMAD2 greatly reversed its effects. Together, these results suggested that NME4 may act as a novel tumor-promoting factor that promotes CRC progression by regulating SMAD2 and mTOR signaling pathways. Research Article Keywords: CYBA, Colorectal cancer, KCNN4, SMAD2, Invasion Posted Date: May 4th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2876848/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Keywords: CYBA, Colorectal cancer, KCNN4, SMAD2, Invasion Posted Date: May 4th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2876848/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: May 4th, 2023 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Page 1/24 Page 1/24 Page 1/24 2.1. cDNA microarray and analysis Total RNA was isolated from RKO cells transfected with shCtrl and shNME4. The quality assessment of microarray data was done by Shanghai Xiaoyan Biotechnology Company (Shanghai, China) on the microarray data, and all probes with mean values less than 0.005 were filtered out. A linear model based on empirical Bayesian distribution was calculated for the significant difference level P-value and corrected for the significant difference level (FDR) with the Benjamini-Hochberg method [21]. The screening criteria for significantly differential genes were \|Fold Change\| ≥ 1.5 and FDR < 0.05. Total RNA was isolated from RKO cells transfected with shCtrl and shNME4. The quality assessment of microarray data was done by Shanghai Xiaoyan Biotechnology Company (Shanghai, China) on the microarray data, and all probes with mean values less than 0.005 were filtered out. A linear model based on empirical Bayesian distribution was calculated for the significant difference level P-value and corrected for the significant difference level (FDR) with the Benjamini-Hochberg method [21]. The screening criteria for significantly differential genes were \|Fold Change\| ≥ 1.5 and FDR < 0.05. Differentially expressed genes were further computationally simulated by Ingenuity Pathway Analysis (IPA; QIAGEN, Valencia, CA, USA) online tool to predict downstream regulators and signaling pathways potentially regulated by CYBA. Differentially expressed genes were further computationally simulated by Ingenuity Pathway Analysis (IPA; QIAGEN, Valencia, CA, USA) online tool to predict downstream regulators and signaling pathways potentially regulated by CYBA. 2.2. Patients 92 patients diagnosed with CRC from in the First Hospital of Zhejiang University of Traditional Chinese Medicine were recruited for this study. All patients did not receive chemotherapy or radiotherapy pre- surgery. CRC tissues were collected during surgery. All cases were staged according to the American Joint Committee on Cancer (AJCC) 7th edition cancer staging manual. Clinical information and pathological data were collected from patients, and follow-up studies were performed and recorded for a period of X months after surgery for all patients. The study was approved by the First Hospital of Zhejiang University of Traditional Chinese Medicine. All patients signed a written informed consent before participation in the study. 1. Introduction Colorectal cancer (CRC) has been classified as one of the predominantly malignant tumors worldwide. The latest statistics reveal that it ranks 3rd in terms of cancer prevalence and 2nd in mortality globally [1]. As well, CRC appears to be considered in the list of the most commonest malignant tumors in China recently, with the incidence increasing dramatically. [2, 3]. The causes are linked to diet, nutritional status, genetics, polyp lesions and chronic inflammation [4]. Further, due to the lack of specific early clinical signs or validated biomarkers, CRC patients suffer from poor prognosis [5]. According to global cancer statistics, approximately 930,000 people across the world died from colorectal cancer in 2020 [6]. Although advances in early screening methods and treatment strategies have somewhat improved the overall survival rate of colorectal cancer patients [7, 8]. Nevertheless, the overall efficacy of current treatments on account of early metastasis and terminal rapid progression leads to poor survival and overall prognosis, making it still among the most major medical burdens around the world [9]. Therefore, seeking sensitive diagnostic biomarkers for colorectal cancer will contribute to the early diagnosis of colorectal cancer and enable innovative therapeutic targets [10]. Nucleoside diphosphate kinase 4 (NME4/NDPK-D) is a member of the NM23 family[11]. The expression of human NME4 is widespread, with high expression in the liver, kidney, bladder, and prostate, and low levels in the brain, testis, and peripheral leukocytes [12, 13]. Previous studies showed that since NME4 contains mitochondrial target sequences, which have been implicated in mitosis, apoptosis, invasive potential and inflammatory response [14, 15]. Currently, only a few data are available for the study of NME4 in human cancer. NME4 is differentially expressed in different tumors, with most suggesting overexpression of NME4 for example in esophageal squamous cell carcinoma, and gastric and colon cancer tumors [16, 17]. Additionally, research has reported that NME4 affects tumor cell migration, invasion and lymph node metastasis in a functional manner [18, 19]. However, rarely does the Page 2/24 Page 2/24 relationship between NME4 and CRC been investigated. A previous study showed that NME4 was statistically down-regulated in response to stimuli thought to be beneficial in the treatment of colon cancer [20]. This points to the potential involvement of the NME4 in the development of CRC. relationship between NME4 and CRC been investigated. 1. Introduction A previous study showed that NME4 was statistically down-regulated in response to stimuli thought to be beneficial in the treatment of colon cancer [20]. This points to the potential involvement of the NME4 in the development of CRC. In this study, bioinformatics and biological experiments were conducted to evaluate the expression of NME4 in colorectal cancer tissues and cells. Furthermore, the effects of NME4 on the proliferation and migration ability of colorectal cancer cells and the underlying molecular mechanisms were investigated by cellular and animal experiments. It will propose new biomarkers and therapeutic targets for the early diagnosis and treatment of colorectal cancer. 2.3. Tissue microarrays analysis Microarrays of colorectal and paracancerous tissues were prepared as berore [22]. The expression levels of NME4 and SMAD2 in CRC tissues were examined by immunohistochemical analysis. 2.8. Hematoxylin and eosin (H&E) staining The collected tumor tissue was fixed in 10% formalin and embedded in paraffin. After cutting the tumor tissues into 4-µm-thick sections, wax was removed and processed. Then, the sections were stained with H&E (Baso, BA4041, BA4042). Visualization was performed under a light microscope (Leica DM5000B, Germany). 2.5. Coimmunoprecipitation (Co-IP) assay 2.5. Coimmunoprecipitation (Co-IP) assay For Co-IP assays, cells were lysed with lysis buffer according to the manufacturer's protocol (Invent Biotechnologies, USA). Nuclear protein lysates were immunoprecipitated with antibodies specific for NME4, SMAD2, or IgG negative control in a spinner overnight at 4°C. The lysates were then slowly spun with 50 µL of Protein A/G magnetic beads pre-cleaned with PBST for 2 hours at 4°C. Following washing the magnetic beads 4 times, the immunoprecipitated complexes were isolated and further analyzed by Western blotting. 2.4. Cell culture and treatment Page 3/24 The RKO cell line obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) was cultured in DMEM (Gibco) of 10% fetal bovine serum (FBS) and incubated at 37°C and 5% CO2 in a humidified atmosphere. NME4 and SMAD2 knockdown lentiviral plasmid (shRNA) were constructed. Negative controls and plasmids were transfected into cells with Lipofectamine 3000 (Invitrogen, CA, USA) and incubated for 2 days. 2.6. Cell viability assay Cell viability was measured using the CCK-8 assay kit. Treated RKO cells were inoculated into 96-well culture plates (2000 cells/well) for 12 hours. CCK-8 (100 µL/well) was supplemented and incubated for 2 hours. The absorbance at 450 nm was measured by microplate reader (BioTek, Winooski, VT) and the growth curve was plotted. 2.7. In vivo tumorigenesis Four-week-old female BALB/c-nude mice were utilized for subcutaneous xenograft experiments. shCtrl or shNME4 (1×106) was subcutaneously injected into the mice. Tumor size was measured every 5 days with calipers and calculated with the formula: volume = length × (width2)/2. At the end of the study, mice were executed under deep anesthesia and tumor tissue was removed. After intraperitoneal injection of D- luciferin (150 mg/kg), tumor growth was monitored by bioluminescence imaging by the IVIS system. After imaging several time points, the animals were euthanized. All animal experiments were performed according to institutional guidelines and approved by the First Hospital of Zhejiang University of Traditional Chinese Medicine 2.9. Ki67 staining After dewaxing the tissue sections, the antigen was recovered with citrate buffer (Sangon Biotech, Shanghai, China) under autoclave conditions. Then the sections were blocked with 5% normal goat serum for 1 h at 25℃ and incubated with antibodies against Ki67 (1:100; Abcam, ab16667) overnight at 4°C. Subsequently, sections were treated with HRP anti-rabbit IgG (1:400; Abcam, ab97080). Images were obtained under a light microscope (Leica DM5000B, Germany). Page 4/24 Page 4/24 2.10. Wound healing assay Cells were seeded in 6-well plates (70,000 cells/well) and cultured to 100% confluence. Scrape the cell layer on the surface at the plate with a 200 µL sterile plastic tip and remove the suspended cells with PBS. Culturing the cells in medium without FBS and capturing images of the cells at 0 and 24 hours. 2.12. Western blot analysis Total proteins were extracted from tumor tissues and cells with cell lysis buffer (RIPA, Beyotime) containing protease inhibitors (PMSF, Boster, China). The protein concentration was measured with a BCA assay kit (Thermo Fisher Scientific, Shanghai, China). Proteins were transferred onto polyvinylidene difluoride (PVDF, Millipore, MA, USA) membranes after electrophoresis of 20 µg aliquots of protein in 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). Subsequently, the membranes were incubated with primary antibodies overnight at 4°C after blocking with 5% skim milk for 1 hour at room temperature. Primary antibodies included anti-NME4 (1:3000, #ab228005, Abcam, UK), anti-SMAD2 (1:2000, # ab40855, Abcam, UK), anti-MYC (1:1000, #ab32072, Abcam, UK), anti-SMAD6 (1:1000, #ab273106, Abcam, UK), anti-PTEN (1:2000, #ab170941, Abcam, UK), anti-RPS27A (1:1000, #ab172293, Abcam, UK) and anti-GAPDH (1:3000, #ab8245, Abcam, UK). The following day, membranes were incubated with HRP goat anti-rabbit IgG secondary antibody (1:3000, #A0208, Beyotime, China) for 1 h at room temperature. Chemiluminescence was detected on a FluorChem FC system (Alpha Innotech) using an ECL kit (Millipore, USA) and protein expression was evaluated by ImageJ software. 2.13. Statistical analysis All experimental data are presented as the standard error of the mean (mean ± SD). Statistical analysis was conducted using GraphPad Prism software 8.0 (San Diego, CA, USA). A two-tailed Student's t-test was applied to compare data differences between the two groups. Survival analysis was estimated from the KM Plotter database. Overall survival was estimated using the Kaplan-Meyer method and the logit test. In addition, variables that were statistically significant in the univariate analysis were included in the multifactorial analysis. Correlations between NME4 and SMAD2 expression and clinicopathological characteristics were estimated by chi-square test. For all clinical analyses, * p < 0.05, p < 0.01 and * p < 0.001 were regarded as statistically significant. 2.11. Quantitative Real-Time PCR (qRT-PCR) analysis Total RNA was extracted with TRIzol reagent (Invitrogen) and qRT-PCR was carried out according to the previously described method. qRT-PCR reactions were performed in duplicate and repeated three times. Data analysis was performed with 2−ΔΔCT [23] using GAPDH as an internal reference. The primers are in Table 1. Table 1 Primers sequense. Gene Forward 5′-3′ Reverse 5′-3′ NME4 GCCCTTCTACCCTGCCCTCA CGTGGATGACATTCCTGCTGAT SKI TCTCGCCACTTGCACCCA TCCTCCCTGCTTTCAACTTCC SMAD2 GAAGGCAGACGGTAACAA TGAGCAACGCACTGAAGG SMAD6 CATCACTGCTCCGGGTGAATT GGTCGTACACCGCATAGAGGC MYC CTGCGACGAGGAGGAGAA CCGAAGGGAGAAGGGTGT BMP2 CTGTATCGCAGGCACTCA GAATCTCCGGGTTGTTTT BCL-2 CTGGGAGAACAGGGTACGATAA GGCTGGGAGGAGAAGATGC BMPR1A AAGTTCTGGTAGTGGGTCT CTGGCTTCTTCAGTGGTA RPS27A GATCCAGGATAAGGAAGG ACCACCACGAAGTCTCAA HRAS TTTGCCATCAACAACACCA TCCTGAGCCTGCCGAGAT HSPB1 CAAGGATGGCGTGGTGGA TCTCGTTGGACTGCGTGGC RPSA GGGTTTGATGTGGTGGAT TTGGTCACTGCCTTCTCA PTEN ACCATAACCCACCACAGC CAGTTCGTCCCTTTCCAG GAPDH TGACTTCAACAGCGACACCCA CACCCTGTTGCTGTAGCCAAA Page 5/24 Page 5/24 Table 2 Expression patterns in breast cancer tissues and para-carcinoma tissues revealed in immunohistochemistry analysis. NME4 expression Tumor tissue Para-carcinoma tissue p value Cases Percentage Cases Percentage 0.000* Low 24 26.1% 72 78.2% High 68 73.9% 20 21.8% bl l i Table 2 Table 2 Expression patterns in breast cancer tissues and para-carcinoma tissues revealed in immunohistochemistry analysis. 2.12. Western blot analysis Page 6/24 Page 6/24 The different staining intensities of NME4 in TMA-based IHC are shown in the Fig. 1A. The NME4 protein levels were upregulated in CRC tissues compared to the adjacent tissues (Table. 2). High expression of NME4 was negatively correlated with OS in CRC patients (Fig. 1B). One-way analysis indicated that NME4 expression had a considerable effect with CRC T Infiltrate and tumor size (P = 0.006 and P = 0.041, Table 3). Spearman's correlation analyses verified the considerable correlation between high NME4 expression and CRC T Infiltrate and tumor size correlation (Table 4). The different staining intensities of NME4 in TMA-based IHC are shown in the Fig. 1A. The NME4 protein levels were upregulated in CRC tissues compared to the adjacent tissues (Table. 2). High expression of NME4 was negatively correlated with OS in CRC patients (Fig. 1B). One-way analysis indicated that NME4 expression had a considerable effect with CRC T Infiltrate and tumor size (P = 0.006 and P = 0.041, Table 3). Spearman's correlation analyses verified the considerable correlation between high NME4 expression and CRC T Infiltrate and tumor size correlation (Table 4). The different staining intensities of NME4 in TMA-based IHC are shown in the Fig. 1A. The NME4 protein levels were upregulated in CRC tissues compared to the adjacent tissues (Table. 2). High expression of NME4 was negatively correlated with OS in CRC patients (Fig. 1B). One-way analysis indicated that NME4 expression had a considerable effect with CRC T Infiltrate and tumor size (P = 0.006 and P = 0.041, Table 3). Spearman's correlation analyses verified the considerable correlation between high NME4 expression and CRC T Infiltrate and tumor size correlation (Table 4). Page 7/24 Table 3 Relationship between NME4 expression and tumor characteristics in patients with colorectal cancer. Features No. Page 6/24 of patients NME4 expression p value low high All patients 158 83 75 Age (years) 0.130 ＜55 68 40 28 ≥ 55 84 39 45 Grade 0.642 II 74 40 34 III 60 30 30 AJCC stage 0.095 0 1 1 0 1 26 16 10 2 79 40 39 3 36 15 21 T Infiltrate 0.006 0 1 1 0 1 39 25 14 2 87 42 45 3 15 4 11 4 1 0 1 lymphatic metastasis(N) 0.455 0 75 41 34 1 39 18 21 2 18 8 10 3 13 7 6 Tumor size 0.041* < 3cm 61 37 24 Table 3 Page 8/24 Features No. of patients NME4 expression p value low high ≥ 3cm 83 36 47 ER nuclear test results 0.738 ≤ 5 65 28 37 ＞5 63 29 34 PR nuclear test results 0.396 =0 78 33 45 ＞0 50 25 25 HER2 testing results of membrane 0.055 ≤ 6 71 37 34 ＞6 57 20 37 KI67 nuclear test results 0.859 ≤ 2.25 64 29 35 ＞2.25 64 28 36 P53 nuclear test results 0.616 ≤ 4 74 35 39 ＞4 56 24 32 FISH data 0.503 negative 90 43 47 positive 41 17 24 Page 9/24 Page 9/24 Table 4 Relationship between NME4 expression and tumor characteristics in patients with colorectal cancer Table 4 Table 4 Relationship between NME4 expression and tumor characteristics in patients with colorectal cancer NME4 Tumor size Spearman's correlation 0.171 Significance (two-tailed) 0.041* N 144 T Infiltrate Spearman's correlation 0.230 Significance (two-tailed) 0.006 N 143 Table 5 Expression patterns in colorectal cancer tissues and para-carcinoma tissues revealed in immunohistochemistry analysis. SMAD2 expression Tumor tissue Para-carcinoma tissue p value Cases Percentage Cases Percentage 0.000* Low 39 42.9% 60 65.9% High 52 57.1% 31 34.1% Page 10/24 Table 6 Relationship between SMAD2 expression and tumor characteristics in patients with colorectal cancer. Features No. Page 6/24 Since cell proliferation is closely related to cell cycle progression, we analyzed the effect of NME4 deletion on cell cycle distribution. The results indicated that shNME4 treatment resulted in a marked arrest of cell cycle progression, characterized by the accumulation of cells in the G1 phase (Fig. 2E). The migration and invasion ability of CRC cells transfected with shNME4 was verified using wound healing assay and transwell assay. The healing rate of scratches in CRC cells was lower in the shNME4 group than in the shCtrl group (Fig. 3A). In vitro cell invasion indicated that the number of cells penetrating the basement membrane was significantly lower in the shNME4 group than in the shCtrl (Fig. 3B). 3.3. Loss of NME4 suppresses CRC tumor growth in vivo Next, we explored the effect of NME4 on tumor progression in vivo in a subcutaneous xenograft tumor mouse model. Bioimaging of animals observed that the bioluminescence of mice injected with NME4 lentiviral cells were all weaker than that of the vector control group (Fig. 4A). Also, as shown in Fig. 4B-D, tumors in the shNME4 group developed more slowly and mildly than those in the vector group. Additionally, tumor tissues were identified by H&E staining (Fig. 4E). Ki-67 staining resulted in clearly more Ki-67 positive cells in the control group than in the shNME4 group (Fig. 4F). Page 6/24 of patients SMAD2 expression p value low high All patients 91 45 46 Age (years) 0.389 ＜61.5 42 16 20 ≥ 61.5 45 20 16 Tumor size 0.580 ＜4.75 44 17 27 ≥ 4.75 45 20 25 AJCC stage 0.033 1 8 2 6 2 44 15 29 3 34 19 15 4 2 1 1 Positive number 0.048 0 29 10 19 1 15 8 7 2 4 2 2 3 8 5 3 4 1 0 1 6 1 1 0 7 1 1 0 10 1 1 0 Table 6 Relationship between SMAD2 expression and tumor characteristics in patients with colorectal cancer. Page 11/24 Page 11/24 Page 11/24 Table 7 Relationship between SMAD2 expression and tumor characteristics in patients with colorectal cancer SMAD2 AJCC stage Spearman's correlation 0.229 Significance (two-tailed) 0.032* N 88 Positive number Spearman's correlation 0.258 Significance (two-tailed) 0.047* N 60 3.2. Loss of NME4 promoted apoptosis and inhibited the activity and migration of CRC cell activity Transfection with shNME4 was effectively reduced NME4 expression as reported by both wb and qpcr results (Fig. 2A-B). The results of CCK-8 assay indicated that shNME4 transfection caused a downregulation of CRC cell proliferation (Fig. 2C). Flow cytometry results indicated that shNME4 transfection elevated the total apoptosis rate of CRC cells (Fig. 2D). Since cell proliferation is closely related to cell cycle progression, we analyzed the effect of NME4 deletion on cell cycle distribution. The results indicated that shNME4 treatment resulted in a marked arrest of cell cycle progression, characterized by the accumulation of cells in the G1 phase (Fig. 2E). Table 7 Relationship between SMAD2 expression and tumor characteristics in patients with colorectal cancer SMAD2 AJCC stage Spearman's correlation 0.229 Significance (two-tailed) 0.032* N 88 Positive number Spearman's correlation 0.258 Significance (two-tailed) 0.047* N 60 Table 7 Relationship between SMAD2 expression and tumor characteristics in patients with colorectal cancer 3.2. Loss of NME4 promoted apoptosis and inhibited the activity and migration of CRC cell activity Transfection with shNME4 was effectively reduced NME4 expression as reported by both wb and qpcr results (Fig. 2A-B). The results of CCK-8 assay indicated that shNME4 transfection caused a downregulation of CRC cell proliferation (Fig. 2C). Flow cytometry results indicated that shNME4 transfection elevated the total apoptosis rate of CRC cells (Fig. 2D). 3.6. NME4 promoted CRC cell proliferation and migration i SCAD2 To investigate the role of SMAD2 in the development of CRC, we conducted functional probes. Figure 8A indicated that overexpression of NME4 increased cell activity and knockdown of SMAD2 decreased it, while simultaneous overexpression of NME4 and knockdown of SMAD2 decreased cell activity compared with overexpression of NME4 alone. Moreover, wound healing and cloning experiments revealed that simultaneous overexpression of NME4 and knockdown of SMAD2 effectively reduced cell migration and proliferation compared to overexpression of NME4 alone (Fig. 8B-C). Page 12/24 3.4. Knockdown of NME4 suppressed SMAD2 expression and mTOR pathway Page 12/24 The cDNA microarray analysis suggested that cells in the shNME4 group had significantly differentially expressed genes compared to cells in the shCtrl group (Fig. 5A). In the classical IPA-based pathway analysis, we noted interactions between NME4 and ERK/MAPK Signaling, PI3K/AKT Signaling, NF-KB Signaling, mTOR Signaling, and TGF-β Signaling pathway gene molecules (Fig. 5B). The results of the biomimetic analysis also suggest a significant enrichment of mTOR Signaling (Fig. 5C). Meanwhile, we also screened several genes with strong association with NME4, among which MLST8, NPRL3 and TELO2 were positively associated, and KRAS, MAPK1, ATP6V1A, CAB39, CHUK and PIK3CA were negatively associated (Fig. 5D). Naturally, the predicted results were verified next. WB outcomes showed that knockdown of NME4 effectively inhibited mTOR pathway activation and reduced SMAD2 expression (Fig. 6A-B). The qpcr results also clearly demonstrate that knockdown of NME4 effectively decreases SMAD2, SMAD6, MYC, BMP2, HPAS, HSPB1 and PTEN expression, and increases SKI and RPSA expression (Fig. 6C). 3.5. SMAD2 is positively correlated with NME4 CO-ip assay verified that SMAD2 interacts with NME4 (Fig. 7A). After confirmation that both shSMAD2 1– 3 effectively reduced SMAD2 expression (Fig. 7B), the expression of both NME4 and SMAD2 was upregulated after overexpression of NME4; knockdown of SMAD2 downregulated both NME4 and SMAD2 expression; simultaneous overexpression of NME4 and knockdown of SMAD2 decreased in NME4 and SMAD2 expression compared to overexpression of NME4 (Fig. 7C-E). Furthermore, the SMAD2 protein levels were upregulated in CRC tissues compared to the adjacent tissues (Table. 5). One-way analysis indicated that SMAD2 expression had a considerable effect with AJCC stage and positive number (P = 0.033 and P = 0.048, Table. 6). Spearman's correlation analyses verified the considerable correlation between high SMAD2 expression and CRC AJCC stage and positive number (Table. 7). 4. Discussion Despite the serious burden CRC poses to society, there are currently no available effective biological targets for its diagnosis and treatment [24, 25]. In the present study, we investigated the specific Page 13/24 expression of NME4 in CRC tissues and proved its pro-carcinogenic effect. Furthermore, we investigated the possible mechanism by which NME4 may act via regulating SMAD2. NME4, as well as naming Nm23-H4, is an abbreviation for NME/NM23 nucleoside diphosphate kinase 4, and its primary role is the synthesis of nucleoside triphosphates [26]. Current studies on its function are mostly focused on mitochondrial function and its role in cancer. It was determined that NME4 interacts with CL to target dysfunctional mitochondria for lysosomal degradation during mitochondrial inner membrane potential collapse [11]. NME4 has also been characterized as a novel metastasis suppressor gene [26]. In this study, we first clarified the specific high expression of NME4 in CRC tissues by tissue microarray analysis. It was also found that high expression of NME4 was relevant to pathological staging and poor prognosis of CRC. That established an important role of NME4 in CRC. Earlier research evidenced that NME4 serves as a possible novel tumor promoter that enhances non-small cell lung cancer progression by overcoming cell cycle arrest and promoting proliferation [18]. Our experimental results were similar in that knockdown of NME4 expression inhibited the proliferation and migration of CRC cells and promoted apoptosis. It demonstrated the pro-carcinogenic role of NME4 in CRC. Similarly, subcutaneous injection of colon cancer cells for subcutaneous tumorigenesis assay confirmed that knockdown of NME4 gene in mice also inhibited tumor growth. Knockdown of NME4 was also evaluated in esophageal squamous cell carcinoma, and similar to our findings, inhibited the proliferation and invasion of cancer cells [16]. To dive deeper into the mechanism of NME4 action, IPA and bioinformatics were performed to analyze possible downstream genes and pathways. The mTOR signalling and SMAD2 were selected to further study. mTOR activity is frequently dysregulated in a variety of human cancers, and upregulation of mTOR signaling facilitates tumor growth and progression through multiple mechanisms [27]. Study evidenced that negative regulation of AKT/mTOR/HIF1α signaling inhibits proliferation of colorectal cancer [28]. Additionally, activation of Smad2/3 promotes metastasis in colorectal and breast cancers [29]. These findings are consistent with our results. Besides, we verified the interaction relationship between NME4 and SMAD2. 4. Discussion In CRC cells, overexpression of NME4 elevated NME4 and SMAD2 expression, and knockdown of SMAD2 expression decreased NME4 and SMAD2 expression; while knockdown of SMAD2 after overexpression of NME4 also efficiently decreased NME4 and SMAD2 expression. Subsequently, we demonstrated that NME4 promotes proliferation and migration of CRC cells through regulation of SMAD2. 5. Conclusions In conclusion, this study identifies a key role for NME4 in CRC development and explores in detail the mechanisms that positively regulate SMAD2. the critical role of NME4 in CRC development may provide evidence for the development of new therapies targeting NME4 for CRC. Author Contributions F.ZY and Z.BA conceived and designed the study. F.ZY and G.QJ performed the majority of the experiments, performed preliminary analysis and drafted the manuscript. C.Z and Q.HX participated in the completion of the experiments. F.ZY and Z.BA reviewed the data analysis and revised the manuscript. All the authors read and approved the final manuscript and agree to be accountable for all aspects of the work. Data Availability Statement The data supporting the findings of this study are available from the corresponding author upon reasonable request. Declarations Page 14/24 Page 14/24 Conflicts of Interest The authors have no conflict of interest. Ethics Statement N/A. Informed Consent N/A. Registry and the Registration No. of the study/trial N/A. Animal Studies N/A. Conflicts of Interest The authors have no conflict of interest. Animal Studies Animal Studies References 1. Kossenas K, Constantinou C. Epidemiology, Molecular Mechanisms, and Clinical Trials: an Update on Research on the Association Between Red Meat Consumption and Colorectal Cancer[J]. Curr Nutr Rep. 2021;10(4):435–67. 2. Li N, Lu B, Luo C, et al. Incidence, mortality, survival, risk factor and screening of colorectal cancer: A comparison among China, Europe, and northern America[J]. Cancer Lett. 2021;522:255–68. 3. Xia C, Dong X, Li H, et al. Cancer statistics in China and United States, 2022: profiles, trends, and determinants[J]. Chin Med J (Engl). 2022;135(5):584–90. 4. Lawrence BJ, Alexander E, Grant H, et al. Colorectal cancer and absolute risks[J]. Gut. 2021;70(11):2215–6. Page 15/24 5. Mahmoud NN. Colorectal Cancer: Preoperative Evaluation and Staging[J]. 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Hua H, Kong Q, Zhang H, et al. Targeting mTOR for cancer therapy[J]. J Hematol Oncol. 2019;12(1):71. 28. Weng ML, Chen WK, Chen XY, et al. Fasting inhibits aerobic glycolysis and proliferation in colorectal cancer via the Fdft1-mediated AKT/mTOR/HIF1alpha pathway suppression[J]. Nat Commun. 2020;11(1):1869. 28. Weng ML, Chen WK, Chen XY, et al. Fasting inhibits aerobic glycolysis and proliferation in colorectal cancer via the Fdft1-mediated AKT/mTOR/HIF1alpha pathway suppression[J]. Nat Commun. 2020;11(1):1869. 29. Zhang L, Zhu Z, Yan H, et al. Creatine promotes cancer metastasis through activation of Smad2/3[J]. Cell Metab. 2021;33(6):1111–23. e1114. Figures Page 17/24 Figure 1 Figure 1 Page 17/24 Page 17/24 NME4 protein expression in CRC tissue samples and corresponding non‐cancer tissue samples. (A) Representative TMA‐IHC images of different staining intensities of NME4. (B) Patients with high NME4 expression showed lower OS compared to those with low NME4 expression. p<0.001. ** epresentative TMA‐IHC images of different staining intensities of NME4. (B) Patients with high NME4 xpression showed lower OS compared to those with low NME4 expression. p<0.001. gure 2 ffect of shNME4 on CRC cell activity, apoptosis and cell cycle progression A) WB detected NME4 protein expression in shCtrl and shNME4 groups. (B) qpcr detection of NME4 RNA expression in shCtrl and shNME4 groups. (C) CCK-8 assay of OD450 in shCtrl and shNME4 roups.(D) Flow cytometry assay for apoptosis rate in shCtrl and shNME4 groups. (E) Flow detection of ell cycle progression in shCtrl and shNME4 groups. Error bars represent mean±SEM and represent the lt f th i t 0 05 0 01 * Figure 2 Effect of shNME4 on CRC cell activity, apoptosis and cell cycle progression (A) WB detected NME4 protein expression in shCtrl and shNME4 groups. (B) qpcr detection of NME4 mRNA expression in shCtrl and shNME4 groups. (C) CCK-8 assay of OD450 in shCtrl and shNME4 groups.(D) Flow cytometry assay for apoptosis rate in shCtrl and shNME4 groups. (E) Flow detection of cell cycle progression in shCtrl and shNME4 groups. Error bars represent mean±SEM and represent the Effect of shNME4 on CRC cell activity, apoptosis and cell cycle progression (A) WB detected NME4 protein expression in shCtrl and shNME4 groups. (B) qpcr detection of NME4 mRNA expression in shCtrl and shNME4 groups. (C) CCK-8 assay of OD450 in shCtrl and shNME4 groups.(D) Flow cytometry assay for apoptosis rate in shCtrl and shNME4 groups. (E) Flow detection of cell cycle progression in shCtrl and shNME4 groups. Error bars represent mean±SEM and represent the results of three experiments. p<0.05, p<0.01. * (A) WB detected NME4 protein expression in shCtrl and shNME4 groups. (B) qpcr detection of NME4 mRNA expression in shCtrl and shNME4 groups. (C) CCK-8 assay of OD450 in shCtrl and shNME4 groups.(D) Flow cytometry assay for apoptosis rate in shCtrl and shNME4 groups. (E) Flow detection of cell cycle progression in shCtrl and shNME4 groups. Error bars represent mean±SEM and represent the results of three experiments. p<0.05, p<0.01. * Page 18/24 Page 18/24 Figure 3 Effect of shNME4 on CRC cell migration and invasion. A) Representative images and quantitative analysis of wound healing of cells in the shCtrl and shNME4 groups were taken after scratching the CRC cell surface for 0 and 24 h. (B) Number of migrating cells in he shCtrl and shNME4 groups. Error bars represent the mean ± SEM and are representative of the result of three experiments p<0 05 p<0 001 ** Figure 3 Effect of shNME4 on CRC cell migration and invasion. Effect of shNME4 on CRC cell migration and invasion. (A) Representative images and quantitative analysis of wound healing of cells in the shCtrl and shNME4 groups were taken after scratching the CRC cell surface for 0 and 24 h. (B) Number of migrating cells in the shCtrl and shNME4 groups. Error bars represent the mean ± SEM and are representative of the results of three experiments. p<0.05, p<0.001. Page 19/24 igure 4 ffect of shNME4 on the growth of tumor in vivo. A) Quantification of bioluminescence images and fluorescence expression in the shCtrl and shNME4 roups of the exosome mouse model. (B) Tumor images. (C) Quantification of tumor weight. (D) Quantitative analysis of tumor volumes. (E) Representative images of HE staining. (F) Representative mages of Ki67 staining. Figure 4 Effect of shNME4 on the growth of tumor in vivo. (A) Quantification of bioluminescence images and fluorescence expression in the shCtrl and shNME4 groups of the exosome mouse model. (B) Tumor images. (C) Quantification of tumor weight. (D) Quantitative analysis of tumor volumes. (E) Representative images of HE staining. (F) Representative images of Ki67 staining. Page 20/24 Figure 5 Bioconductivity analysis of pathways and genes strongly associated with NME4. (A) Volcano plot. (B) Significant enrichment of differential genes in classical pathways. (C) KEGG i h (D) Si ifi i h f diff i l i l i l h Figure 5 Bioconductivity analysis of pathways and genes strongly associated with NME4. (A) Volcano plot (B) Significant enrichment of differential genes in classical pathways (C) KEGG Figure 5 Bioconductivity analysis of pathways and genes strongly associated with NME4. Bioconductivity analysis of pathways and genes strongly associated with NME4. (A) Volcano plot. (B) Significant enrichment of differential genes in classical pathways. (C) KEGG enrichmenr. (D) Significant enrichment of differential genes in classical pathways. (A) Volcano plot. (B) Significant enrichment of differential genes in classical pathways. (C) KEGG enrichmenr. (D) Significant enrichment of differential genes in classical pathways. Page 21/24 gure 6 nockdown of NME4 inhibited mTOR signaling and SMAD2. A) WB detected NME4-related pathway-associated protein expression. (B) WB detected protein xpression associated with NME4 (C) qpcr detected protein expression associated with NME4 Figure 6 Knockdown of NME4 inhibited mTOR signaling and SMAD2. Knockdown of NME4 inhibited mTOR signaling and SMAD2. (A) WB detected NME4-related pathway-associated protein expression. (B) WB detected protein expression associated with NME4. (C) qpcr detected protein expression associated with NME4. Page 22/24 Page 22/24 Page 22/24 Figure 7 NME4 and SMAD2 were positively correlated. Figure 7 Figure 7 NME4 and SMAD2 were positively correlated. (A) CO-ip assay. (B) qpcr detection of shSMAD2-1, shSMAD2-2 and shSMAD2-3 efficiency. (C) NME4 expression detected by qpcr. (D) SMAD2 expression detected by qpcr. (E) NME4 and SMAD2 expression detected by WB. NME4 and SMAD2 were positively correlated. NME4 and SMAD2 were positively correlated. (A) CO-ip assay. (B) qpcr detection of shSMAD2-1, shSMAD2-2 and shSMAD2-3 efficiency. (C) NME4 expression detected by qpcr. (D) SMAD2 expression detected by qpcr. (E) NME4 and SMAD2 expression detected by WB. Page 23/24 Page 23/24 Figure 8 NME4 promoted CRC cell proliferation and migration via SCAD2. (A) CCK-8 assay of OD450. (B) Representative images and quantitative analysis of wound healing of cells were taken after scratching the CRC cell surface for 0 and 24 h. (C) Number of migrating cells. Error bars represent the mean ± SEM and are representative of the results of three experiments. p<0.05, p<0.01. * Figure 8 NME4 promoted CRC cell proliferation and migration via SCAD2 Figure 8 Figure 8 NME4 promoted CRC cell proliferation and migration via SCAD2. (A) CCK-8 assay of OD450. (B) Representative images and quantitative analysis of wound healing of cells were taken after scratching the CRC cell surface for 0 and 24 h. (C) Number of migrating cells. Error bars represent the mean ± SEM and are representative of the results of three experiments. p<0.05, p<0.01. * (A) CCK-8 assay of OD450. (B) Representative images and quantitative analysis of wound healing of cells were taken after scratching the CRC cell surface for 0 and 24 h. (C) Number of migrating cells. Error bars represent the mean ± SEM and are representative of the results of three experiments. p<0.05, p<0.01. * Page 24/24
https://openalex.org/W4296640286	https://www.intechopen.com/citation-pdf-url/82666	English	null	Pollution Evaluation of Industrial Effluents from Consolidated Breweries: A Case Study from Benue State, Nigeria	IntechOpen eBooks	2,023	cc-by	8,012	Abstract Industrial effluent discharged into surface water is an environmental concern, as it affects the esthetics, water quality as well as microbial and aquatic flora. Brewery effluents were analyzed for physicochemical parameters (pH, temperature, conduc- tivity, turbidity, total dissolved solids (TDS), dissolved oxygen (DO), biological oxy- gen demand (BOD), chemical oxygen demand (COD), nitrate, and sulfate, chloride) and heavy metals (As, Cd, Co, Cr, Fe, Mn, Ni, Pb, and Zn). Atomic absorption spectrophotometer was used to characterize heavy metals using standard analytical methods and compared with WHO standards. The result showed that pH (6.2–6.98), conductivity (137–273 μS/cm), chloride (31–53 mg/l), nitrate (7.53–10.72 mg/l), BOD, and DO were within the WHO limit. However, turbidity, sulfate, and phosphate were above the WHO limit. Heavy metal concentrations Cr, Ni, Pb, Mn, As, and Cd were higher than the WHO limit and vice versa for Fe, Zn, and Co. Ecological risk assess- ment revealed that effluent samples pose low to moderate ecological risk, for As, Pb, and Ni. Therefore, there is a need for proper treatment and continual monitoring before discharge into the environment. Keywords: brewery effluents, heavy metal, ecological risk, contamination factor, pollution load index 1. Introduction Due to an increase in industrial activities, environmental pollution is one of the most critical problems in developing countries. More challenging is the unsafe dis- posal of solid wastes/industrial wastes into the ambient environment. Industries that use large amounts of water in their processes include chemical manufacturing, steel plants, metal processors, etc. Effluents and most products from industries create 1 1 River Basin Management - Under a Changing Climate serious pollution to water bodies and soils. Water bodies especially freshwater reser- voirs, and rivers are the most affected. This has rendered underground and surface waters unsafe for human, recreational, and agricultural use. Biotic life is destroyed and natural ecosystems are infected. Human life is at risk and the principle of sus- tainable development is compromised [1]. Moderately or untreated industrial effluents may contain high levels of pollutants which in water body systems cause an increase in BOD, COD, Total Dissolved Solids (TDS), Total Suspended Solids (TSS), toxic metals such as Cd, As, Cr, Ni, and Pb, and fecal coliform. Hence make such water unsuitable for drinking, irrigation, and aquatic life support. Industrial wastewater impacts include high BOD from biodegradable wastes such as those from human sewage, pulp and paper industries, slaughterhouses, tanneries, and chemical industries [2–4]. Brewery wastewater effluent is highly variable in quality and composition. The products of the brewery operations include large volumes of wastewater from liquors pressed from grains and yeast recovery, from the Clean-in-place system located in the brewing house, cellar house, and bottling house, which is discharged into the nearby River. These industrial wastewaters are the main source of heavy metals since nearly all industrial by-products consist of some level of heavy metals [5]. y y Wastewater shows different degrees of environmental nuisance and contamina- tion hazards due to its chemical and microbiological characteristics. Excessive nutri- ents (primarily, nitrogen and phosphorus) in wastewater, sludge, and excreta may contaminate surface waters and cause eutrophication, which affects the esthetics of water bodies (lakes, rivers), and results in odor and appearance problems, which was evident in the physiochemical evaluation of brewery effluents in Enugu State [6] and Edo State [7] both in Nigeria. 1. Introduction Previous research has shown that the release of untreated effluents has the poten- tial to negatively impact aquatic organisms, by decreasing pH to acid level, increasing conductivity, temperature turbidity, and total solids in such an environment leading to a decrease in dissolved oxygen with microbial bloom from rich nutrients (nitro- groups, sulfur-groups, and phosphors) [8–12]. Heavy metals are also released from these effluents. Studies have shown that long- term exposure to low concentrations of some heavy metallic anions can result in the development of sub-chronic to chronic illnesses and diseases in a given population, usually between 1 in 1000 to 1 in 1,000,000 as institutionalized by the US. Environ- mental Protection Agency (EPA) [13, 14]. The forms in which metal pollutants exist in wastewater discharges determine their release into the aquatic ecosystem. Some metals become bio-available when soluble airborne solids are dissolved by weak acids such as carbonic acid. Their concentration became enhanced by the abundance of metals in road dust and tire residues [15]. The physicochemical properties and selected heavy metals of industrial effluents from consolidated Breweries in Benue State, Nigeria were studied. Therefore, the study aimed to assess the concentration levels, and ecological and health risks of industrial effluents discharged daily into the nearby River, a primary source of fishing activity and domestic purposes in the neighboring community. Information from the present study will be helpful to the relevant government agencies and policymakers in preparing preventive action to control the direct discharge of efflu- ents from chemical industries, agro-based activities, and domestic waste to the rivers and the sea. 2 Pollution Evaluation of Industrial Effluents from Consolidated Breweries: A Case Study… DOI: http://dx.doi.org/10.5772/intechopen.105955 Pollution Evaluation of Industrial Effluents from Consolidated Breweries: A Case Study… DOI: http://dx.doi.org/10.5772/intechopen.105955 Pollution Evaluation of Industrial Effluents from Consolidated Breweries: A Case Study… DOI: http://dx.doi.org/10.5772/intechopen.105955 2.1 Location/study area Consolidated breweries plc Makurdi, Benue state is located at kilometer 5 Gboko Road, pm 102,339 Makurdi, Benue State, Nigeria. The locality is predominantly an agrarian community where farming and animal husbandry takes place with a few industrials such as small and medium scale enterprise (mechanic workshop, local markets, construction, and mining) activities. Figure 1 shows the map of the study location. 2.3.5 Biological oxygen demand (BOD) BOD measures the amount of dissolved oxygen used by microorganisms in the oxidation of organic matter in a water sample. The BOD was determined by collecting the water sample in a sealed bottle, incubating for a standard period in the dark (usually 5 days at 200°C), and determining the residual oxygen in the water at the end of incubation (Model H1 9146, HANNA). BOD was determined using the following formula as described by AOAC [16]. BOD ¼ DO1–DO5 ð Þ mg=l (1) Where: DO1 = Sample before incubation. DO1 = Sample before incubation. DO5 = Sample after 5 days of incubation. DO5 = Sample after 5 days of incubation. 2.3.2 Turbidity The turbidity of the samples was determined using the turbidity meter (Labtech digital model), and EPA 180 was selected as the measurement mode. 2.3.4 Dissolved oxygen (DO) DO is the amount of gaseous oxygen dissolved in the water. It was determined using a dissolved oxygen meter (Model H1 9146, HANNA) as described by AOAC [16]. 2.2 Sample collection and preparation Four samples of effluent were collected with a cleaned plastic container at a different location in the brewery. The plastic containers used were carefully washed with 1% HNO3 acid, rinsed with tap water, and then distilled water. The samples were labeled appropriately; Sample A-untreated effluent, B- treated effluent, C- contact point of the treated effluent with the river, and D- 10 kilometers away from the contact point. These samples were transported to the laboratory for analysis in an ice- packs container and protected from direct sunlight. They were stored in the refriger- ator at 20°C. Figure 1. Study map showing the study locations. Figure 1. Study map showing the study locations. Figure 1. Study map showing the study locations. Figure 1. gu . Study map showing the study locations. 3 River Basin Management - Under a Changing Climate 2.3.1 pH The pH values of the samples were determined at the point of sampling using a portable pH meter after calibrating against buffer solution (pH 4.7 and 9.2). 2.3.3 Total dissolved solids (TDS) TDS was determined by an electrometric method using a TDS meter (Jenway, model 4076). 2.3 Physicochemical analysis 2.3.1 pH 2.3.8 Sulfate Ten ml of water sample was pipetted into a conical flask plus 5 ml of 2 N HCl and 0.05 N BaCl2. The solution was boiled for 5 minutes and allowed to cool. Two ml of ammonium (NH4 +) and 5 ml of 0.01 N EDTA were added to the solution and boiled for 5 minutes. Five ml of pH buffer 10 and 3 drops of Eriochrome black) indicators were added. The solution was titrated with 0.01 N MgCl2. Sulfate was determined by using the following formula: 10 Tv 0:93 ð Þ 96:01484 10 mg=l (4) Where: Tv = Titrate value of the sample. 96.01484 = molecular weight of sulfate. 0.93 = Constant. 10 Volume of water sample used (4) 10 p 96.01484 = molecular weight of sulfate. 10 = Volume of water sample used. 2.3.6 Chemical oxygen demand (COD) COD is the amount of oxygen consumed under specified conditions of organic and oxidizable inorganic matter in water and wastewater. The COD is determined first by pipetting 10 ml of the water sample into a conical flask and adding 5 ml of 0.025 N potassium dichromate (K2Cr207). 15 ml of sulfuric acid (H2S04) was added to it, and the solution was diluted with 40 ml of distilled water to get a 70 ml solution. Seven drops of phenolphthalein ferrous sulfate indicator were added, and the solution was allowed to cool. The solution was titrated with 0.025 N ferrous ammonium sulfate. A blank solution was also titrated. COD was determined by using the following formula: 4 Pollution Evaluation of Industrial Effluents from Consolidated Breweries: A Case Study… DOI: http://dx.doi.org/10.5772/intechopen.105955 Pollution Evaluation of Industrial Effluents from Consolidated Breweries: A Case Study… DOI: http://dx.doi.org/10.5772/intechopen.105955 Pollution Evaluation of Industrial Effluents from Consolidated Breweries: A Case Study… DOI: http://dx.doi.org/10.5772/intechopen.105955 T1 T2 ð Þ N 8000 C Volume of water sample used mg=l ð Þ (2) (2) Where: T1 = Titer value for blank. T2 = Titer value for effluent sample. N = Normality of the ferrous ammonium Sulfate used is 0.025. C = Chloride correction which is in Milligram per liter of chloride 0.03. 8000 = Milliequivalent weight of oxygen x 1000 mL/L. T1 = Titer value for blank. 2.3.7 Chloride Ten ml of the water sample was pipetted into a conical flask with 3 drops of potassium chromate (K2Cr02) and the solution was titrated with 0.1 N silver nitrate (AgNO3). Chloride was determined by using the following formula: Tv x 0:003546 x 105 ð Þmg l (3) Tv x 0:003546 x 105 ð Þmg l (3) Tv x 0:003546 x 105 ð Þmg l (3) Tv x 0:003546 x 105 ð Þmg l (3) Where: Tv = Titer value of sample. p 0.003546 = equivalent weight of chloride 2.4.2 Degree of contamination (Cdeg) This is the summation of the contamination factor of all chemical contaminants in the study site. It is calculated as follows: Cdeg ¼ X CF ð Þ ¼ CF1 þ CF2 þ CF3 þ ∙∙∙þ CFn (6) (6) For the description of contamination degree (Cdeg), the following terminologies have been used: i. Cdeg <8: low degree of contamination; i. Cdeg <8: low degree of contamination; i. Cdeg <8: low degree of contamination; ii. 8 ≤Cdeg <16: moderate degree of contamination; iii. 16 ≤Cdeg <32: considerable degree of contamination; iv. Cdeg ≥32: very high degree of contamination. iv. Cdeg ≥32: very high degree of contamination. 2.4.1 Contamination factor (CF) 2.4.1 Contamination factor (CF) CF is the extent of pollution of the contaminant of interest, it is expressed: CF ¼ chemical contaminant of interest background value using WHO standard (5) (5) The following terminology was used to describe the contamination factor: The following terminology was used to describe the contamination factor: The following terminology was used to describe the contamination factor: i. CF < 1: low contamination factor; ii. 1 ≤CF < 3: moderate contamination factor; iii. 3 ≤CF < 6: considerable contamination factor; iv. CF ≥6 _ very high contamination factor. 2.4.2 Degree of contamination (Cdeg) 2.4.2 Degree of contamination (Cdeg) 2.3.9 Analysis of effluent samples for heavy metals concentration An effluent sample of 200 mL was measured into a 500 mL beaker, and 5 mL of concentrated nitric acid was carefully added. This solution was concentrated to 20 mL by heating in a water bath for a few hours. The concentrated extract was cooled and transferred into a 50 mL standard flask, then made up to mark with distilled water. Heavy metal (Pb, Cd, Zn, As, Cr, Fe, Mn, Co, and Ni) contents of the samples were determined using atomic absorption spectrophotometer (Spectra AA Varian 400 plus) involving direct aspiration of the aqueous solution into air-acetylene flame. A 5 River Basin Management - Under a Changing Climate reagent blank was prepared and analyzed. Heavy metal concentrations of a series of standards were determined and a calibration graph was developed. From the graph, the concentrations of heavy metals in the sample were calculated as described by Braid et al. [17]. 2.4 Ecological risk assessment Ecological risk assessment 1 Contamination factor (CF) 2.4.1 Contamination factor (CF) 2.4.3 Modified degree of contamination (mCdeg) This is the average effect of all chemical contaminants of interest, the advantage of mCdeg is that it quantifies the chemical contaminants into a composite aggregate to derive salient information about the study site using the formula: 6 Pollution Evaluation of Industrial Effluents from Consolidated Breweries: A Case Study… DOI: http://dx.doi.org/10.5772/intechopen.105955 Pollution Evaluation of Industrial Effluents from Consolidated Breweries: A Case Study… DOI: http://dx.doi.org/10.5772/intechopen.105955 mCdeg ¼ 1 n X CF ð Þ (7) (7) Where: n is the total chemical contaminant and CF is the contamination factor. mCdeg is classified as: i. mCdeg <4: low moderate contamination; ii. 4 ≤mCdeg <16: medium moderate contamination; iii. 12 ≤mCdeg <20: high moderate contamination; iv. mCdeg ≥20: extreme moderate contamination. i. mCdeg <4: low moderate contamination; ii. 4 ≤mCdeg <16: medium moderate contamination; iv. mCdeg ≥20: extreme moderate contamination. iv. mCdeg ≥20: extreme moderate contamination. 2.4.4 Pollution load index (PLI) This is the geometric mean of CF value to the nth number of chemical contami- nants of interest, it is given as described by Tomlinson et al. [18]: PLI ¼ CF1 CF2 CF3 ∙∙∙ CFn ð Þ1=n (8) PLI ¼ CF1 CF2 CF3 ∙∙∙ CFn ð Þ1=n (8) Where: n is the total chemical contaminant. CF is the contamination factor. The PLI gives the level of pollution classified as: Where: n is the total chemical contaminant. CF is the contamination factor. The PLI gives the level of pollution classified as: Where: i. PLI < 1: no pollution; ii. 1 < PLI < 2: modest pollution; iii. 2 < PLI < 3: high pollution; iii. 2 < PLI < 3: high pollution; iv. 3 < PLI: extremely high pollution. iv. 3 < PLI: extremely high pollution. 2.4.5 Potential ecological risk index (PERI) Assesses the toxicity factor of a particular chemical contaminant of interest, where the definite contamination status is evaluated concerning the ecosystem. It is expressed as shown in Eq. (9) and (10). A methodology to assess ecological risks for aquatic pollution was developed by Hakanson [19]. The ecological risk index (RI) is calculated as a sum of eight elements of heavy metals (As, Cd, Cr, Ni, Mn, Pb, Co, and Zn). PERI ¼ X Er ¼ TF CF (9) Ri ¼ X Er (10) (9) (9) (10) Where: : Er is the ecological risk index of different chemical contaminants, Er is the ecological risk index of different chemical contaminants, 7 River Basin Management - Under a Changing Climate TF is the toxicity factor of each chemical contaminant of interest as described by Hussain et al. [20] and Umeh et al. [21]. CF is the contamination factor in Eq. (5), CF is the contamination factor in Eq. (5), RI is the risk index calculated as the sum of the potential ecological risk factors for RI is the risk index calculated as the sum of the potential ecological risk factors for heavy metals in the wastewater. y Er and RI values are categorized using: y Er and RI values are categorized using: i. Er <40 and RI <150: low ecological risk; ii. 40 < Er ≤80 and 150 < RI <300: moderate ecological risk; iii. 80 < Er ≤160, appreciable ecological risk; iv. 160 < Er ≤320 and 300 < RI <600: high ecological risk; v. Er > 320 and RI ≥600: extremely high ecological risk. i. Er <40 and RI <150: low ecological risk; ii. 40 < Er ≤80 and 150 < RI <300: moderate ecological risk; iii. 80 < Er ≤160, appreciable ecological risk; i. Er <40 and RI <150: low ecological risk; ii. 40 < Er ≤80 and 150 < RI <300: moderate ecological risk; iii. 80 < Er ≤160, appreciable ecological risk; iv. 160 < Er ≤320 and 300 < RI <600: high ecological risk; v. 160 < Er ≤320 and 300 < RI <600: high ecological risk; v. Er > 320 and RI ≥600: extremely high ecological risk. v. Er > 320 and RI ≥600: extremely high ecological risk. Table 1. Physical parameters of the studied effluents. Table 1. Physical parameters of the studied effluents. 3. Results The values of the physical and chemical parameters of effluent samples from the Brewery are presented in Table 1 and Figure 2 respectively. The pH values were 6.98, 6.20, 6.48, and 6.72 for sampling points A, C, D, and E respectively. In all sampling locations, the highest pH value of 6.98 was obtained at sampling point A and the lowest value of 6.20 was obtained at sampling point C. p g p The TDS values in the present work ranged from 4.0 to 48.0 mg/l. Dissolved oxygen values ranged from 4.70 to 23.60 mg/l for all sampling points. The highest (15.90) and lowest (0.40) biological oxygen demand values were recorded at sampling points C and A, respectively. The chemical oxygen demand in the present work ranged from 18.88 to 19.24.32 mg/l. The values of nitrate varied between 1.28–1.95 mg L-1. The highest (7.16) phosphate value was recorded at sampling point C and the lowest (6.93) at sampling point A. Sulfate values ranged from 31.69 to 35.39 mg/l for all sampling points. Chloride values ranged from 28.00 to 53.00 mg/l for all sampling points. g g g Figure 3 shows the results of heavy metals concentration analyses of effluent samples across the study area. Among the 9 elements studied, concentrations of As, Pb, and Ni were higher than WHO recommended limits. In contrast, lower concen- trations of Co, Mn, and Zn were observed in the different sampling locations. Ele- ments displayed wide variations in their distribution, suggesting control of Parameters Sample A Sample B Sample C Sample D WHO pH 6.20 6.98 6.48 6.72 6.50–8.50 Turbidity (NTU) 102.70 115.00 39.90 30.70 50. 00 TDS (mg/l) 48.00 4.00 16.00 10.00 500.00 Conductivity (μS/cm) 272.00 273.00 178.00 187.00 1000.00 A-un treated effluent, B- treated effluent, C- contact point of the treated effluent with the river and D- 10 kilometers away from the contact point. 8 Pollution Evaluation of Industrial Effluents from Consolidated Breweries: A Case Study… DOI: http://dx.doi.org/10.5772/intechopen.105955 Pollution Evaluation of Industrial Effluents from Consolidated Breweries: A Case Study… DOI: http://dx.doi.org/10.5772/intechopen.105955 anthropogenic activities on water chemistry. Overall, concentration of the study ele- ments followed the order: As >Fe > Mn > Zn > Ni > Pb > Cr > Co > Cd. The sampling site C recorded the highest concentration of Fe and As. Figure 3. Heavy metal concentrations. Figure 2. Chemical parameters from the studied effluents. 4. Discussion Water quality in an aquatic environment is very important for the survival of its flora and fauna. Water pH can affect aquatic organisms as their metabolic activities are pH-dependent [22, 23]. The pH across the sampling points ranges from 6.20 in sample A to 6.98 in sample B, indicating slight acidity. A significant (P ≤0.05) difference was observed between the pH values for each sampling point, although, B and D were within the WHO [24] guideline regulatory limit of 6.5–8.5 set for drinking water, while samples A and C were a little below the standard limit. The slight acidity could be attributed to the chemicals used in the treatment processes and the water may serve as a sink for various wastes and chemical preservatives used in the brewery such as oxides of sulfur, nitro, carbon, and phosphor in turn form sulfuric, nitric, carbonic and phosphoric acid on reaction with water leading to microbial bloom from rich nutrients source thereby causing reduction in dissolved oxygen, increase turbid- ity, conductivity, odor and diminish aquatic esthetic respectively. Water pH helps to control metal solubility, and water hardness and serves as an indicator of water pollution [7, 9]. p Nitrate in the present study was all below-recommended limit when compared to the WHO [24] standard for safe drinking water. Nitrate is alleged to be an indicator of pollution in the public water supply [25]. It is the stable form of nitrogen that plays a significant role in the process of eutrophication. The conductivity range of the various sampling points varied considerably across the study area. Point B showed the highest value and, therefore, decreased along with the sampling points, most likely due to the effect of dilution and removal of soluble salts by biological utilization. y g The biological oxygen demand (BOD) and chemical oxygen demand (COD) are useful parameters in water quality analysis. The highest and lowest BOD values were recorded at sampling points A and B, respectively. Biological oxygen demand is the amount of oxygen required by aerobic microorganisms to stabilize the organic mate- rial of wastewater at a standardized temperature (20°C) and time of incubation (usually 5 days). It is used to indicate the organic strength of water. When BOD is less than 4 mg/l, water is deemed to be reasonably clean and unpolluted, while a BOD level greater than 10 mg/l indicates pollution [26]. 3. Results DO: Dissolved oxygen; BOD: Biological oxygen demand; COD: Chemical oxygen demand. DOI: http://dx.doi.org/10.5772/intechopen.105955 Figure 2. Figure 2. Chemical parameters from the studied effluents. DO: Dissolved oxygen; BOD: Biological oxygen demand; COD: Chemical oxygen demand. anthropogenic activities on water chemistry. Overall, concentration of the study ele- ments followed the order: As >Fe > Mn > Zn > Ni > Pb > Cr > Co > Cd. The sampling site C recorded the highest concentration of Fe and As. Figure 3. Heavy metal concentrations. Figure 2. Chemical parameters from the studied effluents. DO: Dissolved oxygen; BOD: Biological oxygen demand; COD: Chemical oxygen demand. 9 anthropogenic activities on water chemistry. Overall, concentration of the study ele- ments followed the order: As >Fe > Mn > Zn > Ni > Pb > Cr > Co > Cd. The sampling site C recorded the highest concentration of Fe and As. Figure 3. Heavy metal concentrations. Figure 2. Chemical parameters from the studied effluents. DO: Dissolved oxygen; BOD: Biological oxygen demand; COD: Chemical oxygen demand. 9 g 3 Heavy metal concentrations. anthropogenic activities on water chemistry. Overall, concentration of the study ele- ments followed the order: As >Fe > Mn > Zn > Ni > Pb > Cr > Co > Cd. The sampling site C recorded the highest concentration of Fe and As. anthropogenic activities on water chemistry. Overall, concentration of the study ele- ments followed the order: As >Fe > Mn > Zn > Ni > Pb > Cr > Co > Cd. The sampling site C recorded the highest concentration of Fe and As. anthropogenic activities on water chemistry. Overall, concentration of the study ele- ments followed the order: As >Fe > Mn > Zn > Ni > Pb > Cr > Co > Cd. The sampling site C recorded the highest concentration of Fe and As. 9 River Basin Management - Under a Changing Climate 4. Discussion g g p Chemical oxygen demand is a measure of organic contamination in water. It is the amount of dissolved oxygen required to cause chemical oxidation of the organic material in water and is a key indicator of the environmental health of surface water [18]. There was a gradual increase in chemical oxygen demand from point B to point D. Chemical oxygen demand values were below the WHO recommended value of 200 mg/l [24]. High chemical oxygen demand COD values indicate pollution due to oxidizable organic matter [27]. Phosphate concentration was high in all sampling points and greater than the WHO recommended value of 2.0 mg/l. Phosphate is known as a limiting nutrient in the aquatic ecosystem [28]. There is little variation in dissolved oxygen values of effluent samples across the study areas. The dissolved oxygen concentration is a function of temperature, pressure, salinity, and biological activities in the water body. The tropical aquatic ecosystem should have a dissolved oxygen concentration of at least 5 mg/l in other to support diversified biota, including fish [29–32]. The level of 4.70 mg/l for point B was within the WHO, [24] standard value of 5 mg/l necessary for aquatic productivity, while other points were above the standard limit of 5 mg/l. 10 Pollution Evaluation of Industrial Effluents from Consolidated Breweries: A Case Study… DOI: http://dx.doi.org/10.5772/intechopen.105955 Pollution Evaluation of Industrial Effluents from Consolidated Breweries: A Case Study… DOI: http://dx.doi.org/10.5772/intechopen.105955 The highest value of sulfate was observed in point C (35.39 mg/l). This value is far below the permissible limit stipulated by World Health Organization WHO, [24]. The present work was in line with the work of Alao [30], who also reported low sulfate levels in the water receiving brewery effluent in Majawe Ibadan. g y j The values of chloride and iron obtained from point B, C, and D falls below the WHO permissible limit, while point C was within the 1 mg/l desirable level from WHO. The result of chloride agrees with Imoobe and Koye [33], who reported the value of chloride in Eruvbi Stream to be below the permissible limit stipulated by the World Health Organization [24]. The discharge of industrial effluents into receiving water bodies invariably results in the presence of a high concentration of pollutants in the water and sediments. The pollutants are present in concentrations that may be toxic to different organ- isms [34–36]. 4. Discussion The concentration of Cadmium across the study area ranged from 0.001 at sampling points B and D to 0.007 at sampling point C. The values recorded were lower than (0.043 mg/l) and (0.072 mg/l) in the water reported by Oguzie and Okhagbuzo [37]. The value of all samples assessed was above the permissible limit of 0.003 ppm set by WHO [24] for drinking water except for sampling points B and D. High concentrations of Cadmium (Cd) have been reported to inhibit the bio-uptake of Phosphorus and Potassium by plants [38]. Specific industries involved in electroplating, pigments production, chemicals, and alloy processing are sources of cadmium to the urban environment. Chromium (Cr) levels in the effluents were relatively low across the different sampling points. The concentrations of chromium in effluents were below the 0.050 mg/l value recommended by the World Health Organization (WHO) [24] in industrial effluents except for sampling point B. A high concentration of nickel (Ni) was recorded in the effluent samples ranging from 0.114 ppm in point D to 0.246 ppm in point A. The concentrations of nickel in effluents are higher than the <1 mg/l value recommended by the WHO [24] in industrial effluents. Ni has wide applications in the manufacture of batteries, fertil- izer, welding products, electroplating, and household appliances and has essential functions in every area of industrial activity [2]. Lead (Pb) and Arsenic (As), a major environmental pollutant is a multi-organ poison that, in addition to well-known toxic effects, depresses immune status and causes damage to the central nervous system, kidney, and reproductive system [39]. The lead (Pb) values were quite low in all the sampling points except in point E where it was not detected. All the points showed a lead value above the maximum acceptable concentration. 4.1 Contamination factor/pollution index The contamination factor (CF) values were revealed in Table 2. Arsenic (As) can be categorized as a very high contamination factor across all the sampling locations. The highest values of CF of As at location C (48.46) and the lowest at location B (33.12), indicating severe anthropogenic contribution to the contamination load of rivers at this site. The CF of Cd (Cadmium) can be categorized as low to moderate. Two locations (B and D) can be categorized as having low CF of Cd, and two locations (A and C) can be categorized as having moderate CF of Cd. Lead (Pb) can be categorized as a very high CF across all the sampling locations. The highest values of CF of Pb at location A (32) and the lowest at location B (12.1). The CF of Ni (Nickel) can be categorized as considerable to very high contamination. Two locations 11 River Basin Management - Under a Changing Climate Sample locations/elements A B C D Arsenic (As) 48.26 33.12 48.46 45.20 Cadmium (Cd) 1.67 0.33 2.33 0.33 Cobalt (Co) 0.00 0.085 0.10 0.00 Chromium (Cr) 0.64 2.48 0.26 0.38 Iron (Fe) 3.42 0.624 0.83 0.53 Manganese (Mn) 0.51 1.035 0.73 0.30 Nickel (Ni) 8.20 7.17 4.96 3.80 Lead (Pb) 32.00 12.1 13.8 0.00 Zinc (Zn) 0.063 0.038 0.033 0.012 PLI 2.10 1.25 1.16 0.70 C.deg 94.7 25.14 71.50 50.55 M-C.deg 40.17 21.05 29.56 5.616 A-un treated effluent, B- treated effluent, C- contact point of the treated effluent with the river and D- 10 kilometers away from the contact point. PLI: pollution load index; C.deg.: degree of contamination; M-C.deg.: modified degree of contamination. A-un treated effluent, B- treated effluent, C- contact point of the treated effluent with the river and D- 10 kilometers away from the contact point. PLI: pollution load index; C.deg.: degree of contamination; M-C.deg.: modified degree of contamination. Table 2. Contamination factor, pollution index and contamination index. Table 2. Contamination factor, pollution index and contamination index. (C and D) can be categorized as having considerable CF of Ni, and two locations (A and B) can be categorized as having very high CF of Ni. The CF of cobalt and Zinc can be categorized as low contamination factors of Co and Zn, respectively. Other ele- ments such as Cr (0.26–2.48), Mn (0.29–1.035), and Fe (0.62–3.42) can be categorized as low to moderate CF. The result indicates that contamination of effluents from Nigeria Brewery contributed to As and Pb [21]. g y The pollution Load Index (PLI) is a resourceful tool to measure and compare contamination. Analyzed effluents samples discharged into rivers at locations A (2.1), B (1.25), and C (1.16) displayed higher PLI values (PLI > 1) and progressive deterio- ration in quality. Location D was observed to have a low pollution index value of 0.7. The order for PLI was A > B > C > D. Higher PLI values in rivers demonstrated substantial anthropogenic impacts on the river quality signifying the need for imme- diate intervention to prevent pollution. In contrast, lower PLI values pointed to no considerable anthropogenic activities, signifying no need for intervention but requir- ing constant monitoring [31]. g g Degrees of contamination (Cdeg) values of effluents from Nigeria’s brewery are revealed in Table 2. The degree of contamination across the sampling locations can be categorized into four categories according to the Patil et al. [28] classification. Sam- pling locations A, C, and D can be categorized as having a very high degree of contamination (Cd value = 94.7, 71.50, and 50.54, respectively), this indicates very severe anthropogenic pollution at these sampling sites. Location B indicates a consid- erable degree of contamination with a Cd value of 25.137. The present study revealed Pb and Ni as the most severe component causing moderate to very high river con- tamination. A similar pattern was noted for contamination degree (Cdeg), where sampling locations having dominant anthropogenic activities displayed a high Con- tamination degree. Regular monitoring of the river for the presence of trace elements, especially Arsenic, lead, and nickel, is required [34]. 4.2 Potential ecological risk index method The evaluation results on the potential ecological risk factor (Eir) and the potential ecological risk index (RI) are summarized in Table 3. The order of potential ecological risk coefficient (Eir) of heavy metals in discharge effluents was As > The evaluation results on the potential ecological risk factor (Eir) and the potential ecological risk index (RI) are summarized in Table 3. The order of potential ecological risk coefficient (Eir) of heavy metals in discharge effluents was As > Pb > Cd > Ni > Cr > Mn > Co > Zn. The mean potential ecological risk coefficient of Cd, Cr, Mn, Ni, Co, and Zn were all lower than 40, which is low ecological risk. At the same time, the mean potential ecological risk coefficient of Pb and As were greater than 80 and 320, respectively, which indicates moderate to extremely high ecological risk. All the sampling locations were at High to very high-risk levels where the RI values were much greater than 600. However, because most samples are contami- nated with As, Pb, and Ni, their impact on the ERI became very obvious and predom- inant. Therefore, the present study indicates that As, Pb, and Ni were the major heavy metal posing an ecological risk in the study area [21, 31, 35]. Table 2. Contamination factor, pollution index and contamination index. 12 Sample Locations/Elements A B C D Mean Arsenic (As) 482.6 331.2 484.6 452 437.6 Cadmium (Cd) 50.1 9.9 69.9 9.9 34.9 Cobalt (Co) 0 0.425 0.5 0 0.231 Chromium (Cr) 3.2 12.4 1.3 0.76 4.42 Manganese (Mn) 2.55 5.175 3.65 1.475 3.212 Nickel (Ni) 41 35.83 24.8 19 30.15 Lead (Pb) 160 60.5 69 0 72.37 Zinc (Zn) 0.038 0.063 0.0326 0.012 0.036 Risk index 739.48 455.5 653.78 483.14 582.9 Risk grade Extremely High High Extremely High High High A- un treated effluent, B- treated effluent, C- contact point of the treated effluent with the river and D-10 kilometers away from the contact point. Table 3. Potential ecological risk index. Pollution Evaluation of Industrial Effluents from Consolidated Breweries: A Case Study… DOI: http://dx.doi.org/10.5772/intechopen.105955 Pollution Evaluation of Industrial Effluents from Consolidated Breweries: A Case Study… DOI: http://dx.doi.org/10.5772/intechopen.105955 Pollution Evaluation of Industrial Effluents from Consolidated Breweries: A Case Study… DOI: http://dx.doi.org/10.5772/intechopen.105955 Acknowledgements The authors acknowledge and extend their sincere gratitude to all who assisted in realizing this present work. 5. Conclusion The laboratory analysis results of the effluent samples indicated that metals and other contaminants from the effluents have compromised the River quality. The results of the physicochemical analysis showed that sulfate, phosphate, COD, and heavy metals such as Cr, Ni, Pb, Mn, As, and Cd were slightly higher than WHO and FEPA standards for drinking water, while the pH, Chloride, Nitrate, Nitrite, TDS, Conduc- tivity, BOD, COD and some heavy metals such as Fe, Zn, Co were within the standard of WHO and FEPA, set for drinking water. The study, however, showed that some contaminants sampled were within statutory limits. It was also observed that sample A (Untreated effluent) and sample B (Treated effluent) had lower mean differences than sample C and sample D. Contamination factors follow a similar trend in metal contam- ination. At the same time, PLI index models confirmed that the effluents from the 13 River Basin Management - Under a Changing Climate different sampled locations were polluted, except for location D, which is unpolluted. The mean anthropogenic input for the sampled effluents for the individual metals followed the order As> Fe > Mn > Zn > Ni > Pb > Zn > Cr > Co > Cd. The ecological risk assessments for the heavy metals were at high ecological risk. Furthermore, the potential ecological index depicts As at extremely high risk, Pb at appreciable risk, Ni at moderate risk, and Cr, Mn, Co, Zn, and Cd at a low ecological risk level. Hence any significant increase would persuade environmental challenges. However, the present study recommends proper treatment of effluents before discharging to reduce their mean difference from the WHO standard and protect the health of the local population. Conflict of interest All the authors declare no conflict of interest regarding this manuscript. Notes/thanks/other declarations We are thankful for the invitation to contribute to River Basin Management and other anonymous reviewers for their assistance. This research work was self-funded, as the authors declare no conflict of interest. Author details Eucheria N. Nweke1,2, Victor U. Okechukwu1, Daniel O. Omokpariola1, Theresa C. Umeh1 and Nwanneamaka R. Oze3 1 Department of Pure and Industrial Chemistry, Nnamdi Azikiwe University, Awka, Nigeria 2 Department of Chemistry, College of Education Umunze, Nigeria 3 Department of Chemistry, Federal University of Technology, Owerri, Nigeria Address all correspondence to: omeodisemi@gmail.com © 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 14 Author details Eucheria N. Nweke1,2, Victor U. Okechukwu1, Daniel O. Omokpariola1, Theresa C. Umeh1 and Nwanneamaka R. Oze3 Eucheria N. Nweke1,2, Victor U. Okechukwu1, Daniel O. Omokpariola1, Theresa C. Umeh1 and Nwanneamaka R. Oze3 1 Department of Pure and Industrial Chemistry, Nnamdi Azikiwe University, Awka, Nigeria 2 Department of Chemistry, College of Education Umunze, Nigeria 3 Department of Chemistry, Federal University of Technology, Owerri, Nigeria Address all correspondence to: omeodisemi@gmail.com © 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. References Midwestern Nigeria. Journal of Scientific Research and Reports. 2014;3(3):514-531 [1] Fakayode SO. Impact assessment of industrial effluent on water quality of the receiving Alaro River in Ibadan, Nigeria. The Journal of African Medical Association. 2005;10:1-13 [8] Teshome JY, Gelanew A, Abayneh B. 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https://openalex.org/W4307427353	https://link.springer.com/content/pdf/10.1007/s10802-022-00968-4.pdf	English	null	Reciprocal Relations Between Conflicted Student-teacher Relationship and Children’s Behavior Problems: Within-person Analyses from Norway and the USA	Research on child and adolescent psychopathology	2,022	cc-by	10,391	Reciprocal Relations Between Conflicted Student-teacher Relationship and Children’s Behavior Problems: Within-person Analyses from Norway and the USA Silje Merethe Husby1,2 · Věra Skalická1 · Zhi Li3 · Jay Belsky1,4 · Lars Wichstrøm1,2 Accepted: 16 August 2022 / Published online: 27 October 2022 © The Author(s) 2022 Accepted: 16 August 2022 / Published online: 27 October 2022 © The Author(s) 2022 Research on Child and Adolescent Psychopathology (2023) 51:331–342 https://doi.org/10.1007/s10802-022-00968-4 Research on Child and Adolescent Psychopathology (2023) 51:331–342 https://doi.org/10.1007/s10802-022-00968-4 Abstract Current evidence suggests that conflicted student-teacher relationships may increase behavior problems in children and vice-versa, but this may be due to confounding. We therefore analyzed their relation applying a within-person approach that adjusts for all time-invariant confounding effects, involving samples from Norway (n = 964, 50.9% females) and the USA (n = 1,150, 48.3% females) followed from age 4–12 years with similar measures. Increased parent-reported behavior prob lems forecasted increased student-teacher conflict to a similar extent in both countries (β = 0.07, p = .010), whereas teacher- reported behavior problems predicted increased student-teacher conflict more strongly in Norway (β = 0.14, p = .001) than in the US (β = 0.08, p = .050). Increased teacher-child conflict also predicted increased parent-reported (β = 0.07, p = .010), but not teacher-reported, behavior problems in both countries. Findings underscore the reciprocal relation between behav ior problems and a conflictual student-teacher relationship. Keywords Behavior problems · Cross-national · Longitudinal · Random intercept · Student-teacher relationship · Within person Keywords Behavior problems · Cross-national · Longitudinal · Random intercept · Student-teacher relationship · Within-person Scholars contend that a good student-teacher relationship may prevent mental health problems in children, whereas a problematic relationship may increase the risk of develop ing such problems (e.g., Silver et al., 2005). These claims are supported by observational research, both cross-sec tional and longitudinal in design, and with particular refer ence to student-teacher conflict and child behavior problems (e.g., Pianta & Stuhlman 2004; Silver et al., 2005). There are also theoretical reasons to expect evocative child effects, and evidence from diverse samples indicates that this may be the the case (Nurmi, 2012). In sum, existing prospective studies converge in suggesting a reciprocal relation between student-teacher conflict and behavior problems. If these associations reflect student-teacher conflict being part of the etiology of behavior problems, preventative measures and treatment efforts targeting such conflicts should be devel oped and evaluated for efficacy. However, if such findings are an artifact of underlying confounding factors, such inter ventions are unlikely to prove effective. We therefore test whether prospective and reciprocal associations between student-teacher conflict and children’s behavior problems remain when all unobserved time-invariant confounding effects (e.g., genetics, gender) are adjusted for, using a within-person approach. Grants ES662241 and 238026, obtained from the Research Council of Norway, and a grant from the Liaison Committee between Central Norway RHA and NTNU partially funded this study. Abstract Grants ES662241 and 238026, obtained from the Research Council of Norway, and a grant from the Liaison Committee between Central Norway RHA and NTNU partially funded this study. Silje Merethe Husby silje.husby@ntnu.no Lars Wichstrøm lars.wichstrom@ntnu.no 1 Department of Psychology, Norwegian University of Science and Technology, Trondheim, Norway 2 Department of Child and Adolescent Psychiatry, St. Olav’s Hospital, Trondheim, Norway 3 School of Psychological and Cognitive Sciences and Beijing Key Laboratory of Behavior and Mental Health, Peking University, Beijing, China 4 Department of Human Ecology, University of California, Davis, USA 1 1 3 Research on Child and Adolescent Psychopathology (2023) 51:331–342 332 Why Teachers Matter for Students and Students Matter for Teachers been inflated in prior research. Longitudinal studies have identified a range of predictors of behavioral problems, no doubt due to the fact that the etiology of disruptive behav ior problems is multifaceted. These include psychological and biological characteristics of the child, such as tempera ment (e.g., negative emotionality (Wichstrøm et al., 2018); effortful control (Atherton et al., 2019; Wichstrøm et al., 2018); inattention (Bellanti et al., 2000); language disorders (Menting et al., 2011); executive functions (Hobson et al., 2011); physiology (e.g., heart-rate variability and skin con ductance (Fanti et al., 2019); brain abnormalities (Thijssen et al., 2015); and genetics (Loeber et al., 2009). Also influ ential are features of the family, including parenting (Loe ber et al., 2009), family structure (Rowe et al., 2002), and socioeconomic circumstances (Bradley & Corwyn, 2002; Hill, 2002). As children grow older, peer relationships also become a source of influence via processes of, for example, rejection and association with deviant peers (Hill, 2002). Factors related to pregnancy have also been tied to the development of behavior problems, perhaps most especially prenatal exposure to drugs, alcohol or tobacco (D’Onofrio et al., 2007), as well as maternal stress (O’Connor et al., 2002). Just as notably, the interactions among any of the effects of these multiple determinants likely contribute to the etiology of child externalizing problems, thus helping to explain why some exposed children develop behavior prob lems whereas other do not (Loeber et al., 2009). In sum, the myriad of identified predictors of behavior problems out side of the school suggests that conflict with the teacher is unlikely to prove decisive for a child to develop behavior problems. As children spend a substantial amount of their weekday hours in school, the school context, including their rela tionships with teachers, is presumed to contribute signifi cantly to their development (Baker et al., 2008; O’Connor et al., 2011). Indeed, these relationships may be particularly important for children with externalizing behavior prob lems, as they may be in especial need for close and support ive relationships with sensitive adults in order to develop adaptive emotional and social skills and competences (e.g., Baker et al., 2008; Sabol & Pianta, 2012; Silver et al., 2005). Unfortunately, children displaying externalizing behavior are more likely than others to experience harsh and critical interactions with teachers, and be subject to teaching that is less warm, nurturing and responsive (Hughes et al., 1999). Unobserved Confounding Collectively, the observations just noted also raise the issue of unobserved confounding when seeking to illuminate reciprocal effects linking student-teacher relationships and externalizing problems. To illustrate, high levels of negative emotionality and limited effortful control have been found to predict behavior problems (Wichstrøm et al., 2018), as well as undermine student-teacher relationships–beyond what may be caused by prior, and even covaried, behav ior problems, in efforts to predict change in problems over time (Rudasill et al., 2010). Problematical parenting is another potential source of confounding, given its links to problem behavior (Miner & Clarke-Stewart, 2008) and dif ficult teacher-child relationships, one that is not entirely dis counted by controlling for prior behavior problems (Hygen et al., 2017). From a demographic stance, low socio-economic status is associated with more behavior problems (Bradley & Corwyn, 2002) as well as greater student-teacher conflict There is also the possibility that externalizing behavior and student-teacher conflict may mutually reinforce each other over time, consistent with a transactional model (Mejia & Hoglund, 2016). Support for such a reciprocal relation can be found in studies chronicling externalizing behavior and conflict mutually predicting each other over time (Roorda & Koomen, 2021; Roorda et al., 2014; Ska licka, Belsky, et al., 2015; Skalicka, Stenseng, et al., 2015). Why Teachers Matter for Students and Students Matter for Teachers Disruptive behavior creates a disturbance in class set tings, making teacher interventions necessary (Birch & Ladd, 1998). As with coercive cycles of parent-child inter action, some teachers may respond to problematic student behavior with ineffective, inconsistent, and harsh classroom management tactics (Hughes et al., 1999), thereby engen dering a conflictual student-teacher relationship. Consistent with this claim is evidence indicating that externalizing behavior at one point is predictive of a more conflictual rela tionship with teachers later on (e.g., Birch & Ladd 1998; Jerome et al., 2009). This has been conceptualized as a child-driven model (Mejia & Hoglund, 2016).l A relationship characterized by high conflict may also promote later disruptive behavior, reflected in findings link ing student-teacher conflict with later externalizing prob lems (Pianta & Stuhlman, 2004; Silver et al., 2005). In this case, the relationship between behavior problems and conflict is viewed as a relationship-driven model (Mejia & Hoglund, 2016). 1 3 Why Teachers and Students May Not Matter for Each Other Despite the evidence and causal processes just considered, it remains the case that teachers’ impact on the development of children’s behavior problems may have, in some respects, 1 3 Research on Child and Adolescent Psychopathology (2023) 51:331–342 333 (Rudasill et al., 2010). Additionally, genetically informed research indicates that aggressive behavior is substantially heritable (Brendgen et al., 2011; Rhee & Waldman, 2002), quite possibly contributing to a conflicted student-teacher relationship (Brendgen et al., 2011). Although a small-scale twin-study (n = 217) failed to find evidence that common genes explained both conflicted student-teacher relation ships and behavior problems (Brendgen et al., 2011), the fact that this is the only genetically informed inquiry addressing this issue raises the possibility that both prob lem behavior and conflictual student-teacher relationships may be influenced by the same genes. Although investiga tors have made considerable strides towards more precise estimates of the relations between student-teacher conflict and behavior problems by controlling for many relevant confounders (Sabol & Pianta, 2012), a range of factors not adjusted for can still affect any detected associations. (Rudasill et al., 2010). Additionally, genetically informed research indicates that aggressive behavior is substantially heritable (Brendgen et al., 2011; Rhee & Waldman, 2002), quite possibly contributing to a conflicted student-teacher relationship (Brendgen et al., 2011). Although a small-scale twin-study (n = 217) failed to find evidence that common genes explained both conflicted student-teacher relation ships and behavior problems (Brendgen et al., 2011), the fact that this is the only genetically informed inquiry addressing this issue raises the possibility that both prob lem behavior and conflictual student-teacher relationships may be influenced by the same genes. Although investiga tors have made considerable strides towards more precise estimates of the relations between student-teacher conflict and behavior problems by controlling for many relevant confounders (Sabol & Pianta, 2012), a range of factors not adjusted for can still affect any detected associations. In addition to such methodological advance—and given concerns about the replication of findings (Aarts & al., 2015)—we utilize data from two large community stud ies, conducted in two countries with significant differences in educational systems and rates of externalizing behavior (Heiervang et al., 2007), Norway and the USA, to investi gate reciprocal relations between problematic student behav ior and conflictual student-teacher relationships. Why Teachers and Students May Not Matter for Each Other Based on previous findings, we hypothesize that increased student- teacher conflict at one wave will forecast increased external izing problems at the next wave, and vice-versa, when using a traditional autoregressive cross-lag approach. As shown, we believe that there are both theoretical and methodologi cal reasons to believe that these associations may be smaller in magnitude at the within-person level—i.e. change in one person predicting later change in that person—than those obtained from traditional cross-lagged models which uti lize both within and between-person information—i.e., a persons’ level relative to others predicting ones’ future level relative to others. We therefore remain open to the ques tion of whether these predictions hold when all unmeasured time-invariant confounding effects at the between-person level are adjusted for. Finally, we test whether the identified paths differ by country. To be appreciated is that current evidence linking stu dent-teacher conflict and child externalizing problems stems from observational research applying regression- type approaches, including cross-lagged panel models. These approaches produce results which are a mixture of between- and within-person variance. The behavior of stu dents or teachers unknown to a specific child–a between- person effect–cannot be involved in the etiology of behavior problems or student-teacher conflicts of the particular child. Rather, causality can only be inferred from within-person results. Several recent statistical approaches are able to tease apart between-person effects from within-person sources of variance (Usami et al., 2019) and by the same token adjust for all unmeasured time-invariant confounding effects.f Participants and Procedure The Trondheim Early Secure Study (TESS) consists of members of the 2003 and 2004 birth cohorts in Trondheim, Norway (N = 3,456) (Steinsbekk & Wichstrøm, 2018). A let ter of invitation along with the Strengths and Difficulties Questionnaire (SDQ) 4–16 version (Goodman et al., 2000) was sent to their homes. The SDQ is a brief and valid screen for emotional, behavioral and social problems in the general population (Sveen et al., 2013). Parents were asked to bring the completed SDQ to their child’s scheduled routine 4-year health check-up at their local well child clinic (n = 3,358 attended). Here, parents were informed about TESS using procedures approved by the Regional Committee for Medi cal and Health Research Ethics Mid-Norway, and written consent to participate was obtained. Parents who were not sufficiently proficient in Norwegian to complete the SDQ screening were excluded (n = 176). Of those who were asked to participate (n = 3,016), 82.2% consented. Despite differences between methods, they converge in doing this by including random intercept latent factors which account for the respective levels of the constructs in question (e.g., behavior problems and the student-teacher relationship) during the study period. This random inter cept then captures what causes one child’s overall levels of the constructs (i.e., a time-invariant effect), and these dif ferences in levels between children are adjusted for in the analyses. Although results from within-person analysis of student-teacher relationships and behavior problems do not warrant the strongest claims of causality, as time-varying confounding effects can still influence the results (e.g., changes in maternal depression), they can certainly aid in moving observational science closer to providing causal insight. For this reason, we have drawn upon the possibili ties within the Random Intercept Cross-lagged Panel Model (RI-CLPM; Hamaker et al., 2015). It affords illumination of within-person effects, measuring each individual’s change from each person’s own average level, thereby moving the study of reciprocal effects of student-teacher relationships and child externalizing problems closer to a causal analysis. To increase statistical power, children with or likely to develop emotional or behavioral problems were overs ampled by dividing children into four strata according to their SDQ scores (cut-offs: 0–4, 5–8, 9–11 and 12–40). Measures The Student-Teacher Relationship Scale Short Form (STRS-SF) is a short form of the original STRS, a fre quently used and validated measure that gauges the teacher- perceived relationship quality with individual children (Pianta, 2001). The STRS-SF consists of 15 items and two factors, closeness and conflict, and has been found to have good psychometric properties in differing cultural contexts (Drugli & Hjemdal, 2013; Tsigilis & Gregoriadis, 2008). In both samples we made use of the 7-item conflict scale (scale range: 7–35) that measures the degree to which a teacher feels that his or her relationship with the student in question is characterized by negativity (Tsigilis & Gregoriadis, 2008) (α = 0.81-0.89). The items are rated on a 5-point scale where higher value represents more conflict. The Student-Teacher Relationship Scale Short Form (STRS-SF) is a short form of the original STRS, a fre quently used and validated measure that gauges the teacher- perceived relationship quality with individual children (Pianta, 2001). The STRS-SF consists of 15 items and two factors, closeness and conflict, and has been found to have good psychometric properties in differing cultural contexts (Drugli & Hjemdal, 2013; Tsigilis & Gregoriadis, 2008). In both samples we made use of the 7-item conflict scale (scale range: 7–35) that measures the degree to which a teacher feels that his or her relationship with the student in question is characterized by negativity (Tsigilis & Gregoriadis, 2008) (α = 0.81-0.89). The items are rated on a 5-point scale where higher value represents more conflict. The mean age in kindergarten was 5.6 years (SD = 0.30). Retesting occurred in first grade (T2): Mage = 7.0 years, SD = 0.30; third grade (T3): Mage = 9.0, SD = 0.31; fifth grade (T4): Mage = 10.7, SD = 0.33 and sixth grade (T5) Mage = 11.9, SD = 0.35. A total of 1,150 cases comprised the ana lytical sample (48.3% female). A total of 77.0% of the moth ers were married and mothers had on average 14.2 years of education, 80.0% of the children were white and 12.9% were African American. Family income-to-needs was cal culated by dividing the family’s gross annual income by the poverty threshold for each family. The average income- to-needs ratio was 3.59 and 16.9% of the families had a score < 1 (below the poverty threshold). Participants and Procedure Retesting occurred at 6 years (T2): Mage = 6.7 years, SD = 0.25; 8 years (T3): Mage = 8.8, SD = 0.24; and 10 years (T4): Mage = 10.5 years, SD = 0.16, and 12 years (T5) Mage = 12.5, SD = 0.67. Overall, 964 participants had information from at least one wave of data collection and comprised the analytical sample. 1 3 Participants and Procedure Using a random number generator, drawing probabilities 1 3 334 Research on Child and Adolescent Psychopathology (2023) 51:331–342 T5 was predicted by teacher-rated problem behavior at T3 and T4 (OR = 1.06, CI = 1.00-1.11 and OR = 1.06, CI = 1.00- 1.12, respectively). Response rates among teachers were 83.9–98.1% at T1 through T5. to participate increased with increasing SDQ scores. The drawing probability increased with increasing SDQ scores of 0.37, 0.48, 0.70, and 0.89 in the four strata, respectively. Of the 1,250 children randomly drawn, 995 were enrolled at T1 (50.9% female). Most parents were either married (56.3%) or had been cohabitating for > 6 months (32.6%). Mothers were predominantly of Norwegian (93.0%) or Western (2.7%) origin. In all, 58.3% had at least a bach elor’s degree whereas 6.7% had not completed senior high school (13 years of education). Drop-out rate after consent did not differ according to SDQ score [(t = 0.17, df = 1, p =. 86) or gender (χ² = 1.02, df = 1, p = .31]. The mean age at the first assessment was 4.7 years (SD = 0.30, 49.9% males). Retesting occurred at 6 years (T2): Mage = 6.7 years, SD = 0.25; 8 years (T3): Mage = 8.8, SD = 0.24; and 10 years (T4): Mage = 10.5 years, SD = 0.16, and 12 years (T5) Mage = 12.5, SD = 0.67. Overall, 964 participants had information from at least one wave of data collection and comprised the analytical sample. to participate increased with increasing SDQ scores. The drawing probability increased with increasing SDQ scores of 0.37, 0.48, 0.70, and 0.89 in the four strata, respectively. Of the 1,250 children randomly drawn, 995 were enrolled at T1 (50.9% female). Most parents were either married (56.3%) or had been cohabitating for > 6 months (32.6%). Mothers were predominantly of Norwegian (93.0%) or Western (2.7%) origin. In all, 58.3% had at least a bach elor’s degree whereas 6.7% had not completed senior high school (13 years of education). Drop-out rate after consent did not differ according to SDQ score [(t = 0.17, df = 1, p =. 86) or gender (χ² = 1.02, df = 1, p = .31]. The mean age at the first assessment was 4.7 years (SD = 0.30, 49.9% males). Measures The Child Behavior Checklist (CBCL) and The Teacher Report Form (TRF). Behavior problems were reported by parents and teachers on the Externalizing Problems scale of the CBCL and the TRF (Achenbach & Rescorla, 2000). The CBCL is a standardized questionnaire designed to be filled out by parents to describe children’s behavioral and emo tional problems. The CBCL 4–18 version was used at all time points in the US sample (Achenbach, 1991). In Norway, the 1.5–5 year version was applied at T1, whereas the 6–18 year version was administered at T2 through T5 (Achen bach & Rescorla, 2000, 2001). Norwegian scores were har monized with the version applied in the USA by summing only the items matching the earlier version (i.e., 25 items at T1—scale range 0–50, 34 items at T2 and later—scale range 0–68). Correlations between the original version scores and the newly created scores were 0.94 at T1 and 0.93 at later time points (α = 0.83-0.88). Day-care centers and schools were requested to select the teacher who knew the child best. In the US, the 5–18 version (Achenbach, 1991) was used. In Norway, the 1.5-5 year version was administered at T1, and the 6–18 year version at T2-T5 (Achenbach & Rescorla, 2000, 2001). Following the procedure described above, the harmonized scores evinced high correlations with original scores (r = .92-0.97; α = 0.92-0.94). Attrition analyses showed that parent-rated behav ior problems at T1 predicted dropout at T3 (odds ratio (OR) = 1.06, 95% confidence interval (CI) = 1.02–1.11). Teacher-rated problem behavior at T1 and T2 predicted dropout at T5 (OR = 1.05, CI = 1.00-1.09, and OR = 1.05, CI = 1.01–1.09, respectively). Response rates among teach ers were 91.8-99.1% at T1 through T5. NICHD Study of Early Child Care and Youth Devel opment (NICHD SECCYD) consists of participants recruited from 24 hospitals from 10 data locations in the USA, during the first 11 months of 1991 (NICHD Early Child Care Research Network, 2005) Participants were selected according to a random sampling plan designed to ensure that the sample reflected the demographic diversity of each site’s catchment area. In all, 8,986 women who gave birth during a selected 24 h interval were screened for eligi bility. From those, 1,364 families of healthy newborns were included via a home interview one month after birth. Analysis Plan problem behavior; a cross-lagged effect). A conceptual model is presented in the Online material (Figure S1). We used the scaled chi-square difference (Satorra & Bentler, 2001) to test (i) whether cross-lagged paths were equal over time in each country and (ii) whether there were differences between the countries in a multigroup comparison apply ing procedures described by Mulder & Hamaker (2020). Because multi-group testing of path equality is based on comparisons of unstandardized path coefficients, we report unstandardized estimates. Because standardized estimates cannot be constrained to be equal, we report mean values of the respective standardized path coefficients, to enable com parisons with other studies. Even though the standardized estimates will be specific to this sample, we follow general guidelines to interpret effect sizes in RI-CLPM, considering 0.03, 0.07, and 0.12 to represent small, medium and large effects, respectively (Orth et al., 2022). Although the RI- CLPM cannot illuminate which unmeasured time-invariant factors prove influential, it is possible to stratify analyses according to measured time-invariant variables. Because behavioral problems as well as student-teacher conflict are more prevalent in boys than girls, we performed gender spe cific analyses and compared the cross-lagged paths with the procedure just described. Potential confounding effects can be divided into time-vary ing and time-invariant ones. The former change their impact over the time-period under observation (e.g., schooling, peer relationships), whereas the latter do not (e.g., stable effects of genetics, gender) (Wichstrøm et al., 2018). To distinguish within and between person effects we applied structural equation modeling (SEM) in Mplus 7.4, employing a robust maximum likelihood estimator which yields robust standard errors. As attrition analyses suggested that data were miss ing at random (MAR), missingness was handled with a full information maximum likelihood (FIML) procedure. Due to the aforementioned stratification of the Norwegian sample, its data were weighted with a factor corresponding to the number of children in the stratum divided by the number of participating children in that stratum. Analyses were conducted in two steps. First, we examined reciprocal relations between student-teacher conflict and child behavior problems by means of traditional autoregres sive cross-lagged analysis in which the level of an earlier- measured predictor vis-à-vis others (‘rank-order’) is used to forecast the level of a later-measured outcome (‘rank order’, without regard for individual change). This will allow us to simultaneously test child-driven, relationship-driven and reciprocal models. Measures Dropout at T3 was predicted by teacher-rated problem behavior at T1 (OR = 1.04, CI = 1.00-1.09), and dropout at 1 3 335 Research on Child and Adolescent Psychopathology (2023) 51:331–342 Analysis Plan Because the magnitude of relations between teacher-rated behavior problems and teacher-rated student-teacher conflict might be inflated by common rater bias, two separate autoregressive cross-lagged models were run, one for teacher and one for parent ratings of behav ior problems. Because short-term issues might affect par ent- and teacher ratings of children (e.g., mood-of-the-day effects or recent conflicts) that are not likely to influence the rating years later, we expected sleeper effects in stabil ity, which pertains to a delayed change in a dependent vari able, bypassing intermediate time points (Cook et al., 1979). All measures were therefore auto-regressed on all preced ing measures. Error terms of problem behavior and conflict were allowed to correlate at each time point. The scaled chi- square difference test (Satorra & Bentler, 2001) was applied to test equality of cross-lagged paths, and to test equality between countries in a multigroup analysis. In Norway, the children came from the same city, and thus some belonged to the same school class, and whether mul tilevel analyses should be performed or not was therefore considered. There are about 250 elementary school classes in Trondheim. Accordingly, the design effect in grades 1–3 varied between 1.2 and 1.6, which is well below the recom mended cut-off of 2.0 for when multilevel analyses should be conducted. Results Higher scores on student-teacher conflict and behavior problems were observed in the USA than in Norway (Table S1, available online), and variance in all three measures was also higher in the USA (Table S2). Even so, behavior prob lems and student-teacher conflict were both concurrently and prospectively positively correlated in both countries, meaning that more conflict was associated with more prob lems, with no apparent systematic or major differences in magnitude.i Second, to control for all unmeasured time-invariant effects we employed RI-CLPM for Norway and USA, respectively. This model extends the autoregressive cross- lagged model by separating variance into a stable between- person part (consisting of two latent random intercepts loading on behavior problems and conflict, respectively) and a within-person part; in so doing, it estimates changes from one’s own mean level in a variable (e.g., conflict) as a function of changes in that variable at the previous mea surement point (autoregression) and in the predictor (e.g., Cross-lagged Panel Models. We first ran a pair of CLPM, one for teacher-rated behavior problems and one for parent- rated behavior problems in each country, resulting in four models altogether. Then, the cross-lagged paths in the two countries were compared. Based on the Sattora-Bentler test, all cross-lagged paths between conflict and problem behav ior in the traditional autoregressive cross-lagged models 1 3 Research on Child and Adolescent Psychopathology (2023) 51:331–342 336 Table 1 Model fit of the random intercept cross lagged panel models and χ² difference test Countries Model Fits χ² - difference test across time χ² - difference test across countries χ² RMSEA SRMR CFI TLI CBCL Norway 47.93 (27), p = .008 0.028 0.041 0.982 0.970 5.78 (6), p = .45 CBCL USA 48.10 (27), p = .008 0.026 0.034 0.993 0.989 9.49 (6), p = .15 CBCL Norway and USA 60.16 (27), p < .001 0.024 0.036 0.992 0.987 0.65 (2), p = .72 TRF Norway 56.85 (27), p < .001 0.034 0.049 0.981 0.969 8.49 (6), p = .20 TRF USA 50.74 (27), p = .004 0.028 0.051 0.993 0.989 4.08 (6), p = .67 TRF Norway and USA 108.52 (54), p < .001 0.031 0.050 0.989 0.981 8.55 (2), p = .014 Note: RMSEA = Root mean square error of approximation, SRMR = Standardized root mean residual, CFI = Comparative fit index, TLI = Tucker- Lewis index, CBCL = Child behavior checklist, TRF = Teacher report form Fig. Results 1 RI-CLPM showing within-person cross-lagged paths between behavior problems as measured by the CBCL and student-teacher conflict Note: CBCL = Child Behavior Checklist; CO = Student-teacher conflict; Unstandardized path coefficients and p-values, Norway first, USA second. Results from multigroup analyses, where cross-lagged paths were held equal across time and across countries Fig. 1 RI-CLPM showing within-person cross-lagged paths between behavior problems as measured by the CBCL and student-teacher conflict Note: CBCL = Child Behavior Checklist; CO = Student-teacher conflict; Unstandardized path coefficients and p-values, Norway first, USA second. Results from multigroup analyses, where cross-lagged paths were held equal across time and across countries confounding effects at the between-person level, RI-CLPM- analyses were employed. First, we ran separate analyses for teacher-rated and parent-rated behavior problems for each country, respectively. Constraining the cross-lagged paths to be equal over time did not worsen the model fit (Table 1), and all such constrained models in each country proved to evince good fit (Table 1). At the between-person level, ran dom intercepts of conflict and problem behavior were mod erately (CBCL) to highly (TRF) correlated and consistent in direction with associations described in the preceding paragraph (Norway: CBCL: r = .45 (p < .001), TRF: r = .84 (p < .001); USA: CBCL: r = .51 (p < .001), TRF: r = .97 (p < .001)). At the next step, the cross-lagged paths could be constrained to be equal across countries without deteriorat ing model fit in the parent-reported model, while they could not be in the model involving teacher-reported behavior in which they proved different (Table 1). were found to be equal across all time points, and also equal across both countries for both parent-reported and teacher- reported behavior problems (Table S3). Thus, they indi cated that greater student-teacher conflict at an immediately preceding measurement occasion predicted more external izing problems, irrespective of whether rated by teachers or parents, by the time of the next measurement occasion; and, reciprocally, that greater externalizing problems at an earlier time of measurement, irrespective of whether rated by teachers or parents, similarly predicted higher teacher- student conflict at the next measurement occasion (Supple mentary figures S2 and S3). In sum, a transactional model was supported in the CLPM analyses. 1 3 Random Intercept Cross-lagged Panel Models Relationship-driven Model: Student-teacher Con flict Predicting Behavior Problems. Prospective cross- lagged within-person results (Fig. 1) revealed that increased student-teacher conflict predicted increased parent-rated Initial Model Testing: Constraining Paths Across Devel opment and Countries. To test whether predictions con veyed in the CLPM could be attributed to time-invariant 1 3 Research on Child and Adolescent Psychopathology (2023) 51:331–342 337 Fig. 2 RI-CLPM showing within-person cross-lagged paths between behavior problems as measured by the TRF and student-teacher conflict Note: TRF = Teacher Report Form; CO = Student-teacher conflict. Unstandardized path coefficients and p-values, Norway first, USA second Fig. 2 RI-CLPM showing within-person cross-lagged paths between behavior problems as measured by the TRF and student-teacher conflict Note: TRF = Teacher Report Form; CO = Student-teacher conflict. Unstandardized path coefficients and p-values, Norway first, USA second student-teacher conflict at age 8, Δχ2 = 8.66 (1), p = .003. However, it should be acknowledged that these two signifi cant results emerged after testing 32 paths. Adjusting for the false discovery rate (Benjamini & Hochberg, 1995), these p-values were rendered insignificant, p = .63 and p = .096 respectively. In effect, as the identified cross-lagged effects were the same in girls and boys, gender was not among the time-invariant variables captured by the random intercepts. behavior problems (standardized β = 0.06), and the path coefficients did not differ between countries (Table 1). However, increased student-teacher conflict did not forecast increased teacher-rated behavior problems in either country (Fig. 2). Child-driven Model: Behavior Problems Predicting Student-teacher Conflict. Cross-lagged paths involving parent-rated behavior problems predicting later conflict did not differ between countries (Table 1), and a medium effect of increased behavior problems on later increased conflict was identified (β = 0.07). In contrast, multigroup analy sis of the model involving teacher-rated behavior prob lems revealed that the cross-lagged paths from increased behavior problems to later increased conflict were stronger in Norway (β = 0.14) compared to the estimates in the US (β = 0.08; Table 1; Fig. 2). Of note, path coefficients were typically smaller than in the ordinary cross-lagged panel models and medium in magnitude. For example, as can be seen in Fig. 1, when the student-teacher conflict increased with 1 point on the STRS above its average for that relation ship, behavior problems increased with 0.05 points on the CBCL scale. Random Intercept Cross-lagged Panel Models One notable exception to these medium effects was the prediction of increased student-teacher conflict from increased teacher-rated behavior problems in Norway. Discussion Potential reasons for why increased teacher-rated conflict does forecast increased behavior problems as seen by par ents, but not teachers, remain unclear. previous findings (Jerome et al., 2009; Mejia & Hoglund, 2016; Pakarinen et al., 2018; Roorda & Koomen, 2021; Roorda et al., 2014), and in sum a transaction model was hence supported irrespective of whether parents or teachers rated the behavior problems. previous findings (Jerome et al., 2009; Mejia & Hoglund, 2016; Pakarinen et al., 2018; Roorda & Koomen, 2021; Roorda et al., 2014), and in sum a transaction model was hence supported irrespective of whether parents or teachers rated the behavior problems. In the within-person RI-CLPM analysis, that evaluates whether and how change in the predictor forecasts change in the outcome, the support for a relationship-driven model was maintained—increased student-teacher conflict still predicted increased parent-rated behavior problems in both countries when parents assessed the behavior problems. However, when teachers assessed the behavior problems, no effect was seen in either country, perhaps because teachers see children in a more limited setting than parents do, for less time, and only during school hours. As regards a child- driven model, we detected a medium effect of increased parent-rated behavior problems on later increased conflict which was similar in both countries. Such a child-driven effect was also found for teacher-rated behavior problems, and this effect was slightly stronger in Norway than in the US. In sum, a transactional model was supported with respect to parent-rated behavior problems, whereas a child- driven model was supported for teacher-rated behavior problems. Notably, coefficients obtained in the RI-CLPM were generally smaller than in the traditional cross-lagged panel model, the exception being the path between increased teacher-rated behavior problems on later increased student- teacher conflict in Norway. These findings imply that any suggestive causal inferences from traditional approaches risk being overstated and may also be context specific. In the following we advance some possible explanations for the obtained pattern of results. One possible explanation could be that children experi encing conflict in the student-teacher relationship respond with an increase in problematic behavior at home, in a sort of spillover effect. Previous research indicates that children’s negative experiences at school predict more aversive interac tions with parents (Flook & Fuligni, 2008), and higher school stress predicts more aversive family interactions even three years later (Flook & Fuligni, 2008). Discussion Evidence from observational studies suggests that student- teacher conflict contributes to externalizing behavior prob lems in children and that the reverse is true as well. Yet it remains the case, even when covariates are included in prediction models and prior measurements of the outcome are statistically controlled, that substantial threats to causal inference remain. For this reason, we sought not only to discount time-invariant confounding effects, but also to compare results when these are and are not taken into con sideration. The fact that we were not positioned to control for all unmeasured time-varying confounding effects limits, of course, any strong causal inferences that can be drawn from this work. Reciprocal Model. Taken together, the RI-CLPM analy ses supported a transactional model with respect to parent rated behavior problems. However, the lack of support for a relationship-driven model when teachers rated behav ior problems, prevented support for a transactional model involving teacher ratings of behavior problems.if In the traditional cross-lagged analysis, we found sup port for a relationship-driven model—more student-teacher conflict at an earlier measurement occasion predicted later behavior problems, as rated by both parents and teachers, in both countries, in line with previous findings (Pianta & Stuhlman, 2004; Roorda & Koomen, 2021; Roorda et al., 2014; Silver et al., 2005). Conversely, a child-driven model was also supported—more behavior problems as rated by both parents and teachers at an earlier measurement occa sion, in both countries, predicted more student-teacher con flict at the next measurement occasion. This also aligns with Gender-specific analyses revealed no differences in magnitudes, with two exceptions. In Norway, increased student-teacher conflict at age 4 predicted more parent rated behavioral problems at age 6 to a stronger degree in boys than in girls, Δχ2 = 4.05 (1), p = .04, and increased par ent rated behavior problems at age 6 predicted increased 1 3 338 Research on Child and Adolescent Psychopathology (2023) 51:331–342 In contrast to CLPM results, the effect of student-teacher conflict on teacher-rated behavior problems were rendered insignificant in the RI-CLPM—in both countries. This accords with a view that a seeming effect of student-teacher conflict on behavior problems is due to unmeasured time- invariant confounding. Similar to CLPM findings, however, an effect of student-teacher relationship on parent-rated behavior problems was retained in the RI-CLPM analysis. Discussion Thus, it is conceivable that increased student-teacher conflict at school may play out as an increase in externalizing behavior at home. Moreover, parents and teachers usually communicate about the student and their behavior and functioning in school, for example in parent – teacher meetings; and it is possible that should the teacher convey a conflictual or problematic relationship with the child, this may influence parents’ overall impression of their child’s behavior, as a kind of negative halo-effect. Admittedly, there is no ready explanation for why these effects do not emerge as behavior problems at school. On a methodological note, although the teacher-rated effect fell short of being statistically significant, the effect was almost identical in magnitude to the parent-rated one. Therefore, caution is called for before breathing too much meaning into the parent versus teacher difference. A Relationship-driven Model This report is the first of its kind analyzing data from a large and diverse population, encompassing children, par ents and teachers from two different countries with differing school-systems and differing rates of behavior problems. Methodologically, this may have better situated us to pro vide accurate estimates of the relations between student- teacher conflict and behavior problems. In sum, the findings suggest that previously reported effects of student-teacher conflict on later behavior problems appear to have been inflated due to failure to take into account all time-invariant confounding effects. Regardless of whether effects turned out statistically significant or not, the effects were medium. Even so, these effects emerged at each time point, so effects may accumulate across development. Moreover, the effects appeared at the population level and even medium, aver aged effects may prove important for a subset of children. Such moderational effects should be addressed in prospec tive studies given ever-increasing evidence that children Although our traditional CLPM results concurred with those of many studies chronicling a predictive effect of student- teacher conflict on later behavior problems as rated by teach ers, the present effects were not large, and smaller than many of the ones previously reported. However, these larger effects generally stem from smaller sized studies (Birch & Ladd, 1998; Mejia & Hoglund, 2016), whereas studies with sample sizes approaching the ones involved here typically report smaller estimates, comparable to the ones we found (Pianta & Stuhlman, 2004; Roorda & Koomen, 2021). Smaller sam ple sizes result in larger confidence intervals, implying more uncertainty around the precision of their estimates. With its combination of two large samples from two different coun tries, the present study has, to the best of our knowledge, the largest and most diverse sample to date, and the medium CLPM estimates therefore line up with previous trends. 1 3 339 Research on Child and Adolescent Psychopathology (2023) 51:331–342 is exposed to several teachers, so that the relationship with a specific teacher becomes less important. But contrary to such an analysis, the concurrent correlation between a negative student-teacher relationship and children’s exter nalizing problems has been found to be somewhat stronger in the upper primary grades than in kindergarten and the lower primary grades (Lei et al., 2016). Such correlational evidence does not afford much leverage for drawing con clusions about developmental trends in the importance of teachers to students’ externalizing behavior. A Child-driven Model An effect of teacher-rated behavior problems on conflict was retained in the RI-CLPM analyses in both countries, being stronger in Norway than in the US. In Norway, students are typically taught by the same teacher from grades 1 to 4 and from grades 5 to 7, making for much greater continuity in the student-teacher relationship than in the US. Hence, there is time to detect a prospective build-up of conflicts in a spe cific student-teacher relationship when behavior problems increase. Such a build-up may be disrupted in the US by the end of the year, so that—to the extent that previous classes or teachers’ do not forewarn—students and teachers may start with a clean slate each year.if In the RI-CLPM, we also detected a reduction in esti mates of prospective relations relative to the traditional cross-lagged models. Once again, this would seem to be the result of controlling for time invariant between-person effects from the CLPM estimates so that only actual within- person processes are estimated. Of course, we were not positioned to identify these confounding factors, but there is no shortage of influential suspects at the level of the child, such as genetics (Brendgen et al., 2011), attention problems (Bellanti et al., 2000), temperamental traits (Valiente et al., 2003) and personality traits (Zee et al., 2013); the level of the teacher, such as teacher stress (Yoon, 2002), and class room management strategies (Korpershoek et al., 2016); or other contextual factors, such as parenting practices (Hygen et al., 2017) and low socio-economic status (Bradley & Cor wyn, 2002). This is a task awaiting future research. That said, we did find evidence for a predictive effect of change in teacher-rated behavior problems on change in teacher-student conflict in Norway even at those grade-based transitions when teachers change (i.e., from day-care to 1st grade, from elementary to middle school). This is consistent with Brendgen et al.’s (2006) findings that the probability of experiencing teacher verbal abuse proved highly stable even when teachers changed from one year to the next. One pos sible explanation advanced by Brendgen et al. is that teach ers often talk to each other during staff-meetings—and thus that some students develop reputations as troublemakers, deservedly or not. This might help to explain why results proved different in the USA where teacher-teacher commu nication practices seem likely to be different. A Relationship-driven Model The present prospective, within-person associations, however, do pro vide some support for continued importance of the student- teacher relationship at least from preschool age through middle childhood. vary in their susceptibility to environmental effects (Belsky & Pluess, 2009). Strengths and Limitations In Norway it is also commonplace for teachers to hold transitional meetings or write transitional reports when chil dren move from day-care (there is no kindergarten in Nor way) to school, and when children move from elementary to middle school. This is particularly true in cases in which the child has shown behavioral, social or academic difficul ties. The stability of behavior problems was also moderate to high, thus increasing the risk of continuing conflict with a new teacher (Pakarinen et al., 2018).f Despite using prospective and multisource data from two large population studies in countries with differing educa tional systems, and strong analytical techniques that discount one important set of confounding effects—time-invariant ones—this report is not without limitations. The student- teacher relationship is a two-way street, and teacher-reports could be affected by personal characteristics, appraisals, experiences and expectations on part of the teacher, thereby reflecting teacher-characteristics rather than actual conflict (Thijs & Koomen, 2009). However, many of these biases could be considered more or less time-invariant and thus are adjusted for in the RI-CLPM analyses. Also, we were only able to capture the teacher’s appraisal of the amount of student-teacher conflict, and there is some evidence to indi cate that student and teacher ratings of their relationship do not correspond well (Murray et al., 2008). An observational measure might have provided a more unbiased account of conflict-level. Even so, the STRS’ conflict dimension and When child-driven and relationship-driven effects were detected, for both parent and teacher rating of behavior problems and in both Norway and the USA, cross-lagged paths in RI-CLPM did not differ between ages. This lack of developmental differences is notable, as it might have been expected that the importance of the teacher may change over time. Conceivably, the student’s primary teacher may stand out more distinctly as a non-parental socializing agent in preschool and the lower primary grades, thus making conflicts with this teacher especially detrimental. This might seem especially so given that in middle school the student 1 3 Research on Child and Adolescent Psychopathology (2023) 51:331–342 340 conflict ratings of external observers have at least moderate agreement (Thijs & Koomen, 2009).f no doubt gains to be made from avoiding student-teacher conflict on many parameters relevant to both student and teacher adjustment and well-being, but the effect on behav ior problems is likely smaller than previously assumed. Conclusion Aarts, A. A., et al. (2015). Estimating the reproducibility of psycho logical science [Article]. Science, 349(6251), 8, Article aac4716. A large body of research into the teacher-child relation ship clearly suggests that this is a relationship of significant developmental importance. Prior observational research has chronicled reciprocal effects linking increased student- teacher conflict and exacerbated behavior problems. Causal interpretations of such associations have led to suggestions that the student-teacher relationship may be a target of inter vention to help prevent behavior problems (e.g., O’Connor et al., 2011). Capitalizing on the possibilities of within-per son analysis, we find that these anticipated statistical asso ciations are evident in both Norway and the USA, and from preschool or day-care through middle childhood. These findings render support for a reciprocal relationship between a conflictual student-teacher relationship and child behavior problems, as assessed by parents, and from teacher-rated behavior problems to conflicts in the student-teacher rela tionship. However, even though the effects are smaller than previously portrayed when time-invariant effects were not adjusted for, they still lend some support to the importance of the student-teacher relationship for the development of behavior problems and vice-versa. Future work should seek to determine whether some children prove more sus ceptible to the effects under consideration, with the same perhaps worth considering in the case of teachers. There are Aarts, A. A., et al. (2015). Estimating the reproducibility logical science [Article]. Science, 349(6251), 8, Arti https://doi.org/10.1126/science.aac4716 https://doi.org/10.1126/science.aac4716 Achenbach, T. (1991). Manual for the child behaviour checklist/4–18 and 1991 profile Achenbach, T. M., & Rescorla, L. A. (2000). Manual for the ASEBA preschool forms and profiles (Vol. 30). University of Vermont, research center for children youth & families Achenbach, T. M., & Rescorla, L. A. (2001). Manual for the ASEBA school-age forms & profiles: child behavior checklist for ages 6–18, teacher’s report form, youth self-report: an integrated system of multi-informant assessment. University of Vermont, research center for children youth & families Atherton, O. E., Zheng, L. 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Nevertheless, research indicates that teacher’s perceptions, interpretations and expectations regarding children’s behavior are somewhat culture specific, and behaviors that are condu cive to the relationship in one culture may actually be associated with higher levels of conflict in another (Gregoriadis & Tsigilis, 2008). Hence, generalization to locales with different educa tional systems or sociocultural conditions or different ethnicities should be done with utmost care. Subgroups of children—such as those with disabilities or those doing poorly academically (Hamre et al., 2008; Murray & Murray, 2004)—may also be differentially at risk for, and sensitive to, conflict in the student- teacher relationship (McGrath & Van Bergen, 2015). Hence, although we find medium to small overall effects, for some children—or teachers—the conflict-behavior problems link may be stronger than is portrayed here. It is also possible, given evidence of differential susceptibility to many environmental effects (Belsky & Pluess, 2009), that the general or average effect sizes evaluated herein mask meaningful variation in the degree to which both individual teachers and children proved susceptible to relational processes at school. Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s10802- 022-00968-4. Funding Open access funding provided by NTNU Norwegian Univer sity of Science and Technology (incl St. Olav’s Hospital - Trondheim University Hospital) Declaration The authors have no conflict of interest to disclose. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/. 1 3 Conclusion Disentangling the impact of low cognitive ability and inattention on social behav ior and peer relationships. 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STRS: Student-Teacher Relationship Scale: Pro fessional manual Thijssen, S., Ringoot, A. P., Wildeboer, A., Bakermans-Kranen burg, M. J., Marroun, E., Hofman, H., Jaddoe, A., Verhulst, V. W. V., Tiemeier, F. C., van Ijzendoorn, H., M. H., & White, T. (2015). Brain morphology of childhood aggressive behavior: A multi-informant study in school-age children. Cognitive Affec tive & Behavioral Neuroscience, 15(3), 564–577. https://doi. org/10.3758/s13415-015-0344-9 Pianta, R. C., & Stuhlman, M. W. (2004). Teacher-child relationships and children’s success in the first years of school. School Psychol ogy Review, 33(3), 444–458. ://WOS:000224564100011 Rhee, S. H., & Waldman, I. D. (2002). Genetic and environmen tal influences on antisocial behavior: A meta-analysis of twin and adoption studies. Conclusion Psychological Bulletin, 128(3), 490–529. https://doi.org/10.1037//0033-2909.128.3.490 Tsigilis, N., & Gregoriadis, A. (2008). Measuring Teacher-Child Relationships in the Greek Kindergarten Setting: A Validity Study of the Student-Teacher Relationship Scale-Short Form. Early Education and Development, 19(5), 816–835. https://doi. org/10.1080/10409280801975826i Roorda, D. L., & Koomen, H. M. Y. (2021). Student-Teacher Relation ships and Students’ Externalizing and Internalizing Behaviors: A Cross-Lagged Study in Secondary Education. Child Develop ment, 92(1), 174–188. https://doi.org/10.1111/cdev.13394 Usami, S., Murayama, K., & Hamaker, E. L. (2019). A Unified Frame work of Longitudinal Models to Examine Reciprocal Relations. Psychological Methods, 24(5), 637–657. https://doi.org/10.1037/ met0000210 Roorda, D. L., Verschueren, K., Vancraeyveldt, C., Van Craeyevelt, S., & Colpin, H. (2014). Teacher-child relationships and behavioral adjustment: Transactional links for preschool boys at risk. 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Explaining the relationship between temperament and symptoms of psychiatric disorders from preschool to middle childhood: hybrid fixed and random effects models of Norwegian and Spanish children. Journal of Child Psychology and Psychia try, 59(3), 285–295. https://doi.org/10.1111/jcpp.12772 Rudasill, K. M., Reio, T. G., Stipanovic, N., & Taylor, J. E. (2010). A longitudinal study of student-teacher relationship quality, difficult temperament, and risky behavior from childhood to early adoles cence. Journal of School Psychology, 48(5), 389–412. https://doi. org/10.1016/j.jsp.2010.05.001 Yoon, J. S. (2002). Teacher characteristics as predictors of teacher- student relationships: Stress, negative affect, and self-efficacy. Social Behavior and Personality, 30(5), 485–493. https://doi. org/10.2224/sbp.2002.30.5.485 Sabol, T. J., & Pianta, R. C. (2012). Recent trends in research on teacher-child relationships. Attachment & Human Development, 14(3), 213–231. https://doi.org/10.1080/14616734.2012.672262f Zee, M., Koomen, H. M. Y., & Van der Veen, I. (2013). Student- teacher relationship quality and academic adjustment in upper elementary school: The role of student personality. Journal of School Psychology, 51(4), 517–533. https://doi.org/10.1016/j. jsp.2013.05.003 Satorra, A., & Bentler, P. M. (2001). Publisher’s Note Springer Nature remains neutral with regard to juris dictional claims in published maps and institutional affiliations. Conclusion A scaled difference chi-square test statistic for moment structure analysis. Psychometrika, 66(4), 507–514. https://doi.org/10.1007/bf02296192 Silver, R. B., Measelle, J. R., Armstrong, J. M., & Essex, M. J. (2005). Trajectories of classroom externalizing behavior: Contributions of child characteristics, family characteristics, and the teacher-child relationship during the school transition. 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https://openalex.org/W4388279368	https://www.nature.com/articles/s41598-023-46036-9.pdf	English	null	Structure–activity studies of Streptococcus pyogenes enzyme SpyCEP reveal high affinity for CXCL8 in the SpyCEP C-terminal	Scientific reports	2,023	cc-by	8,930	Structure–activity studies of Streptococcus pyogenes enzyme SpyCEP reveal high affinity for CXCL8 in the SpyCEP C‑terminal Max Pearson 1,2, Carl Haslam 3, Andrew Fosberry 3, Emma J. Jones 3, Mark Reglinski 1, Lucy Reeves 1, Robert J. Edwards 4, Richard Ashley Lawrenson 1, Jonathan C. Brown 1, Danuta Mossakowska 3,5, James Edward Pease 6* & Shiranee Sriskandan 1,2* OPEN The Streptococcus pyogenes cell envelope protease (SpyCEP) is vital to streptococcal pathogenesis and disease progression. Despite its strong association with invasive disease, little is known about enzymatic function beyond the ELR+ CXC chemokine substrate range. As a serine protease, SpyCEP has a catalytic triad consisting of aspartate (D151), histidine (H279), and serine (S617) residues which are all thought to be mandatory for full activity. We utilised a range of SpyCEP constructs to investigate the protein domains and catalytic residues necessary for enzyme function. We designed a high-throughput mass spectrometry assay to measure CXCL8 cleavage and applied this for the first time to study the enzyme kinetics of SpyCEP. Results revealed a remarkably low Michaelis-Menton constant (KM) of 82 nM and a turnover of 1.65 molecules per second. We found that an N-terminally- truncated SpyCEP C-terminal construct containing just the catalytic dyad of H279 and S617 was capable of cleaving CXCL8 with a similar KM of 55 nM, albeit with a reduced substrate turnover of 2.7 molecules per hour, representing a 2200-fold reduction in activity. We conclude that the SpyCEP C-terminus plays a key role in high affinity substrate recognition and binding, but that the N-terminus is required for full catalytic activity. Group A Streptococcus (GAS) or Streptococcus pyogenes is a leading human pathogen that manifests clinically as a broad spectrum of diseases, ranging from less severe, usually self-limiting infections, to life-threatening invasive diseases such as necrotizing fasciitis and toxic shock syndrome. While much of the global health burden of S. pyogenes can be attributed to auto-immune sequelae such as rheumatic heart disease, invasive diseases contribute greatly to S. pyogenes-associated global mortality. At least 663,000 new cases of invasive infection arise per year, accounting for approximately 163,000 deaths1, with the global incidence increasing in both high and low-income countries2. Mortality rates remain high despite intervention; indeed, 20% of patients with invasive S. pyogenes disease die within 7 days of infection onset3. Several virulence factors contribute to pathogenesis in invasive S. pyogenes disease, chief among which is Streptococcus pyogenes cell envelope protease (SpyCEP), an immune-modulatory cell wall-associated protease. SpyCEP is responsible for the rapid and efficient cleavage of a distinct group of CXC chemokines comprising CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8, both locally at the site of an infection, and systemically4–7. The ELR+ chemokines, named for their conserved N-terminal glutamate-leucine-arginine motifs, specifically act upon neutrophils eliciting their recruitment and activation. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ such, SpyCEP cleavage of CXC chemokines results in a reduction of CXCR1 and CXCR2-mediated neutrophil chemotaxis and subsequent paucity of neutrophils at the site of S. pyogenes infection4. SpyCEP has recently been shown to cleave the human anti-microbial peptide, LL-37 in addition to chemokines10. Whilst cleavage does not affect the antimicrobial action of the peptide, it was reported to reduce LL-37 specific neutrophil chemotaxis10. f p p p pi p SpyCEP is expressed by S. pyogenes as a 1647 amino acid, 180 kDa subtilisin-like serine protease, found both anchored in the bacterial cell wall and shed into the local enviroment. The crystal structure of SpyCEP was recently solved to 2.8 Å resolution11 and further refined to 2.2 Å resolution12. It is a member of the S8 subtilase family, members of which are characterised by a catalytic triad consisting of an aspartate, histidine, and serine residue, each surrounded by a region of highly conserved amino acids13. SpyCEP is unique among streptococcal proteases in that, during maturation, it is autocatalytically cleaved between residues Q244 and S245 into 2 distinct polypeptides, a 30 kDa N-terminal polypeptide and a 150 kDa C-terminal polypeptide. The two polypeptides harbour the separate residues required for the formation of the catalytic site7; the N-terminal fragment contains the catalytic D151 residue, and the C-terminal fragment contains the catalytic H279 and S617 residues. Upon cleavage, the two polypeptides re-associate non-covalently to reconstitute the active enzyme7. The recent crystal structures have shed further light upon the structure of SpyCEP, describing 9 separate domains, the first 5 of which contain the catalytic triad necessary for enzymatic activity11. y y y y SpyCEP has been included as a target antigen in several recent S. pyogenes vaccine designs due to its cell surface expression, highly conserved nature, and central role in S. pyogenes pathogenesis. Immunisation with SpyCEP successfully elicits a SpyCEP-specific neutralising antibody response, providing protection against sys- temic bacterial dissemination and reducing disease severity in S. pyogenes intramuscular, skin infection models and non-human primate infection models14–19. Data that demonstrate vaccine dependence on enzyme inhibition highlight the importance of understanding the enzymatic activity of SpyCEP and the potential to improve upon vaccine or inhibitor design14. Despite this, little is known about the catalytic properties of the enzyme, except preliminary structure–func- tion studies7,11,12,20. Published data largely focus on the impact of SpyCEP on streptococcal pathogenesis and disease progression. www.nature.com/scientificreports/ In this study we generated multiple SpyCEP constructs to confirm domains necessary for catalytic activity. We then used mass spectrometry to determine the enzyme kinetics of two SpyCEP constructs for the natural substrate CXCL8. Methods Cl i Cloning and purification of recombinant SpyCEP constructs in Escherichia coli Codon-optimised SpyCEP gene constructs were expressed in Escherichia coli using synthetic gene sequences (GenScript) from Spy_0416 in the SF370 S. pyogenes M1 genome7,21 representing full-length enzyme, SpyCEP34–1613, the N-terminal polypeptide, SpyCEP34–244, and C-terminal polypeptide SpyCEP245–1613. Con- structs were also generated with alanine substitutions to replace catalytic residues in the N-terminal fragment (SpyCEP34–244 D151A), and the C-terminal fragment (SpyCEP245–1613 S617A). To enable downstream protein purification, both the full-length enzyme and C-terminal polypeptides were expressed with a C-terminal 6-his- tidine tag and the N-terminal polypeptides were expressed with an N-terminal FLAG tag and TEV linker. g p yp p p g All SpyCEP constructs were cloned into the vector pET-24B and expressed in BL21 (DE3) competent E. coli, cultured in Terrific Broth medium supplemented with 50 µg/mL kanamycin at 37 °C and shaken at 200 rpm, for 3 h. The cultures were induced with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), cooled to 15 °C and shaken at 200 rpm for 16 h before lysis by sonication on ice. Full-length and C-terminal constructs were purified by Ni-IDA affinity chromatography (GenScript) as per the manufacturer’s instructions. N-terminal constructs were purified by anti-flag M2 agarose resin chromatography (Sigma-Aldrich) as per manufacturer’s instruction, and further concentrated and purified by SP ion-exchange (Sigma-Aldrich) and Q FF ion-exchange chromatography (GE Healthcare) as per manufacturer’s instruction. All SpyCEP constructs were subsequently concentrated and purified by size exclusion chromatography on a HiLoad Superdex 200 or 75 prep grade column (GE Healthcare) dependent on molecular weight.h The resultant panel of recombinantly expressed SpyCEP constructs is shown in Table 1. All of the resultant protein preparations were found to be > 80% pure as determined following analysis by SDS-PAGE (Figure S1). The re-association of the SpyCEP N and C-termini to create full-length constructs was carried out by equimolar co- incubation at 37 °C for 30 min in 40 mM Tris–HCl pH 7.5, 0.1 mM CHAPS, 1 mM DTT, 0.1% BSA, 75 mM NaCl. Table 1. SpyCEP constructs used in this study. Constructs were expressed recombinantly in E. coli except for s.pSpyCEP that was expressed in, and purified from S. pyogenes. SpyCEP construct SpyCEP construct description SpyCEP Full-length, SpyCEP34–1613-6His SpyCEPD151A, S617A Full-length double mutant, SpyCEP34–1613 D151A, S617A-6His N-terminal N-terminal fragment, FLAG-TEV-SpyCEP34–244 C-terminal C-terminal fragment, SpyCEP245–1613-6His N-terminalD151A N-terminal mutant fragment, FLAG-TEV-SpyCEP34–244 D151A C-terminalS617A C-terminal mutant fragment, SpyCEP245–1613 S617A-6His s.pSpyCEP SpyCEP from S. Structure–activity studies of Streptococcus pyogenes enzyme SpyCEP reveal high affinity for CXCL8 in the SpyCEP C‑terminal Max Pearson 1,2, Carl Haslam 3, Andrew Fosberry 3, Emma J. Jones 3, Mark Reglinski 1, Lucy Reeves 1, Robert J. Edwards 4, Richard Ashley Lawrenson 1, Jonathan C. Brown 1, Danuta Mossakowska 3,5, James Edward Pease 6* & Shiranee Sriskandan 1,2* OPEN SpyCEP inactivates these chemokines by cleaving the chemokine C-terminal α-helix, releasing a 13 amino acid peptide in the case of CXCL84. Although chemokine specificity for binding neutrophil chemokine receptors, CXCR1 and CXCR2, is conferred to a large extent by the chemokine N-terminal ELR motif8, the chemokine C-terminus is necessary for efficient recep- tor binding and activation9, in addition to chemokine translocation from tissues to endothelial lumen8. As 1Department of Infectious Disease, Imperial College London, London W12 0NN, UK. 2Centre for Bacterial Resistance Biology, Imperial College London, London SW7 2AZ, UK. 3GlaxoSmithKline R&D, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK. 4Department of Medicine, Imperial College London, London W12 0NN, UK. 5Malopolska Centre of Biotechnology, Jagiellonian University, 30‑387 Kraków, Poland. 6National Heart and Lung Institute, Imperial College London, London SW7 2AZ, UK. email: j.pease@imperial.ac.uk; s.sriskandan@ imperial.ac.uk \| https://doi.org/10.1038/s41598-023-46036-9 Scientific Reports \| (2023) 13:19052 www.nature.com/scientificreports/ Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‑page) y p p y y g p p g To visualise chemokine cleavage, 50 pmol of recombinant human CXCL1 or CXCL8 (R&D Systems) was incu- bated with C-terminal SpyCEP in a final volume of 20 μl reaction buffer (40 mM Tris-HCL pH 7.5, 0.1% BSA, 75 mM NaCl), at a molar ratio of 1:5, 1:50, 1:500, 1:5000 in favour of chemokine for 2 h at 37 °C. C-terminalS617A and N-terminal constructs34–244 were included as controls and assayed at the highest 1:5 molar ratio. Reactions were halted by the addition of Dithiothreitol (DTT) at a final concentration of 100 mM, 4X Bolt LDS sample buffer and heating to 70 °C for 10 min. Samples were separated on pre-cast 4–12% MES buffered Bolt Bis–Tris gradient gels (Invitrogen) by SDS-PAGE gel electrophoresis at 165 V for 35 min with SeeBlue Plus 2 (Invitrogen) used for molecular weight ladder. Gels were stained with PageBlue protein staining solution (Thermo Fisher Scientific) overnight and de-stained in deionised water. Expression of histidine‑tagged SpyCEP in S. pyogenes Expression of histidine‑tagged SpyCEP in S. pyogenes l f l bl d b p gg py py g To permit release of soluble His-tagged SpyCEP by S. pyogenes, the C-terminal anchor domain of SpyCEP was replaced by a stop codon in S. pyogenes strain H292, an emm81 strain that makes abundant SpyCEP6, with activity shown by an ELISA-based method12 to be: KM 101 nM, Kcat 3.19. A 549 base pair region immediately upstream of the SpyCEP cell wall anchor motif was amplified from S. pyogenes H292 genomic DNA using the primers 5′GGGAATTCTGTTGTCAGGTAACAGTCTTATCTTGCC-3′ and 5′CCGAATTCACAACACTAGGCTTTT GCTGAGGTCGTTG-3′. EcoRI restriction sites were incorporated at the terminal ends of the primer sequences. The amplified DNA was cloned into the homologous recombination plasmid pUCMUT to produce the vector pUCMUTCEP which was transformed into One Shot TOP10 Chemically Competent E. coli (Thermo Fisher Scientific) according to the manufacturer’s instructions. The nucleotide sequence of the SpyCEP C-terminus was subsequently further modified to encode a hexa-histidine tag by inverse PCR using pUCMUTCEP as the template and the primers 5′-TATCCTAGGTAGTGTTGTGAATTCGTAATCATGGTCATAG-3′ and 5′-TATCCT AGGATGATGATGATGATGATGGGCTTTTGCTGAGGTCGTTG-3′. The amplification was performed using GoTaq Long PCR Master Mix (Promega) according to the manufacturer’s instructions. AvrII restriction sites, incorporated at the terminal ends of the primer sequences, facilitated re-circulation of the amplified plasmid (denoted pUCMUTCEP-HIS). The pUCMUTCEP-HIS construct was introduced into H292 by electroporation and crossed into the chromosome by homologous recombination as previously described22 to generate strain H1317. Secretion of 6-His-tagged SpyCEP into the culture supernatant of H1317 was confirmed by western blotting (data not shown). To purify SpyCEP, S. pyogenes H1317 was grown in Todd-Hewitt broth (Oxoid) overnight at 37 °C with 5% CO2 The culture was pelleted at 2500 × g for 10 min and the supernatant sterilised using Amicon 0.22 µm filters, then concentrated using 15 ml Amicon 100 kDa centrifuge filter columns and purified by nickel column affinity chromatography (Novagen His-Bind Resin) as per the manufacturer’s instructions. SDS-PAGE analysis of the resultant s.pSpyCEP construct showed a pure enzyme preparation (Figure S2). Multiplex fluorescent western blotting l g To visualise generation of both intact CXCL8 and the larger (N-terminal) CXCL8 cleavage product, a rabbit anti- serum was raised against the CXCL8 neo-epitope (anti-ENWVQ) that is exposed following SpyCEP cleavage of CXCL823. SpyCEP constructs were incubated at 37 °C with 18.75 pmol of recombinant human CXCL8 in a final volume of 20 μl, at a 1:50 molar ratio in favour of CXCL8. Digests were halted and separated by SDS-PAGE gel electrophoresis as described above. Proteins were transferred by iBlot2 (Thermo Fisher Scientific) onto 0.2 μm nitrocellulose membranes as per manufacturer’s instructions. Membranes were subsequently blocked for 1 h at room temperature in blocking buffer (PBS with 5% skimmed milk powder (Sigma-Aldrich) and 0.1% Tween), then blotted overnight at 4 °C with 2 primary antibodies, 1 µg/ml mouse anti-human CXCL8 (R&D Systems) and 1:1000 rabbit anti-ENWVQ. Membranes were washed in wash buffer (PBS with 0.05% Tween) and incubated with 1:7500 goat anti-rabbit IgG A680nm and 1:7500 goat anti-mouse IgG A790nm for 1 h at room temperature before being visualised on LiCor Odyssey Fc (Invitrogen). g y y g SpyCEP activity against LL-37 was assessed by a 16 h, 37 °C incubation of 111.1 pmol human LL-37 (R&D Systems) with SpyCEP constructs at a 1:10 molar ratio in favour of LL-37 in a final volume of 20 μl. Reactions were stopped, separated and blotted onto 0.2 μm nitrocellulose as above and incubated overnight at 4 °C in blocking buffer supplemented with 2 μg/ml polyclonal sheep IgG anti-LL-37 (R&D Systems). Membranes were washed in wash buffer (PBS with 0.05% Tween) and incubated in blocking solution with rabbit anti-sheep IgG (Abcam) at 1:40,000 dilution for 1 h at room temperature and visualised on LiCor Odyssey Fc (Invitrogen). Methods Cl i pyogenes supernatant, SpyCEP34–1613-6His Table 1. SpyCEP constructs used in this study. Constructs were expressed recombinantly in E. coli except for s.pSpyCEP that was expressed in, and purified from S. pyogenes. https://doi.org/10.1038/s41598-023-46036-9 Scientific Reports \| (2023) 13:19052 \| www.nature.com/scientificreports/ Expression of histidine‑tagged SpyCEP in S. pyogenes Enzyme linked immunosorbent assay (ELISA) analysis of chemokine cleavage Enzyme linked immunosorbent assay (ELISA) analysis of chemokine cleavage Catalytic activities of SpyCEP constructs were measured through detection of remaining intact CXCL8 or CXCL1 substrate following incubation with SpyCEP by ELISA (R&D Systems human CXCL8 and CXCL1 DuoSet ELISA) as per manufacturer’s instructions. For cleavage time courses of CXCL8 by C-terminal SpyCEP, 5 pmol CXCL8 were incubated with C-terminal constructs at a range of enzyme: chemokine molar ratios, 1:5–1:250, in a final volume of 100 μl at room temperature for 60 min. Full-length SpyCEP and C-terminal S617A were included as controls at a molar ratio of 1:1000 and 1:5, respectively. To compare SpyCEP cleavage rates of CXCL8 and CXCL1, 5 fmol or 10 fmol SpyCEP were incubated with 2 pmol human CXCL8 and CXCL1 respectively (R&D Systems), in a final volume of 100 μl and incubated at room temperature for 30 min. All reactions were halted at defined timepoints with the addition of concentration of Pefabloc (Sigma-Aldrich) to a final concentration of 2 mg/ml (8.34 mM).i g Linear regression analyses of the initial five time points of CXCL1 and CXCL8 cleavage (0, 1, 2, 3 and 4 min) were utilised to determine the maximal rate of SpyCEP activity. Scientific Reports \| (2023) 13:19052 \| https://doi.org/10.1038/s41598-023-46036-9 www.nature.com/scientificreports/ Mass spectrometry analysis of CXCL8 cleavage p y y g Analysis of CXCL8 cleavage was assayed on a SCIEX API6500 triple quadrupole electrospray mass spectrometer coupled to a high-throughput robotic sample preparation and injection system, RapidFire200 (Agilent Technolo- gies). CXCL8 substrate, the 13 amino acid CXCL8 SpyCEP cleavage product RVVEKFLKRAENS, and a heavy atom substituted internal standard of CXCL8 SpyCEP cleavage product RV [U13C5 15N-VAL]-EKF-[U-13C6 15N-Leu]- KRAENS) were monitored by mass spectrometry. The mass spectrometer was operated in positive electrospray MRM mode, and transitions (Q1/Q3) for each species were optimised to give m/z as follows: CXCL8, 1048.7/615.3, CXCL8 cleavage product 526.2/211.1, internal standard 530.5/211.1. A dwell time of 50 ms was used for the MRM transitions. The mass spectrometer was operated with a spray voltage of 5500 V and at a source temperature of 650 °C. p To assay CXCL8 cleavage dynamically, chemokine and SpyCEP constructs were loaded into a 384 well plate to the following final concentrations: 6.25–2000 nM chemokine, 250 pM SpyCEP or 40 nM C-terminal SpyCEP in a 20 μl reaction volume. Enzyme linked immunosorbent assay (ELISA) analysis of chemokine cleavage Reactions were carried out at 21 °C and stopped at desired time points, 0–240 min, by the addition of stop solution (1% formic acid) supplemented with 1 µM heavy atom substituted CXCL8 internal standard and centrifuged 2000 × g for 10 min. Assay plates were transferred onto the RapidFire200 integrated to the API6500 mass spectrometer. Samples were aspirated under vacuum directly from 384-well assay plates for 0.6 s. The samples were then loaded onto a C18 solid-phase extraction cartridge to remove non-volatile buffer salts, using HPLC -grade water supplemented with 0.1% (v/v) formic acid at a flow rate of 1.5 mL/min for 4 s. The retained analytes were eluted to the mass spectrometer by washing the cartridge with acetonitrile HPLC- grade water (8:2, v/v) with 0.1% (v/v) formic acid at 1.25 mL/min for 4 s. The cartridge was re-equilibrated with HPLC-grade water supplemented with 0.1% (v/v) formic acid for 0.6 s at 1.5 mL/min. g Results were expressed as a ratio of CXCL8 cleavage product (526.2/211.1) intensity areas compared with the internal standard (530.5/211.1) instensity areas. The results were interpolated from a standard curve constructed using known amounts of cleaved CXCL8 over the range of 25–2000 nM where there was a linear relationship (R2 = 0.99) and with a coefficient of variation < 9% for all concentrations. Where interpolated values were below 25 nM, the assay LOQ, values were assigned an arbitrary value of LOQ/2, 12.5 nM. Kinetic analysis Linear regression analyses from five time points of full-length SpyCEP (1, 2, 3, 4, 5 min) and C-terminal SpyCEP (15, 30, 45, 60, 75 min) CXCL8 reactions were plotted against substrate concentration and kinetics derived from the Michaelis-Menton equation Y = VmaxX/(KM + X) and the Kcat equation Y = ETkcatX/(KM + X) where Et = enzyme concentration as fitted by GraphPad Prism v 8.0.2. Screening of CXCL8 cleavage activity using fluorescent western blotting Differential cleavage of CXCL8 and CXCL1 by full‑length and C‑terminal SpyCEP To determine whether the catalytic activity of the C terminal SpyCEP fragment was reproducible Screening of CXCL8 cleavage activity using fluorescent western blotting Screening of CXCL8 cleavage activity using fluorescent western blotting To initially assess the activity of recombinant SpyCEP constructs we screened constructs for specific CXCL8 cleavage activity using two-colour multiplex western blotting. Human CXCL8 was incubated for 2 h at 37 °C with the different SpyCEP constructs, and the reaction products were separated by SDS-PAGE, then immunoblotted using separate antibodies that detect either intact or cleaved CXCL8. Detection of full-length CXCL8 (8 kDa, green bands) or the CXCL8 neo epitope (ENWVQ), exposed after SpyCEP cleavage, (6 kDa, red bands) was evi- dent with this system (Fig. 1). Recombinant full-length SpyCEP expressed in E. coli successfully cleaved CXCL8 to completion, with no difference observed between CXCL8 cleavage with S. pyogenes SpyCEP and E. coli SpyCEP (Fig. 1). As expected, no CXCL8 cleavage was observed when using the catalytically inactive mutant, SpyCEPD151A, S617A. As has been previously reported7, the SpyCEP N- and C-termini, when independently expressed and purified, can be re-associated to form an active enzyme R.E SpyCEP, which successfully cleaved CXCL8. The N-terminal fragment of SpyCEP alone could not cleave CXCL8. Unexpectedly, however, the C-terminal frag- ment of SpyCEP was observed to cleave CXCL8, albeit not to completion. This independent catalytic activity was negated by mutation of the catalytic S617 to alanine (C-terminalS617A construct, Fig. 1). Cleavage of CXCL8 by the SpyCEP C-terminal fragment was enhanced when re-associated with the catalytically inert N-terminal mutant (N-terminalD151A construct); indeed, activity was equivalent to that observed with the re-associated enzyme under these conditions, with cleavage of CXCL8 to near completion. However, the N-terminalD151A construct was unable to restore catalytic activity to the C-terminalS617A construct when the two were combined.hifi g g y gl g To initially assess the activity of recombinant SpyCEP constructs we screened constructs for specific CXCL8 cleavage activity using two-colour multiplex western blotting. Human CXCL8 was incubated for 2 h at 37 °C with the different SpyCEP constructs, and the reaction products were separated by SDS-PAGE, then immunoblotted using separate antibodies that detect either intact or cleaved CXCL8. Detection of full-length CXCL8 (8 kDa, green bands) or the CXCL8 neo epitope (ENWVQ), exposed after SpyCEP cleavage, (6 kDa, red bands) was evi- dent with this system (Fig. 1). Recombinant full-length SpyCEP expressed in E. coli successfully cleaved CXCL8 to completion, with no difference observed between CXCL8 cleavage with S. pyogenes SpyCEP and E. coli SpyCEP (Fig. 1). Screening of CXCL8 cleavage activity using fluorescent western blotting As expected, no CXCL8 cleavage was observed when using the catalytically inactive mutant, SpyCEPD151A, S617A. As has been previously reported7, the SpyCEP N- and C-termini, when independently expressed and purified, can be re-associated to form an active enzyme R.E SpyCEP, which successfully cleaved CXCL8. The N-terminal fragment of SpyCEP alone could not cleave CXCL8. Unexpectedly, however, the C-terminal frag- ment of SpyCEP was observed to cleave CXCL8, albeit not to completion. This independent catalytic activity was negated by mutation of the catalytic S617 to alanine (C-terminalS617A construct, Fig. 1). Cleavage of CXCL8 by the SpyCEP C-terminal fragment was enhanced when re-associated with the catalytically inert N-terminal mutant (N-terminalD151A construct); indeed, activity was equivalent to that observed with the re-associated enzyme under these conditions, with cleavage of CXCL8 to near completion. However, the N-terminalD151A construct was unable to restore catalytic activity to the C-terminalS617A construct when the two were combined. The results demonstrated for the first time that the SpyCEP C-terminal fragment alone is sufficient for cata- lytic cleavage of CXCL8 in this assay system. The data also established that, although the presence of the SpyCEP N-terminal fragment enhanced enzymatic activity, the N-terminal residue D151 was dispensable for enzymatic activity. We further sought to examine whether the C-terminal displayed activity against the newly described substrate, LL-3710. Western blot analysis confirmed cleavage of LL-37 by full-length recombinant SpyCEP, dem- onstrated by a reduction in band size, however the C-terminal fragment was unable to cleave LL-37 despite a high molar ratio of enzyme to LL-37 (1: 10) and a prolonged 16-h incubation at 37 °C (Figure S3). The results demonstrated for the first time that the SpyCEP C-terminal fragment alone is sufficient for cata- lytic cleavage of CXCL8 in this assay system. The data also established that, although the presence of the SpyCEP N-terminal fragment enhanced enzymatic activity, the N-terminal residue D151 was dispensable for enzymatic activity. We further sought to examine whether the C-terminal displayed activity against the newly described substrate, LL-3710. Western blot analysis confirmed cleavage of LL-37 by full-length recombinant SpyCEP, dem- onstrated by a reduction in band size, however the C-terminal fragment was unable to cleave LL-37 despite a high molar ratio of enzyme to LL-37 (1: 10) and a prolonged 16-h incubation at 37 °C (Figure S3). Differential cleavage of CXCL8 and CXCL1 by full‑length and C‑terminal SpyCEP Reactions were halted at specified timepoints by the addition of Pefabloc to a final concentration of 2 mg/ml. N = 6 experimental replicates for each construct, data points show means, error bars represent SD. ns p > 0.05, * p ≤ 0.05, **** p ≤ 0.0001, at 60 min as determined by ordinary one-way ANOVA. Figure 2. Cleavage activity of SpyCEP and C-terminal SpyCEP245–1613 constructs using CXCL8 ELISA. SpyCEP constructs were co-incubated with 5 pmol CXCL8. Graphs show residual CXCL8 after a 60-min room temperature incubation, using full-length SpyCEP at a 1:1000 ratio to CXCL8; the C-terminal SpyCEP construct at a 1:5–1:250 molar ratio to CXCL8; and the C-terminalS617A mutant at a 1:5 molar ratio to CXCL8. Reactions were halted at specified timepoints by the addition of Pefabloc to a final concentration of 2 mg/ml. N = 6 experimental replicates for each construct, data points show means, error bars represent SD. ns p > 0.05, * p ≤ 0.05, **** p ≤ 0.0001, at 60 min as determined by ordinary one-way ANOVA. After 5 min incubation full-length SpyCEP cleaved over 50% of the starting CXCL8 input, with only 6.25% of CXCL8 remaining after 1 h. At the highest concentration of C-terminal SpyCEP tested, a 1:5 molar ratio, the C-terminal fragment alone cleaved 9% of the starting CXCL8 by 5 min, 25% by 30 min and 42% by 60 min. Indeed, at the lowest concentration tested, a 1:250 molar ratio, the C-terminal of SpyCEP cleaved 9% of CXCL8 input by 1 h. As demonstrated by immunofluorescent western blotting, the serine residue at position 617 was vital for SpyCEP catalytic function, as no CXCL8 cleavage was observed using the C-terminalS617A construct, even when employed at the highest enzyme: chemokine ratio. After a 1-h incubation, full-length SpyCEP and C-terminal constructs (assayed using a molar enzyme: chemokine ratio of 1:5, 1:25 and 1:50) cleaved significantly more CXCL8 compared to the C-terminalS617A construct. p SDS-PAGE analysis of the catalytic activity of the C-terminal SpyCEP construct additionally showed that C-terminal activity was not restricted to the CXCL8 substrate. Over 2 h at 37 °C using a 1:5 molar ratio of enzyme: substrate, the SpyCEP C-terminal construct was capable of cleaving human CXCL1 to near completion (Figure S4). Differential cleavage of CXCL8 and CXCL1 by full‑length and C‑terminal SpyCEP f g y g py To determine whether the catalytic activity of the C-terminal SpyCEP fragment was reproducible over a shorter incubation period, we assessed CXCL8 cleavage by ELISA, where CXCL8 cleavage is detected through a reduction in substrate concentration. SpyCEP C-terminal constructs were incubated with CXCL8 at molar ratios ranging from 1:5 to 1:250 (enzyme: chemokine) over a 60-min time course at room temperature. Full-length recombinant SpyCEP and the inactive C-terminal fragment, C-terminalS617A, were included as controls at a molar ratio of 1:1000 and 1:5, respectively. Under these conditions, near complete CXCL8 cleavage was observed for full-length SpyCEP and a dose-dependent increase in catalytic activity was observed for the C-terminal fragment (Fig. 2). https://doi.org/10.1038/s41598-023-46036-9 Scientific Reports \| (2023) 13:19052 \| www.nature.com/scientificreports/ Figure 1. Cleavage activity of recombinant SpyCEP constructs assayed by immunoblot. Two colour immunoblot showing cleavage of 18.75 pmol CXCL8 when incubated for 2 h at 37 °C either alone (2nd lane) or with a panel of SpyCEP constructs at a 1:50 molar ratio (SpyCEP: CXCL8). Green bands represent intact CXCL8 (anti-CXCL8 antibody); red bands represent cleaved CXCL8 (anti-ENWVQ). A 17 kDa molecular weight marker is shown in blue, 8 kDa and 6 kDa molecular weights are highlighted by arrows. Figure has been cropped and the original blot is presented in supplementary Fig. S6. Figure is representative of 2 independent immunoblots. Figure 1. Cleavage activity of recombinant SpyCEP constructs assayed by immunoblot. Two colour immunoblot showing cleavage of 18.75 pmol CXCL8 when incubated for 2 h at 37 °C either alone (2nd lane) or with a panel of SpyCEP constructs at a 1:50 molar ratio (SpyCEP: CXCL8). Green bands represent intact CXCL8 (anti-CXCL8 antibody); red bands represent cleaved CXCL8 (anti-ENWVQ). A 17 kDa molecular weight marker is shown in blue, 8 kDa and 6 kDa molecular weights are highlighted by arrows. Figure has been cropped and the original blot is presented in supplementary Fig. S6. Figure is representative of 2 independent immunoblots. Figure 2. Cleavage activity of SpyCEP and C-terminal SpyCEP245–1613 constructs using CXCL8 ELISA. SpyCEP constructs were co-incubated with 5 pmol CXCL8. Graphs show residual CXCL8 after a 60-min room temperature incubation, using full-length SpyCEP at a 1:1000 ratio to CXCL8; the C-terminal SpyCEP construct at a 1:5–1:250 molar ratio to CXCL8; and the C-terminalS617A mutant at a 1:5 molar ratio to CXCL8. Differential cleavage of CXCL8 and CXCL1 by full‑length and C‑terminal SpyCEP g To further assess the activity of SpyCEP and to interrogate reaction rates against separate chemokines, full- length SpyCEP was incubated with CXCL1 and CXCL8 and the remaining chemokine levels determined by ELISA. Human CXCL1 or human CXCL8 were incubated with SpyCEP at room temperature over a 30-min time course, at 1:200 or 1:400 molar ratios respectively (enzyme: chemokine). 5 fmol of SpyCEP rapidly and efficiently cleaved 2 pmol CXCL8, with ~ 15% of the chemokine input remaining after 10 min of incubation (Fig. 3A). This contrasted with CXCL1 cleavage, that required 10 fmol SpyCEP to cleave just 25% of the chemokine input over the same 10-min period. Indeed, by 30 min SpyCEP had yet to cleave half of the starting amount of CXCL1 https://doi.org/10.1038/s41598-023-46036-9 Scientific Reports \| (2023) 13:19052 \| www.nature.com/scientificreports/ Figure 3. Cleavage of CXCL8 and CXCL1 by recombinant full-length SpyCEP. 30-min, room temperature cleavage time course of: (A) 2 pmol CXCL8 with 5 fmol SpyCEP and (B) 2 pmol CXCL1 with 10 fmol SpyCEP. Cleavage reactions were halted at specified timepoints by the addition of Pefabloc to a final concentration of 2 mg/ml and the remaining chemokine measured by ELISA. N = 6 experimental replicates for each data point, error bars represent SD of mean values. Figure 3. Cleavage of CXCL8 and CXCL1 by recombinant full-length SpyCEP. 30-min, room temperature cleavage time course of: (A) 2 pmol CXCL8 with 5 fmol SpyCEP and (B) 2 pmol CXCL1 with 10 fmol SpyCEP. Cleavage reactions were halted at specified timepoints by the addition of Pefabloc to a final concentration of 2 mg/ml and the remaining chemokine measured by ELISA. N = 6 experimental replicates for each data point, error bars represent SD of mean values. Figure 3. Cleavage of CXCL8 and CXCL1 by recombinant full-length SpyCEP. 30-min, room temperature cleavage time course of: (A) 2 pmol CXCL8 with 5 fmol SpyCEP and (B) 2 pmol CXCL1 with 10 fmol SpyCE Cleavage reactions were halted at specified timepoints by the addition of Pefabloc to a final concentration of 2 mg/ml and the remaining chemokine measured by ELISA. N = 6 experimental replicates for each data poin error bars represent SD of mean values. (Fig. 3B). Mass spectrometry‑derived kinetics of active SpyCEP constructs d l h f ll l h d l To directly compare the active full-length and C-terminal SpyCEP constructs and to understand relative catalytic efficiencies, a mass spectrometry approach to continuously assay the generation of the 13 amino acid peptide cleaved from the native substrate CXCL8, following incubation with enzyme, was employed. A range of CXCL8 concentrations (25–2000 nM) were incubated with a fixed concentration of enzyme, either 250 pM of full-length SpyCEP, or 40 nM of the C-terminal SpyCEP245–1613 and the production of the 13 amino acid peptide was moni- tored over time. 250 pM full-length SpyCEP cleaved CXCL8 to near completion over 30 min when substrate concentrations were less than 250 nM; incomplete CXCL8 cleavage was observed when substrate concentration was in excess of 250 nM (Figure S5A). In contrast, 40 nM of the C-terminal SpyCEP245–1613 cleaved CXCL8 to near completion over 4 h when the CXCL8 concentration was 250 nM or less; incomplete CXCL8 cleavage was again observed when substrate concentrations were over 250 nM (Figure S5B). Under these conditions the C-terminal SpyCEP fragment maintained measurable catalytic activity throughout, though with reduced efficacy compared to the full-length construct. A 160-fold increase in enzyme concentration and additional 3.5 h reaction time were required to cleave a comparable amount of CXCL8. q p Linear regression analyses of 5 time points, where the rate of cleaved CXCL8 production was linear, were used to derive Michaelis-Menton plots (Fig. 4) and KM and Kcat values for each construct (Table 2). KM values for full-length SpyCEP and the C-terminal fragment were 82 nM and 55 nM respectively, suggesting nanomolar affinity of the SpyCEP constructs for CXCL8. The similarity in the KM values between the C-terminal SpyCEP fragment and the full-length enzyme point to a similar ability to bind substrate. The Kcat values (indica- tive of the number of molecules of cleaved CXCL8 produced per second) however, differed substantially. The Figure 4. Kinetic activity of full-length and C-terminal SpyCEP construct cleavage of CXCL8. Data points represent the mean change in cleaved CXCL8 production over time (M s-1) of, (A) Full-length SpyCEP and (B) C-terminal SpyCEP. Error bars represent the standard error of the mean of 5 reactions. Figure 4. Kinetic activity of full-length and C-terminal SpyCEP construct cleavage of CXCL8. Data points represent the mean change in cleaved CXCL8 production over time (M s-1) of, (A) Full-length SpyCEP and (B) C-terminal SpyCEP. Differential cleavage of CXCL8 and CXCL1 by full‑length and C‑terminal SpyCEP Utilising a linear regression of the initial 5 timepoints, where SpyCEP activity is maximal, we found that 5 fmol of SpyCEP was able to cleave 284 fmol of CXCL8 per minute, and 10 fmol of SpyCEP was capable of cleaving 62 fmol of CXCL1 per minute. These data confirmed the activity of recombinant SpyCEP and highlighted differential cleavage efficiency across the CXC substrate range—a feature which has been previously recognised but not quantified9. (Fig. 3B). Utilising a linear regression of the initial 5 timepoints, where SpyCEP activity is maximal, we found that 5 fmol of SpyCEP was able to cleave 284 fmol of CXCL8 per minute, and 10 fmol of SpyCEP was capable of cleaving 62 fmol of CXCL1 per minute. These data confirmed the activity of recombinant SpyCEP and highlighted differential cleavage efficiency across the CXC substrate range—a feature which has been previously recognised but not quantified9. Discussion SpyCEP is a serine protease and a leading virulence factor of S. pyogenes, with a narrow range of substrate specific- ity, restricted to the family of ELR+ CXC chemokines which modulate neutrophil mediated immune responses4,5,9 and LL-3710. Autocatalytic processing of SpyCEP results in the generation of two individual fragments that re- assemble to form an active enzyme7. Here, we describe the enzyme kinetics of full-length SpyCEP and report the KM of the enzyme for its natural substrate to be remarkably low, just 82 nM, consistent with high efficiency. Furthermore, we demonstrate that the C-terminal SpyCEP fragment can catalyse the cleavage of CXCL1 and CXCL8 independent of the N-terminal fragment. Indeed, when KM values were compared, they were found to be similar, suggesting that substrate binding may be confined to the C-terminal domain of SpyCEP. The enzymatic activity of the C-terminal SpyCEP fragment was, however, markedly reduced compared to full-length SpyCEP. When assessed by immunoblot, the N-terminal and N-terminalD151A constructs were equally able to restore full catalytic activity of the C-terminal SpyCEP fragment. Collectively, this suggests that although the aspartate 151 of the N-terminal fragment may be dispensable for catalysis, the domain itself is important for optimal enzyme activity. Serine proteases are ubiquitous and comprise up to one third of all proteolytic enzymes currently described. They are currently categorised by the MEROPS database24 into 13 distinct clans, being differentiated into groups of proteins based on their evolution from the same common ancestor, with SpyCEP belonging to the S8 family of the SB clan. S8 serine proteases are typified by a classical catalytic triad composed of serine, histidine and aspartic acid residues that together contribute to the hydrolysis of a peptide bond within the substrate. It is recognised that a number of serine protease clans employ a variation on the S8 catalytic triad, utilising instead a triad of serine, histidine, and glutamic acid, or serine, glutamic acid, and aspartic acid residues. Other clans utilise catalytic dyads of lysine and histidine or histidine and serine for proteolytic activity. Our data suggest that SpyCEP activity can reside in a catalytic dyad of histidine and serine, albeit at a greatly reduced efficacy. It is likely this large gulf in efficiency explains the failure of previous studies to detect catalytic activity within the isolated C-terminal SpyCEP fragment7. py g Within serine proteases there are additional features, beyond the catalytic triad residues, which can contribute to activity. Discussion The oxyanion hole for example, a pocket in the active site composed of backbone amide NH groups, may provide substrate stabilisation and help drive catalysis25. Additional residues located in close proximity to the catalytic pocket can also mitigate a loss of activity resulting from a missing residue, and water also has the potential to moonlight as a missing functional group25. These ‘stand ins’ can provide a possible substitute machinery to help drive catalytic function. Indeed, some studies have shown that, even with all three catalytic triad residues removed, serine proteases are still capable of catalysis at rates 1000-fold greater than the back- ground rate of hydrolysis25–27. g y y Mass spectrometry-based kinetics showed that recombinant full-length active SpyCEP has a KM of 82 nM and Kcat of 1.65 molecules per second; values which are in agreement with our initial ELISA-based kinetic assessment12 and values obtained from native S. pyogenes SpyCEP, KM 101 nM, Kcat 3.19 molecules per second (not shown). In contrast, the C-terminal fragment of SpyCEP has a KM of 55 nM and Kcat of 0.00075 molecules per second. These constructs both demonstrated low, nanomolar KM values, suggesting a high binding efficiency of SpyCEP for its natural substrate CXCL8, likely conferred by the C-terminal domain. This is in keeping with the fact that low nanomolar concentrations of CXCL8 are optimal for neutrophil recruitment9. Although KM values are often reported in the µM – mM range, nanomolar KM values are not without precedent for other serine proteases; human kallikrein 6 has a reported KM of 300 nM and Factor Xa, a constituent of the prothrombinase complex, has a KM of 150 nM for prothrombin28,29. Enzyme specificity, a constant which measures the cleavage efficiency of enzymes, (Kcat /KM ), for full-length SpyCEP was estimated to be 2.02 × 107 M-1 s-1, a value in the order of magnitude typical for serine proteases25. The specificity constant of the C-terminal fragment was ~ 1500- fold less, 1.36 × 104 M-1 s-1, and Kcat 2200-fold less, a reduction that is in line with previously reported aspartic acid mutants from a systematic mutational study of the Bacillus amyloliquefaciens subtilisin catalytic triad27. Mass spectrometry‑derived kinetics of active SpyCEP constructs d l h f ll l h d l Error bars represent the standard error of the mean of 5 reactions. Scientific Reports \| (2023) 13:19052 \| https://doi.org/10.1038/s41598-023-46036-9 www.nature.com/scientificreports/ Construct Kcat ± SEM (s-1) KM ± SEM (nM) Vmax ± SEM (M s-1) Kcat/KM (M-1 s-1) Full-length 1.65 ± 0.13 82 ± 26 4.14 × 10–10 ± 3.35 × 10–11 2.02 × 107 C-terminal 0.00075 ± 0.00005 55 ± 17 3.00 × 10–11 ± 2.17 × 10–12 1.36 × 104 Table 2. Kinetic parameters of full-length and C-terminal SpyCEP construct activity in cleavage of CXCL8. Kcat, KM and Vmax ± SEM and Kcat/KM for full-length SpyCEP and C-terminal SpyCEP derived from Michaelis-Menton graphs Y = VmaxX/(KM + X) and Kcat equation Y = ETkcatX/(KM + X) where Et = enzyme concentration, 2.5 × 10–10 M for full-length SpyCEP and 4 × × 10–8 M for C-terminal SpyCEP. Table 2. Kinetic parameters of full-length and C-terminal SpyCEP construct activity in cleavage of CXCL8. Kcat, KM and Vmax ± SEM and Kcat/KM for full-length SpyCEP and C-terminal SpyCEP derived from Michaelis-Menton graphs Y = VmaxX/(KM + X) and Kcat equation Y = ETkcatX/(KM + X) where Et = enzyme concentration, 2.5 × 10–10 M for full-length SpyCEP and 4 × × 10–8 M for C-terminal SpyCEP. Table 2. Kinetic parameters of full-length and C-terminal SpyCEP construct activity in cleavage of CXCL8. Kcat, KM and Vmax ± SEM and Kcat/KM for full-length SpyCEP and C-terminal SpyCEP derived from Michaelis-Menton graphs Y = VmaxX/(KM + X) and Kcat equation Y = ETkcat*X/(KM + X) where Et = enzyme concentration, 2.5 × 10–10 M for full-length SpyCEP and 4 × × 10–8 M for C-terminal SpyCEP. full-length enzyme cleaved 1.65 molecules of CXCL8 per second compared to the C-terminal construct which cleaved 2.7 molecules per hour, a 2200-fold reduction in activity. ull-length enzyme cleaved 1.65 molecules of CXCL8 per second compared to the C-terminal construct which leaved 2.7 molecules per hour, a 2200-fold reduction in activity. www.nature.com/scientificreports/ structural contribution to enzyme activity. Our data strongly suggest that substrate binding is likely to be attrib- uted to the C-terminal fragment, a finding consistent with related cell envelope proteinases of Lactococci13 and the closely related streptococcal protein, C5a peptidase30.hi The implications of our findings relating to activity of the C-terminal SpyCEP fragment in S. pyogenes patho- genesis are currently unclear; without the N-terminus, the enzymatic activity detected may be too low to be of consequence for chemokine cleavage in vivo, and in nature both N- and C-terminal fragments are likely to co-exist. SpyCEP has been a focus of S. pyogenes vaccine development since 200631, used either in isolation or combination with other antigenic targets12,15,17,18,32. Vaccine-induced SpyCEP specific antibodies appear not to act through traditional opsonic means14 and so may act through inhibition of SpyCEP activity. Many vaccine preparations evaluated have been based on ‘CEP5’; a polypeptide spanning residues 35–587 of SpyCEP which contains the N-terminal fragment and only part of the C terminal fragment19. Our findings relating to enzyme function suggest that antibodies targeting the C-terminal fragment of SpyCEP are more likely to provide greater neutralizing activity, and potentially improve vaccine efficacy. References Cell 91(3), 385–395 (1997). 8. Middleton, J. et al. Transcytosis and surface presentation of IL-8 by venular endothelial cells. Cell 91(3), 385–395 (1997). 9. Goldblatt, J. et al. A requirement for neutrophil glycosaminoglycans in chemokine: Receptor interactions is revealed by the Strep tococcal protease SpyCEP. J. Immunol. 202(11), 3246–3255 (2019). 10. Biswas, D. et al. LL-37-mediated activation of host receptors is critical for defense against group A Streptococcal infection. Cell Rep. 34(9), 108766 (2021). 10. Biswas, D. et al. LL-37-mediated activation of host receptors is critical for defense against group A Streptococcal infection. Cell Rep. 34(9), 108766 (2021). p 11. Jobichen, C. et al. Structure of ScpC, a virulence protease from Streptococcus pyogenes, reveals the functional domains and maturation mechanism. Biochem. J. 475(17), 2847–2860 (2018). 2. McKenna, S. et al. Structure, dynamics and immunogenicity of a catalytically inactive CXC chemokine-degrading protease SpyCEP from Streptococcus pyogenes. Comput. Struct. Biotechnol. J. 18, 650–660 (2020). coccus pyogenes. Comput. Struct. Biotechnol. J. 18, 650–660 (2020 p py g p 13. Siezen, R. J. Multi-domain, cell-envelope proteinases of lactic acid bacteria. Antonie Van Leeuwenhoek 76(1–4), 139–155 (1999). y g p 13. Siezen, R. J. Multi-domain, cell-envelope proteinases of lactic acid bacteria. Antonie Van Leeuwenhoek 76(1–4), 139–155 (1999). 14 Rivera Hernandez T et al An experimental group A Streptococcus vaccine that reduces pharyngitis and tonsillitis in a nonhuman 13. Siezen, R. J. Multi-domain, cell-envelope proteinases of lactic acid bacteria. Antonie Van Leeuwenhoek 76(1–4), 139–155 (1999). 14. Rivera-Hernandez, T. et al. An experimental group A Streptococcus vaccine that reduces pharyngitis and tonsillitis in a nonhuman i d l MBi 10(2) 10 1128 (2019) . Multi-domain, cell-envelope proteinases of lactic acid bacteria. A d l l h . Siezen, R. J. Multi-domain, cell-envelope proteinases of lactic ac 3. Siezen, R. J. Multi domain, cell envelope proteinases of lactic acid bacteria. Antonie Van Leeuwenhoek 76(1 4), 139 155 (1999). 4. Rivera-Hernandez, T. et al. An experimental group A Streptococcus vaccine that reduces pharyngitis and tonsillitis in a nonhuman primate model. MBio 10(2), 10–1128 (2019).i p 5. Bensi, G. et al. Multi high-throughput approach for highly selective identification of vaccine candidates: the Group A Streptococcus case. Mol. Cell Proteomics 11(6), M111.015693 (2012). 16. Pandey, M. et al. A synthetic M protein peptide synergizes with a CXC chemokine protease to induce vaccine-mediated protection against virulent streptococcal pyoderma and bacteremia. J. Immunol. 194(12), 5915–5925 (2015). 7. Pandey, M. et al. References References 1. Carapetis, J. R. et al. The global burden of group A Streptococcal diseases. Lancet Infect. Dis. 5(11), 685–694 (2005). ph g g p p f 2. Craik, N., et al. Global disease burden of Streptococcus pyogenes, in Streptococcus Pyogenes: Basic Biology to Clinical Manifestations, (eds Ferretti, J.J., Stevens, D.L., & Fischetti, V.A.) University of Oklahoma Health Sciences Center© The University of Oklahoma Health Sciences Center.: Oklahoma City (OK) (2022). 2. Craik, N., et al. Global disease burden of Streptococcus pyogenes, in Streptococcus Pyogenes: Basic Biology to Clinical Manifestations, (eds Ferretti, J.J., Stevens, D.L., & Fischetti, V.A.) University of Oklahoma Health Sciences Center© The University of Oklahoma Health Sciences Center.: Oklahoma City (OK) (2022). y ( ) ( ) 3. Ralph, A. P. & Carapetis, J. R. Group a streptococcal diseases and their global burden. Curr. Top. Microbiol. Immunol. 368, 1–27 (2013).i y ( ) ( ) 3. Ralph, A. P. & Carapetis, J. R. Group a streptococcal diseases and their global burden. Curr. Top. Microbiol. Immunol. 368, 1–27 (2013).i 3. Ralph, A. P. & Carapetis, J. R. Group a streptococcal diseases and their global burden. Curr. Top. Microbiol. Immunol. 368, 1–27 (2013). 4. Edwards, R. J. et al. Specific C-terminal cleavage and inactivation of interleukin-8 by invasive disease isolates of Streptococcus 4. Edwards, R. J. et al. Specific C-terminal cleavage and inactivation of interleukin-8 by invasive disease isolates of Streptococcus pyogenes. J. Infect. Dis. 192(5), 783–790 (2005).fi py g f 5. Kurupati, P. et al. Chemokine-cleaving Streptococcus pyogenes protease SpyCEP is necessary and sufficient for bacterial dissemi- nation within soft tissues and the respiratory tract. Mol. Microbiol. 76(6), 1387–1397 (2010). 5. Kurupati, P. et al. Chemokine-cleaving Streptococcus pyogenes protease SpyCEP is necessary and sufficient for bacterial dissemi- nation within soft tissues and the respiratory tract. Mol. Microbiol. 76(6), 1387–1397 (2010). t p y 6. Turner, C. E. et al. Emerging role of the interleukin-8 cleaving enzyme SpyCEP in clinical Streptococcus pyogenes infection. J. Infect. Dis. 200(4), 555–563 (2009). t p y 6. Turner, C. E. et al. Emerging role of the interleukin-8 cleaving enzyme SpyCEP in clinical Streptococcus pyogenes infection. J Infect. Dis. 200(4), 555–563 (2009). f 7. Zingaretti, C. et al. Streptococcus pyogenes SpyCEP: a chemokine-inactivating protease with unique structural and biochemica features. Faseb J. 24(8), 2839–2848 (2010). ( ) ( ) 8. Middleton, J. et al. Transcytosis and surface presentation of IL-8 by venular endothelial cells. Data availability y All data generated or analysed during this study are included in this published article and its supplementary information files. The datasets used and/or analysed during the current study are available from the correspond- ing auther on reasonable request. Received: 1 August 2023; Accepted: 26 October 2023 Received: 1 August 2023; Accepted: 26 October 2023 Discussion The development of an MS-based assay of SpyCEP activity, that is not dependent on western blotting or ELISA, provides potential for future high-throughput analysis of SpyCEP activity and detection of SpyCEP inhibitors.h The kinetic assays attributed a marked increase in CXCL8 turnover to the additional presence of the N-ter- minal fragment. Although we did not specifically evaluate the role of the aspartate residue at position 151 on the kinetics of SpyCEP activity, this residue did not contribute appreciably to cleavage of CXCL8 when evaluated using immunoblotting. This raises a question as to whether the N-terminal fragment confers some additional https://doi.org/10.1038/s41598-023-46036-9 Scientific Reports \| (2023) 13:19052 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ 29. Luettgen, J. M. et al. Apixaban inhibition of factor Xa: Microscopic rate constants and inhibition mechanism in purified protein systems and in human plasma. J. Enzyme Inhib. Med. Chem. 26(4), 514–526 (2011). 29. Luettgen, J. M. et al. Apixaban inhibition of factor Xa: Microscopic rate constants and inhibition mechanism in purified protein systems and in human plasma. J. Enzyme Inhib. Med. Chem. 26(4), 514–526 (2011). 29. Luettgen, J. M. et al. 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Infect. 72(4), 450–459 (2016). 32. Reglinski, M. et al. Development of a multicomponent vaccine for Streptococcus pyogenes based on the antigenic targets of IVIG. J. Infect. 72(4), 450–459 (2016). 32. Reglinski, M. et al. Development of a multicomponent vaccine for Streptococcus pyogenes based on the antigenic targets of IVIG. J. Infect. 72(4), 450–459 (2016). Acknowledgementsh g The authors are grateful for the support of a GSK Golden Triangle Discovery Partnership in Academia Collabora- tion (DPAC) that permitted development of the mass spectrometry high throughput cleavage assay and reagent provision. SS acknowledges the Imperial College London NIHR Biomedical Research Centre. Fundingh Funding This work was funded by a Biotechnology and Biological Sciences Research Council (BBSRC) NPIF iCASE studentship and a Wellcome Trust Collaborative award 215539: ‘Understanding and exploiting Group A strep- tococcal anti-chemotactic proteases in vaccines for infection’. 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Chem. 281(6), 3116–3126 (2006). https://doi.org/10.1038/s41598-023-46036-9 Scientific Reports \| (2023) 13:19052 \| Competing interests p g Co-authors Carl Haslam, Andrew Fosberry, Emma J Jones and Danuta Mossakowska were employed by Glaxo- SmithKline R&D at the time this work was conducted. All other authors declare no conflict of interest. © The Author(s) 2023 Additional informationh Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-023-46036-9. Correspondence and requests for materials should be addressed to J.E.P. or S.S. Correspondence and requests for materials should be addressed to J.E.P. or S.S. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. 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https://openalex.org/W4284880659	https://research.rug.nl/files/235154237/s12888_022_04098_5.pdf	English	null	“I lost so much more than my partner” – Bereaved partners’ grief experiences following suicide or physician-assisted dying in case of a mental disorder	BMC psychiatry	2,022	cc-by	10,609	University of Groningen “I lost so much more than my partner” – Bereaved partners’ grief experiences following suicide or physician-assisted dying in case of a mental disorder Snijdewind, M. C.; de Keijser, J.; Casteelen, G.; Boelen, P. A.; Smid, G. E. University of Groningen “I lost so much more than my partner” – Bereaved partners’ grief experiences following suicide or physician-assisted dying in case of a mental disorder Snijdewind, M. C.; de Keijser, J.; Casteelen, G.; Boelen, P. A.; Smid, G. E. Abstract Background: There is a lack of existing research on grief following the intentional death of people suffering from a mental disorder. Our study aims to provide insight into grief experiences and social reactions of bereaved persons who lost their life partners, who were suffering from a mental disorder, to physician-assisted dying (PAD) or suicide. Methods: For this mixed-methods research, we conducted a survey and in-depth interviews with 27 persons living in the Netherlands and bereaved by the death of their life partners. The deceased life partners suffered from a mental disorder and had died by physician-assisted dying (n = 12) or suicide (n = 15). Interviews explored grief experiences and social reactions. In the survey we compared self-reported grief reactions of partners bereaved by suicide and PAD using the Grief Experience Questionnaire. Results: Compared to suicide, physician-assisted dying was associated with less severe grief experiences of the bereaved partners. Participants reported that others rarely understood the suffering of their deceased partners and sometimes expected them to justify their partners’ death. Following physician-assisted dying, the fact that the partner’s euthanasia request was granted, helped others understand that the deceased person’s mental suffering had been unbearable and irremediable. Whereas, following suicide, the involvement of the bereaved partners was some- times the focus of judicial inquiry, especially, if the partner had been present during the death. Conclusion: When individuals suffering from a mental disorder die by suicide or PAD, their bereaved partners may experience a lack of understanding from others. Although both ways of dying are considered unnatural, their impli- cations for bereaved partners vary considerably. We propose looking beyond the dichotomy of PAD versus suicide when studying grief following the intentional death of people suffering from a mental disorder, and considering other important aspects, such as expectedness of the death, suffering during it, and partners’ presence during the death. Keywords: Grief, Suicide, Physician-assisted dying, Mental health, Death taboo © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. “I lost so much more than my partner” – Bereaved partners’ grief experiences following suicide or physician‑assisted dying in case of a mental disorder M. C. Snijdewind1,2, J. de Keijser3, G. Casteelen4, P. A. Boelen1,5,6 and G. E. Smid1,5,7* Correspondence: g.smid@arq.org 7 Department of Humanistic Chaplaincy Studies, University of Humanistic Studies, Kromme Nieuwegracht 29, 3512, HD, Utrecht, the Netherlands Full list of author information is available at the end of the article University of Groningen “I lost so much more than my partner” – Bereaved partners’ grief experiences following suicide or physician-assisted dying in case of a mental disorder Snijdewind, M. C.; de Keijser, J.; Casteelen, G.; Boelen, P. A.; Smid, G. E. University of Groningen “I lost so much more than my partner” – Bereaved partners’ grief experiences following suicide or physician-assisted dying in case of a mental disorder Snijdewind, M. C.; de Keijser, J.; Casteelen, G.; Boelen, P. A.; Smid, G. E. “I lost so much more than my partner” – Bereaved partners’ grief experiences following suicide or physician-assisted dying in case of a mental disorder Snijdewind, M. C.; de Keijser, J.; Casteelen, G.; Boelen, P. A.; Smid, G. E. Snijdewind, M. C.; de Keijser, J.; Casteelen, G.; Boelen, P. A.; Smid, G. E. DOI: 10.1186/s12888-022-04098-5 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2022 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Snijdewind, M. C., de Keijser, J., Casteelen, G., Boelen, P. A., & Smid, G. E. (2022). “I lost so much more than my partner” – Bereaved partners’ grief experiences following suicide or physician-assisted dying in case of a mental disorder. BMC Psychiatry, 22(1), Article 454. https://doi.org/10.1186/s12888-022-04098-5 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Snijdewind et al. BMC Psychiatry (2022) 22:454 https://doi.org/10.1186/s12888-022-04098-5 Background Grief, following the loss of a loved one, is influenced by multiple factors, including the circumstances of the death and the bereaved person’s involvement in it [1]. Death from unnatural causes, e.g., suicide, is associated with more severe and prolonged grief compared to that from natural causes [2]. Previous research including people © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Snijdewind et al. BMC Psychiatry (2022) 22:454 Page 2 of 13 of isolation and fear of social stigma [16–19]. However, if they knew that the passing was an autonomous choice, they could accept the death and showed less symptoms of grief and posttraumatic stress disorder [16–19]. How- ever, research on the impact of PAD on the mental health of bereaved relatives is scarce. Moreover, to the best of our knowledge, no former study focused specifically on grief following PAD in the case of a mental disorder. Therefore, we aimed to provide insight into the experi- ence of losing a partner to PAD or suicide in case of a mental disorder. For this, we used mixed methods to con- duct an exploratory qualitative semi-structured in-depth interview study combined with a comparative quantita- tive survey. We hypothesized that the experience of grief would differ between partners of people dying from sui- cide and PAD and that intensity of grief would be higher in case of suicide. bereaved by suicide indicated that feelings of responsi- bility, guilt, and rejection, and experiencing stigma are common [2–4]. Grief is more severe in people bereaved by suicide than those by other forms of sudden death [4]. by suicide than those by other forms of sudden death [4]. Worldwide, around 1.3% of all deaths are caused by sui- cide [5]. Methodsh This research was performed in accordance with relevant guidelines and regulations. We consulted the Medical Ethics Research Committee Utrecht (protocol number 19/596), who exempted the study from formal review because the Medical Research Involving Human Subjects Act (WMO) does not apply to the study. Each participant provided written informed consent before enrolling in the study. In the Netherlands, 1.1% of deaths were caused by suicide in 2020 [6] and in 56.8% of such deaths, sui- cide was motivated by a mental disorder [7]. Worldwide, mental disorders account for a majority of suicides. A meta-analysis regarding psychological autopsies of peo- ple who committed suicide showed that 87.3% of these people had mental disorders [8]. However, a review of psychological autopsy studies found that between 5.5% and 66.7% of suicides occurred in the apparent absence of a mental health condition [9]. In line with the Dutch statistics, the 2018 US Surveillance for Violent Deaths [10] reported that circumstances were identified in 88.3% of suicides, and among them mental health problem was the most common circumstance, with 49.7% of decedents having had a diagnosed mental health problem. Unlike other forms of unnatural death – for example, accident, natural disaster, terrorism – suicide is to some extent intentional and takes perseverance to complete. Death of a loved one by suicide may be anticipated to some degree, based on previous suicide attempts or conversations, as illustrated by studies on people receiving de-medical- ized assistance in suicide [11], e.g., people passing away through self-ingesting self-collected lethal medication. Previous research indicates that when the bereaved antic- ipate the suicide and understand the reasons behind it, they search less for explanations and have less preoccu- pation with it [12]. Suicide may be further divided into violent, i.e., mutilating (e.g., strangulation or high impact collision) or non-violent, i.e., non-mutilating (e.g., physi- cian-assisted dying (PAD), drug overdose, ingestion of a deadly substance, helium inhalation). Violent deaths are more likely to generate distressing intrusive memories in the bereaved than nonviolent deaths [13]. Recruitment and selection Th l f a letter was sent informing them about the research, including a response card, which could be sent to the researchers to indicate willingness to participate. One reminder was sent. Twenty-one potential participants were informed and ten positive responses were received; all met the inclusion criteria. Next, we used social media to recruit participants. An invitation was placed on the website of ARQ Centrum’45 and the University for Humanistic Studies, the Netherlands. In addition, 113 Zelfmoordpreventie (113 Suicide Prevention) tweeted about the research. Moreover, during a symposium for people bereaved through suicide, held on December 11th 2020, the attendees were informed about the research and the possibility to participate. A newsletter of the Vrienden van Expertisecentrum Euthanasie contained information about the research and a call to participate. Through all these, 24 people responded, of which ten were eligible. We also used snowball sampling to recruit participants, by sending out flyers about the research in our professional network and actively asking people if they knew someone who would be eligible to participate. Thus, nine people responded, of which seven met the inclusion criteria. a letter was sent informing them about the research, including a response card, which could be sent to the researchers to indicate willingness to participate. One reminder was sent. Twenty-one potential participants were informed and ten positive responses were received; all met the inclusion criteria. Next, we used social media to recruit participants. An invitation was placed on the website of ARQ Centrum’45 and the University for Humanistic Studies, the Netherlands. In addition, 113 Zelfmoordpreventie (113 Suicide Prevention) tweeted about the research. Moreover, during a symposium for people bereaved through suicide, held on December 11th 2020, the attendees were informed about the research and the possibility to participate. A newsletter of the Vrienden van Expertisecentrum Euthanasie contained information about the research and a call to participate. Through all these, 24 people responded, of which ten were eligible. We also used snowball sampling to recruit participants, by sending out flyers about the research in our professional network and actively asking people if they knew someone who would be eligible to participate. Thus, nine people responded, of which seven met the inclusion criteria. The interviews were conducted by a researcher with extensive experience in this. Since the interviews were semi-structured, a topic list was used (Table 1). Statistical Analyses A l f Analyses were performed using SPSS version 27 for Win- dows. Missing scale item responses were present in a mean of 0.5% of responses per case (range, 0 to 3.6%) and were handled using mean imputation. Prior to the final analyses, we checked normality by verifying that none of the variables had skewness or kurtosis values smaller than -3 or larger than 3. Descriptive analyses of the sam- ple included frequencies and percentages for binary and categorical variables, respectively, and means and stand- ard deviations for continuous variables. Chi-square tests and independent t-tests were conducted to examine sociodemographic and mental health related differences between the participants bereaved by PAD and suicide of their partners. Fisher’s exact tests were performed in addition to chi-square tests if expected counts were < 5 (since the significance levels did not differ, they were not reported separately). For multiple regression analyses, binary variables were dummy coded as follows: yes=1, no=0. Regression analyses evaluated the impact of the mode of partner’s death and time since death on men- tal health related variables, with and without adjustment for the effects of other loss-related variables, specifically, being present during the partner’s death and violent mode of partner’s suicide. For this, independent variables were entered into the regression in two steps, and the sig- nificance of the R2 change was calculated for the second step. Visual inspection of residual plots confirmed homo- scedasticity. There was no evidence of multicollinearity, as indicated by tolerance values > 0.25. Alpha level was set at .05 for statistical significance. Study designh The study combined a quantitative survey with a quali- tative interview. After receiving contact information of people willing to participate, a researcher called or emailed to check if they met the inclusion criteria and if so, appointments were made for the interviews. Two weeks prior to the interview, the respondent received the survey questionnaire by mail. The survey contained back- ground and loss-related questions, the Grief Experiences Questionnaire, and a list of potential stressful life events, experienced after the loss of the partner. p p The Grief Experience Questionnaire (GEQ) [21] was used to measure grief reactions. The GEQ is designed to measure two types of grief: 1) expected in any bereave- ment, 2) specific to suicide. The original 55-item GEQ [21] has been psychometrically evaluated in several stud- ies [12, 22–24]. Kõlves et al. [23] performed confirmatory factor analysis [23] to evaluate the original GEQ 11 sub- scale structure proposed by Barrett and Scott [21] and found that eight original subscales had good reliability. We used these eight validated subscales assessing somatic reactions, search for explanation, loss of support, stigma- tization, guilt, responsibility, shame, and rejection, each consisting of five items [23]. Items were rated on 5-point Likert scales (1 = never to 5 = almost always), with total scores ranging between 5 and 25. We used the translation by Wojtkowiak et al. [12]. Earlier studies established sat- isfactory internal [12, 22] and test-retest reliability [24]. Recruitment and selection Th l f The inclusion criteria for the participants were: (1) loss of a life partner to PAD or suicide in case of a mental dis- order for which the decedent received treatment, or had been on a waiting list, for at least two years; (2) time since the loss was between six months and 10 years. The required number of study participants was guided by the expected number of interviews needed to reach qualitative data saturation. Given the heterogeneity in our study population, we anticipated a larger sample would be needed to achieve this [20]. Since we also wanted to conduct quantitative comparative analyses, we aimed to include at least ten participants each in bereaved by PAD and ten bereaved by suicide of their partners. Partici- pants were recruited through Expertisecentrum Eutha- nasie (EE, Euthanasia Expertise Centre, the Netherlands). While PAD related to a mental disorder is still rare, most of these cases are performed by physicians working for EE – in 2021 a total of 115 cases of PAD based on mental disorders were reported, of which 83 by physicians work- ing for EE [15].h Suicide, however, is not the only cause of death which depends on an intentional act by the person who dies; this is also the case in PAD. In this case, the death of the patient can be anticipated, as the date of death is set in advance. PAD is legally regulated in several countries worldwide [14]. In some of these countries, including the Netherlands, legal criteria of PAD include the medi- cal condition of mental disorder of the patient. Grant- ing such a request is still rare – while in 2021 a total of 7666 people died by PAD in the Netherlands (4.5% of total deaths), only 115 of these (1.5% of all reported PAD- cases) concerned PAD in psychiatric patients (0.07% of total deaths) [15]. The EE provided information regarding all cases of PAD in patients with a mental disorder, performed by EE-physicians where a life partner had been involved. If contact information of these life partners was available, Research on the impact of PAD on the mental health of bereaved relatives showed that they experienced feeling Snijdewind et al. BMC Psychiatry (2022) 22:454 Snijdewind et al. BMC Psychiatry (2022) 22:454 Snijdewind et al. BMC Psychiatry (2022) 22:454 Page 3 of 13 Internal reliability (Cronbach’s alpha) in the current sam- ple was 0.96. Recruitment and selection Th l f In the interviews, participants described the period of time leading up to the death of their partner and the time afterwards, up to their current situation. Most interviews were conducted face-to-face at participants’ homes. However due to COVID-19-related restrictions, three interviews were conducted through video calls. All par- ticipants were informed about the purpose of the study. They provided either written or oral informed consent. Interviews lasted between 46 and 231 minutes and were recorded. All interviews were held between February 2020 and March 2021. Interview Analyses Information about the research project Recording and confidentiality Questions Informed consent Reason(s) to participate Name of the partner General information about partner First experience with partner’s mental disorder(s) and impact Good day/period, bad day/period Awareness/reaction social environment (Professional) care related to the mental disorder(s) Possible death: awareness/topic of discussion/ suicidal thoughts or tendencies Conversations about ending one’s life with care provider Way of dying, meaning for bereaved partner Farewell Period following death Reactions social environment Openness and sharing, support, loneliness Interview experience Memories Emotions Influence loss on current life Difficulties Support and comfort Openness social environment View on death Outlook on future Recap Other important experiences related to the loss? Current situation Ending Ending researchers and these codes were extensively compared and discussed until an agreement was reached. Based on this, a coding scheme was drafted that was further developed through coding of the next seven interviews. Thereafter, codes were frequently grouped and regrouped into overarching themes. Codes were added when new information emerged during analyses. Data analysis was frequently discussed between the authors and agreement was reached by discussing the interpretation of specific quotations in the context of the entire interview. Inter- subjectivity was assured by coding and discussing three interviews with two other researchers and discussing excerpts, codes, and interpretations of other interviews with another researcher. during the death. Being present during the partner’s sui- cide only occurred in non-violent suicides. Table 2 lists the characteristics of the participants and the mode of death of their partners as well as the GEQ-scores. As shown in Table 2, participants whose partners died by suicide were significantly higher educated and more often female compared to those whose partners died by PAD. In addition, they – and their partners – were signif- icantly younger, and the duration of the relationship was shorter. At the moment of study, more time had passed for the participants whose partners died by suicide. Stressful life experiences following the partner’s death did not differ significantly between the two groups except the experience of breakup in a relationship, which occurred more often in the suicide- than the PAD-bereaved group. Mean GEQ scores were lower for the PAD-bereaved group on all subscales. Between-group differences in mean GEQ subscale scores were all significant except for the guilt subscale. Sample characteristics Twenty-seven bereaved participants were included in this study, of whom twelve had experienced the death of their partner by PAD and fifteen by suicide. Using data from the interviews, the cause of death was further divided into violent, i.e., mutilating (e.g., strangulation or high impact collision) or non-violent, i.e., non-mutilating (e.g., PAD, drug overdose, ingestion of a deadly substance, helium inhalation). Fifteen participants were present Interview Analyses All recorded interviews were transcribed. By inductive coding, we conducted a thematic analysis using ATLAS. ti. The first three interviews were open coded by three Page 4 of 13 Snijdewind et al. BMC Psychiatry (2022) 22:454 Snijdewind et al. BMC Psychiatry (2022) 22:454 Snijdewind et al. BMC Psychiatry (2022) 22:454 Table 1 Topic list (translated, original in Dutch) Table 1 Topic list (translated, original in Dutch) Interview introduction Information about the research project Recording and confidentiality Questions Informed consent Introductory questions Reason(s) to participate Name of the partner General information about partner Mental disorder of the deceased partner First experience with partner’s mental disorder(s) and impact Good day/period, bad day/period Awareness/reaction social environment Care providers (Professional) care related to the mental disorder(s) Potential/possible death of the partner Possible death: awareness/topic of discussion/ suicidal thoughts or tendencies Conversations about ending one’s life with care provider Passing of the partner and life afterwards Way of dying, meaning for bereaved partner Farewell Period following death Reactions social environment Openness and sharing, support, loneliness Current situation Interview experience Memories Emotions Influence loss on current life Difficulties Support and comfort Openness social environment View on death Outlook on future Ending Recap Other important experiences related to the loss? Table 1 Topic list (translated, original in Dutch) Interview introduction Information about the research project Recording and confidentiality Questions Informed consent Introductory questions Reason(s) to participate Name of the partner General information about partner Mental disorder of the deceased partner First experience with partner’s mental disorder(s) and im Good day/period, bad day/period Awareness/reaction social environment Care providers (Professional) care related to the mental disorder(s) Potential/possible death of the partner Possible death: awareness/topic of discussion/ suicidal thoughts or tendencies Conversations about ending one’s life with care provider Passing of the partner and life afterwards Way of dying, meaning for bereaved partner Farewell Period following death Reactions social environment Openness and sharing, support, loneliness Current situation Interview experience Memories Emotions Influence loss on current life Difficulties Support and comfort Openness social environment View on death Outlook on future Ending Recap Other important experiences related to the loss? Unnatural death Both suicide and PAD are considered non-natural causes of death. Because of this, the body of the deceased needs to be released following inspection by the medical Snijdewind et al. BMC Psychiatry (2022) 22:454 Page 5 of 13 Table 2 Sociodemographic variables, stressful life events, and grief experiences by mode of partner’s death 1 vs. Unnatural death lower education 2 vs no children Full sample Cause of partner’s death (N = 27) Suicide (N = 15) PAD (N = 12) N % N % N % χ2 (df = 1) Gender Female 13 48.1 10 66.7 3 25.0 4.64 Male 14 51.9 5 33.3 9 75.0 Higher education1 17 63.0 14 93.3 3 25.0 13.35 * Children2 20 74.1 12 80.0 8 66.7 0.62 Filed a complaint3 2 7.4 2 13.3 0 0.0 1.73 Present during the partner’s death4 15 55.6 3 20.0 12 100.0 17.28 * Violent partner’s death5 8 29.6 8 53.3 0 0.0 9.10 Stressful life events after partner’s death Experienced illness, injury 1 3.7 1 6.7 0 0.0 0.83 Family member ill, injured 6 22.2 5 33.3 1 8.3 2.41 Parent, child or sibling died 4 14.8 4 26.7 0 0.0 3.76 Friend or other family member died 2 7.4 0 0.0 2 16.7 2.70 Breakup of relationship 8 29.6 8 53.3 0 0.0 9.09 Relationship problem 8 29.6 6 40.0 2 16.7 1.74 Job loss or inability to find a job 2 7.4 1 6.7 1 8.3 0.03 Fired 0 0.0 0 0.0 0 0.0 - Financial problem 1 3.7 1 6.7 0 0.0 0.83 Contact with the law 1 3.7 1 6.7 0 0.0 0.83 Theft or loss of valuables 2 7.4 1 6.7 1 8.3 0.03 M SD M SD M SD t (df = 25) Age 61.02 13.11 54.27 11.45 69.46 9.99 -3.62 ** Years since death 2.63 2.52 3.52 2.81 1.51 1.59 2.21 * Age partner 58.15 14.61 49.83 11.03 68.56 11.71 -4.27 * Duration of relationship 30.00 18.22 21.20 14.81 42.04 15.58 -3.55 Number of stressful life events 1.30 1.20 1.87 1.25 0.58 0.67 3.21 * Grief Experience Questionnaire Somatic reactions 9.04 3.95 10.67 3.77 7.00 3.25 2.67 * Search for explanation 11.35 3.93 12.85 3.27 9.48 4.01 2.41 * Loss of support 10.17 4.38 12.43 4.07 7.33 2.93 3.65 Stigmatization 9.63 4.82 11.93 5.20 6.75 2.05 3.25 Guilt 9.87 4.07 11.20 4.44 8.21 2.93 2.00 Responsibility 7.72 3.53 9.20 4.05 5.88 1.38 2.71 * Shame 8.04 3.55 9.40 3.79 6.33 2.39 2.44 * Rejection 9.05 4.32 10.68 4.59 7.00 3.02 2.39 * 2 Sociodemographic variables, stressful life events, and grief experiences by mode of partner’s death examiner. Respondents bereaved by PAD mentioned that this was just a formality. It was all taken care of by the physician of the eutha- nasia association [Expertisecentrum Euthanasie]. A medical examiner had to come, had to be called, or the public prosecutor or something like that. That was all taken care of, I was not involved in that. I Unnatural death But then again, if the medical examiner had come and he would’ve had a suspicion and those bottles were still in the kitchen... Should I have thrown them out? No, that only raises suspicion. You shouldn’t do that. (r.18, non-violent suicide, present during the death) Anyone may know. If I talk about his death – when I’m somewhere new, or have new colleagues – I tell them my husband died not so long ago, he had so many complaints, it was no longer doable, he received euthanasia. That I’ll tell them right after. Interviewer (I): And people’s reaction … ? Then it must have been bad. I mean, you don’t receive euthanasia just like that, do you? (r17, PAD, present during the death) Anyone may know. If I talk about his death – when I’m somewhere new, or have new colleagues – I tell them my husband died not so long ago, he had so many complaints, it was no longer doable, he received euthanasia. That I’ll tell them right after. Interviewer (I): And people’s reaction … ? Then it must have been bad. I mean, you don’t receive euthanasia just like that, do you? (r17, PAD, present during the death) Following PAD, contact with the officials was organ- ized by the physician, who was also the liable person. In contrast, following suicide, the respondent was often the one who needed to initiate contact with the officials. How and when this should take place was not always self-evident. The respondents faced questions, such as when should they report their partner missing and how long should they wait to inform officials to be certain that their partner actually died. Though sometimes they did not recognize these moments of choice until later. Respondents often received supportive reactions con- cerning the loss of their partner. The reactions concerning the fact their partners had intentionally ended their lives varied from wondering how this could have happened and feeling that it should have been prevented, to recogniz- ing the tragic situation for which there had been no alter- native solution. Respondents felt they were sometimes blamed for the suicide of their partner, while respondents whose partner died by PAD did not feel this way. Being blamed left respondents feeling vulnerable, isolated and angry, and resulted in loss of friends and family. Unnatural death Often, the medical examiner was informed by the physician about the upcoming PAD beforehand and the examination did not take much time. Snijdewind et al. BMC Psychiatry (2022) 22:454 Page 6 of 13 Page 6 of 13 don’t know if there was a medical examiner, or that she called the public prosecutor. (r.19, PAD, present during the death) don’t know if there was a medical examiner, or that she called the public prosecutor. (r.19, PAD, present during the death) disorder and its impact. They explained this by saying that their partner preferred other people not to know, since they wanted to appear as normal and healthy as possible and not to be treated differently, or because they were ashamed of the condition. Others feared an unsup- portive response from outsiders, based on past experi- ences. The common experience was that other people, even loved ones, did not notice the severity of the dis- order and its impact. This might explain why the social environment often did not understand the intention of the deceased to end his or her life. It resulted in openly questioning this intention and sometimes trying to con- vince to not follow through, or blaming the respondent afterwards for not stopping the partner. Respondents had to face negative emotions and opinions of others instead of feeling supported by them. While these reac- tions were not limited to suicide, the granting of a PAD request sometimes made it easier for the respondents to explain the severity of the disorder and the suffering of their partner to others; it seemed to validate the intention to end one’s life. Respondents bereaved by suicide mentioned that besides a medical examiner, police officers were present to investigate the circumstances of the death. Several respondents were questioned about their involvement in the suicide and were not allowed near their part- ner’s body during investigation. This weighed heavily on them. An extensive examination involving the bereaved respondents seemed to occur more often in non-violent suicides where the partner had been present. However, one of our interviewees mentioned that sometimes a non-violent suicide could almost pass for a natural death – in which no medical examination or police investiga- tion would take place. She [the physician] was about to declare a natural death. Had I only put the note [of the partner, stat- ing she ended her life herself] somewhere else. Unnatural death We had the deal that if something happened, we wouldn’t try to save each other. We didn’t want to end up in a persistent vegetative state. Afterwards I felt regret, I went to the neighbors too soon. Should I not have waited longer? ( … ) I begged them, twice, three times. First to the officer on duty, to the police, to the officers on duty, each time there was a higher ranking... if they would please stop the resuscitation, but they just kept going. They just kept going. (r21, violent suicide, not present during the death) And then [their reaction] ‘oh, suicide?’. It is still taboo. ‘Was he happy?’ I think [partner] was very happy here, but at certain moment … You can’t explain it. Then I’ll leave it. Never mind, because people are so short sighted. ‘Was there no help? Hasn’t there been this [or that]?’ Yeah, as if we hadn’t tried everything ourselves. ( … ) It’s not only suicide, that is a taboo, but also just dealing with grief. (r11, non-violent suicide, not present during the death) Social environment’s reactions to the partner’s mental disorderh The respondents mentioned that their social environ- ment – friends, family, co-workers, or acquaintances – was often not fully aware of the decedent’s mental Page 7 of 13 Snijdewind et al. BMC Psychiatry (2022) 22:454 Snijdewind et al. BMC Psychiatry (2022) 22:454 They [the partner’s parents] blame me for every- thing, they blame me for the death of [partner] (r21, violent suicide, not present during the death) think of anything that I find difficult or have prob- lems with. No, I knew what the situation was and that it would never change and that I would have to continue like this. (r13, PAD, present during the death) His brother turned me in as a suspect. ( … ) His brother wanted my role in this story to be re-exam- ined. So, everything had to be handed in [to the police]: phones, laptops, whatever. What a suspicion, on top of everything that’s already happened. (r27, PAD, present during the death) Mode of death predicting grief experiences and stressful life events Table 3 shows the factors associated with GEQ scores, as well as the number of stressful life events the par- ticipants endured since their partners’ death. In the first step of the multiple regression analyses, dying by PAD was found to be associated with less somatic reactions, search for explanation, loss of support, stigmatization, guilt, responsibility, and lower number of stressful life events. In the second step, we added the predictors vio- lent death and being present during the death. Violent death was associated with increased somatic reactions, loss of support, stigmatization, guilt, and responsibility of the participants. PAD was associated with increased (i.e., decreased loss of) support. The number of stressful life events was associated with longer time since the death and violent death. Some respondents, from both groups, found it impor- tant to be open about the way their partner had died, motivated by a wish to contribute to a society where these topics were talked about more openly. Many respondents advocated that physicians and psy- chiatrists should be more willing to consider and grant a request for PAD in patients with mental disorders. This was often mentioned as one of the reasons to participate in the research. Questions and doubts Some respondents indicated that they began to doubt themselves after the death of their life partners. Self- doubt concerned their choice of partner, but also their own current feelings and thoughts. These questions and insecurities were mostly present in respondents who had not foreseen the death of their partner. Discussionh The aim of this study was to provide insight into grief experiences following the loss of a partner, suffering from mental disorder, to PAD or suicide. Our results show that people bereaved by suicide and PAD of their part- ners not only lose their loved one, but also experience a lack of understanding and support from others. Although both PAD and suicide are considered unnatural causes of death, their implications for bereaved partners vary considerably. Following PAD, all persons involved are supported by the physician, who initiates and handles all contacts with officials. In contrast, following suicide, the bereaved partner has to find out what to do if he or she was present during the time of death or has found the partner’s body. Furthermore, many people bereaved by suicide at some point had to make difficult on-the- spot decisions, regarding their partner, to which they felt ill-prepared. I thought, that’s not right, how can this be? How could this happen, that I’m feeling happy again? My partner died half a year ago. How can I feel happy again, that’s strange? I almost felt ashamed because of it. ( … ) I felt ashamed, I thought I’m doing some- thing weird, I’m weird, I’m the only one. I’m already the only one that this had happened to, and now I’m also the only one who feels this way. That makes you judge yourself. ( … ) When I’m passionate about something … then I just get on with it. And right now, I just can’t. And then I start doubting myself. Am I right? Am I on the right track? Then I’m overcome by doubt and with that my self-confidence [declines] ( … ) It’s not like it was before, I’m not so free any- more, that’s a part of grief. (r20, violent suicide, not present during the death) A good death? A d d h Respondents who were aware of the impending death, did not have self-doubt or unanswered questions, as they were convinced that their partners willingly chose to die and at the time, it was the only option for their partner to find relief. A good death may be seen from the perspective of its impact on grief, mental health, and wellbeing of the bereaved. People bereaved by suicide are at higher risk of developing mental health issues and suicidal behav- ior compared to those bereaved by other modes of death [25]. Notably, it has been shown that loss to violent death is strongly associated with difficulty in accepting the loss, and consequently, with other prolonged grief symptoms Well, you obviously prefer to continue [living] as you’re used to. However, you know that’s not on option anymore, it’s no longer there. And well, I don’t really have a hard time dealing with it. ( … ) I can’t Snijdewind et al. A good death? A d d h BMC Psychiatry (2022) 22:454 Page 8 of 13 Table 3 Multiple regressions predicting grief experiences and stressful life events based on mode of partner’s death and time since death Step 1 Step 2 B 95% CI Beta F R2 B 95% CI Beta F R2 ΔR2 ΔF Somatic reactions 3.65 * 0.23 4.18 * 0.43 0.20 3.84 * Years since death -0.19 -0.82 0.44 -0.12 -0.19 -0.77 0.38 -0.12 Physician-assisted dying1 -4.04 -7.19 -0.90 -0.52 * -2.83 -7.40 1.75 -0.36 Violent death2 5.29 1.18 9.40 0.62 * Present during the death3 2.00 -3.15 7.14 0.26 Search for explanation 4.05 * 0.25 2.50 0.31 0.06 0.97 Years since death -0.43 -1.05 0.19 -0.28 -0.42 -1.05 0.21 -0.27 Physician-assisted dying1 -4.24 -7.33 -1.15 -0.55 -2.59 -7.61 2.42 -0.33 Violent death2 2.34 -2.16 6.85 0.28 Present during the death3 -0.46 -6.10 5.18 -0.06 Loss of support 6.42 0.35 6.21 ** 0.53 0.18 4.27 * Years since death -0.07 -0.72 0.58 -0.04 -0.10 -0.68 0.48 -0.06 Physician-assisted dying1 -5.24 -8.45 -2.02 -0.60 ** -5.58 -10.20 -0.97 -0.65 * Violent death2 5.83 1.69 9.98 0.62 ** Present during the death3 4.25 -0.95 9.44 0.49 Stigmatization 5.14 * 0.30 4.81 ** 0.47 0.17 3.44 * Years since death 0.12 -0.61 0.86 0.06 0.11 -0.57 0.79 0.06 Physician-assisted dying1 -4.94 -8.61 -1.27 -0.52 * -4.31 -9.72 1.11 -0.45 Violent death2 6.10 1.24 10.96 0.59 * Present during the death3 3.24 -2.85 9.33 0.34 Guilt 2.02 0.14 5.71 0.51 0.37 8.19 Years since death -0.13 -0.82 0.56 -0.08 -0.08 -0.63 0.47 -0.05 Physician-assisted dying1 -3.26 -6.68 0.17 -0.41 1.67 -2.72 6.05 0.21 Violent death2 5.14 1.20 9.08 0.59 * Present during the death3 -2.60 -7.53 2.33 -0.32 Responsibility 3.53 * 0.23 5.68 0.51 0.28 6.28 Years since death 0.02 -0.55 0.58 0.01 0.05 -0.43 0.53 0.04 Physician-assisted dying1 -3.29 -6.12 -0.47 -0.47 * 0.34 -3.47 4.16 0.05 Violent death2 4.05 0.63 7.47 0.53 * Present during the death3 -1.75 -6.04 2.53 -0.25 Shame 3.16 0.21 2.07 0.27 0.07 0.99 Years since death -0.20 -0.78 0.38 -0.14 -0.22 -0.80 0.37 -0.15 Physician-assisted dying1 -3.47 -6.34 -0.60 -0.49 * -3.89 -8.54 0.76 -0.56 Violent death2 2.80 -1.38 6.97 0.37 Page 9 of 13 Snijdewind et al. A good death? A d d h BMC Psychiatry (2022) 22:454 Table 3 (continued) Step 1 Step 2 B 95% CI Beta F R2 B 95% CI Beta F R2 ΔR2 ΔF Present during the death3 2.35 -2.88 7.58 0.34 Rejection 2.90 0.19 1.69 0.24 0.04 0.58 Years since death -0.17 -0.88 0.54 -0.10 -0.18 -0.91 0.55 -0.11 Physician-assisted dying1 -4.03 -7.56 -0.51 -0.47 * -3.80 -9.61 2.01 -0.45 Violent death2 2.70 -2.52 7.92 0.29 Present during the death3 1.49 -5.05 8.02 0.17 Number of stressful life events 7.68 0.39 6.10 0.53 0.14 3.14 Years since death 0.16 -0.01 0.34 0.34 0.16 0.00 0.32 0.34 * Physician-assisted dying1 -0.95 -1.81 -0.10 -0.40 * -0.67 -1.94 0.61 -0.28 Violent death2 1.34 0.20 2.48 0.52 * Present during the death3 0.53 -0.90 1.96 0.22 1 i id Snijdewind et al. BMC Psychiatry (2022) 22:454 Page 10 of 13 Fig. 1 A heuristic, hypothetical model of death-related factors influencing bereaved partners’ grief experiences following suicide and PAD related to a mental disorder (e.g., a continued sense of shock, bitterness, emptiness, and yearning), symptoms of posttraumatic stress disor- der, and depression [26]. Given the predictive effects of prolonged grief on reduced mental health over time [27], it is likely that mental health issues in people bereaved through suicide are in part grief related. The analysis of GEQ showed that PAD had a protective effect on the severity of the grief experiences of bereaved partners compared to suicide. It is possible that the protective effect of PAD may be relevant to mental health issues and suicidal behavior of people bereaved by PAD. Future research is needed to further investigate this association. following PAD, the bereaved partner can counter these reactions by the fact that a physician came to the con- clusion that the suffering of the patient was unbearable and without a prospect of improvement. This can not only convince outsiders, but also reinforce the bereaved partner’s own interpretation of the past situation. Feeling isolated and experiencing a lack of understanding is also reflected in the ‘loss of support’ and ‘stigmatization’ sub- scales of the GEQ. A previous interview study on stigma suggested that death taboo still exists in Western society, more specifically concerning sudden deaths [28]. The authors reasoned that this might be related to the “shock- ing or unusual nature; causing others significant unease” [28]. A good death? A d d h Our study findings suggest that stigmatization may be less prevalent after PAD, despite the unnatural cause of the death. Thus, death taboo might be less related to the unnatural cause of death, but more to the violent and unexpected manner of it. g Addition of other independent variables showed that it might be worthwhile to look beyond the PAD-suicide dichotomy. A partner’s violent death impacted the grief experiences of the bereaved in addition to the suicide. The interviews offered an even more nuanced view into grief than did the GEQ results. In addition to the cause of death (PAD or suicide), bereaved partners’ grief experi- ences were influenced by the death being violent or non- violent, irrespective of whether they were present during the death, and anticipated it. A previous study into grief experiences of people bereaved by suicide showed that the more the loved one’s suicide was expected, the less the bereaved seek explanations and meaning after the death [12]. Further similar studies might reveal a pattern in which grief experiences in the context of suicide and PAD might be placed on a scale from the experience of a good death without severe grief, to one of a horrible death followed by severe grief. PAD would be on the one end of this scale, and an unexpected and violent suicide on the other. In between we will find a planned non-violent suicide with the partner present, a planned non-violent suicide without the partner present, and an unexpected non-violent suicide without the partner present. Figure 1 illustrates this hypothesis. Overlapping this spectrum are reactions from outsiders who show little understand- ing of the severity of the mental disorder. Unlike suicide, We have seen that bereaved persons sometimes strug- gle with questions and doubts following the loss of their significant others. Although the subscale ‘search for explanation’ did not show significant outcomes, the inter- views showed that some people were confronted with questions – most often when the suicide was unexpected. The subscales ‘guilt’ and ‘responsibility’ also seem to be associated with these inner questions and doubts. Being confronted with blame and having the feeling of not being understood by the social environment seemed to stir up inner doubts even more. Our findings show that feelings of self-doubt following the partner’s death seem to be related to the death being unanticipated. A good death? A d d h When the death was expected, some of the questions (e.g., about why and how) had been resolved by talking to the partner – questions that bereaved partners following an unexpected death were still struggling with. Anticipating the death of the partner and having conver- sations about it are parts of a process known as antici- patory grief [29]. Conversations about the death seem to Snijdewind et al. BMC Psychiatry (2022) 22:454 Page 11 of 13 Page 11 of 13 (particularly expected in PAD), this could lead to under- representation of severe mental health problems. Since the actual experiences of the participants were not one- sided – they also mentioned difficulties experienced in the process of PAD and positive aspects of suicide – we expected that self-selection bias did not skew our results. strengthen the bond between partners. However, they also confront the healthy partner with the past, present, and future losses [29], as losses are not limited to the death of the partner but may include altered relationships or other social and economic changes. In this context it is interesting to note that a program focused on fam- ily connections in people with suicidal behavior disorder [30] may contribute to reducing grief associated with the mental illness of a loved one, and it would be worthwhile to investigate the effects of the family connection pro- gram on grief if the loved one eventually dies by suicide. p It is noteworthy that our study took place in the Neth- erlands, and the results may not be generalizable to other jurisdictions. We found gender differences between participants bereaved by suicide and PAD. It is known that more women die by PAD and more men by suicide [31]. Given the predominance of heterosexual relationships, gen- der distribution in our sample reflects expected general population patterns, with participants bereaved by sui- cide and PAD being most often women and men, respec- tively. We also found mean age differences between the two groups. Studies of granted euthanasia requests due to mental disorders found that most patients were under treatment for over ten years [32]. Thus, it is possible that people who died by suicide in our sample had a shorter mean duration of treatment compared with people who died by PAD. In addition, the time required to grant a request for PAD may be a contributing factor. A good death? A d d h Patients are likely to direct their initial request for PAD to their treating physician. If the physician refuses, the patient may turn to the EE. For psychiatric patients, the mean waiting time at the EE has been 10 months during the past years [32]. In addition, to meet the due care criteria of irremediable suffering, remaining treatment options sometimes need to be tried before considering PAD as an option. In our sample, participants bereaved by PAD were in the relationship with their partners longer than those bereaved by suicide. In addition to the process leading to PAD, that may support anticipatory grief, the longer duration of the relationship may have helped anticipate the death. It was evident from the study that bereaved partners lost so much more than their partner. Timely conversa- tions may help them prepare for these losses in advance. Concepts of preparedness and anticipatory grief might be helpful to open such conversation between care provid- ers. In case of planned suicides, specifically, in jurisdic- tions that do not allow PAD related to a mental disorder, care providers need to be aware of the challenges of partners having discussions with each other, and try to prevent the other partner from being seen as aiding and abetting the suicide. Study strengths and limitationshi This is the first study on grief experiences of life partners of people who died by PAD or suicide in case of a men- tal disorder. By including partners of people who died by mutilating or non-mutilating suicide, we obtained a nuanced view on grief of losing a loved one who inten- tionally ended his or her life due to a mental disorder. By combining a survey and an interview, we were able to show the broad variety of experiences and provide an indication of the severity of the grief.h Due to the COVID-19 pandemic, three interviews were conducted through video calls. The impact of COVID-19 was addressed during the interviews. Social restrictions related to COVID-19 may have impacted the experience of grief of the participants, such as recent feelings of iso- lation, or a decline in social contacts. Some respondents thought the measures, mainly social distancing, indeed contributed to these feelings. The study had the following limitations. First, for the quantitative analyses, our sample size was small. Although a small sample size is generally associated with a low power to detect statistically significant associations, we found several significant associations to confirm our hypotheses, suggesting that our sample size yielded enough statistical power. However, generalizability of our findings may be limited by the small sample size. In addi- tion, generalizability may be limited due to self-selection of participants in the study. It could bias the results if people were motivated to participate because of nega- tive experiences, thus overrepresenting the severity of mental health problems. Conversely, if people were moti- vated by a desire to advocate for a specific method to die Implications P di f Pending future studies with larger samples, our study provides initial evidence that PAD due to mental disor- der may be associated with an increased understanding of the extent of the suffering of the deceased person by the bereaved and their social environment. In addition, the findings provide a more nuanced view on suicide, which is often considered a traumatic, violent, and sud- den death. Our results show that it might do more jus- tice to the act and its experience to distinguish between ways of suicide, so that non-sudden and non-violent sui- cides are not overlooked. In practice, non-violent suicide Snijdewind et al. BMC Psychiatry (2022) 22:454 Page 12 of 13 Page 12 of 13 Page 12 of 13 of Clinical Psychology and Experimental Psychopathology, Faculty of Behav- ioral and Social Sciences, University of Groningen, Grote Kruisstraat 2/1, 9712, TS, Groningen, The Netherlands. 4 Expertisecentrum Euthanasie, P.O. Box 13480, 2501, EL, Den Haag, The Netherlands. 5 ARQ National Psychotrauma Centre, Nienoord 5, 1112 XE, Diemen, The Netherlands. 6 Department of Clinical Psychology, Faculty of Social Sciences, Utrecht University, P.O. Box 80140, 3508, TC, Utrecht, The Netherlands. 7 Department of Humanistic Chaplaincy Studies, University of Humanistic Studies, Kromme Nieuwegracht 29, 3512, HD, Utrecht, the Netherlands. of Clinical Psychology and Experimental Psychopathology, Faculty of Behav- ioral and Social Sciences, University of Groningen, Grote Kruisstraat 2/1, 9712, TS, Groningen, The Netherlands. 4 Expertisecentrum Euthanasie, P.O. Box 13480, 2501, EL, Den Haag, The Netherlands. 5 ARQ National Psychotrauma Centre, Nienoord 5, 1112 XE, Diemen, The Netherlands. 6 Department of Clinical Psychology, Faculty of Social Sciences, Utrecht University, P.O. Box 80140, 3508, TC, Utrecht, The Netherlands. 7 Department of Humanistic Chaplaincy Studies, University of Humanistic Studies, Kromme Nieuwegracht 29, 3512, HD, Utrecht, the Netherlands. death cases might be reported as a natural deaths, thus limiting the reliability of current registrations. Specifi- cally, planned suicides may be underreported. However, further research is needed on this. According to the guideline of the Dutch Association for Psychiatry (NVvP), the physician considering PAD in case of a mental disorder should involve significant others of the patients in the process towards PAD and provide care to them afterwards [33]. Our study results support this recommendation. Abbreviations EE E i EE: Expertisecentrum Euthanasie (Euthanasia Expertise Centre, The Nether- lands); GEQ: Grief Experience Questionnaire; PAD: Physician-assisted dying. g j j 3. Scocco P, Preti A, Totaro S, Corrigan P, Castriotta C. Stigma, grief and depressive symptoms in help-seeking people bereaved through suicide. J Affect Disord. 2019;244:223–30. https://doi.org/10.1016/j.jad.2018.10.098. Consent for publication Not applicable. Not applicable. Funding 8. Arsenault-Lapierre G, Kim C, Turecki G. Psychiatric diagnoses in 3275 suicides: a meta-analysis. BMC Psychiatry. 2004;4:37. https://doi.org/10. 1186/1471-244X-4-37. 8. Arsenault-Lapierre G, Kim C, Turecki G. Psychiatric diagnoses in 3275 suicides: a meta-analysis. BMC Psychiatry. 2004;4:37. https://doi.org/10. 1186/1471-244X-4-37. This work was supported by ARQ Centrum’45, Vrienden van Expertisecentrum Euthanasie, and “Stichting Stimuleringsfonds Rouw” – a foundation stimulat- ing attention for and knowledge development about grief. 9. Milner A, Sveticic J, De Leo D. Suicide in the absence of mental disorder? A review of psychological autopsy studies across countries. Int J Soc Psychiatry. 2013;59:545–54. https://doi.org/10.1177/0020764012444259. 9. Milner A, Sveticic J, De Leo D. Suicide in the absence of mental disorder? A review of psychological autopsy studies across countries. Int J Soc Psychiatry. 2013;59:545–54. https://doi.org/10.1177/0020764012444259. Competing interests 15. Regionale Toetsingscommissies Euthanasie. Jaarverslag 2021. Welzijn en Sport: Ministerie van Volksgezondheid; 2022. 15. Regionale Toetsingscommissies Euthanasie. Jaarverslag 2021. Welzijn en Sport: Ministerie van Volksgezondheid; 2022. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 16. Andriessen K, Krysinska K, Castelli Dransart DA, Dargis L, Mishara BL. Grief after euthanasia and physician-assisted suicide. Crisis. 2020;41:255–72. https://doi.org/10.1027/0227-5910/a000630. Implications P di f In addition, the results suggest that involvement of significant others should include attention for their perspective on a good death and their grief in order to further reduce the risk of prolonged grief and other negative grief experiences. Received: 15 March 2022 Accepted: 23 June 2022 Availability of data and materials The datasets generated and/or analysed during the current study are not publicly available, due to the ethically sensitive nature of the research and potential participant identifiers in the datasets. Selected data are available from the corresponding author on reasonable request. 10. Sheats KJ. Surveillance for Violent Deaths — National Violent Death Reporting System, 39 States, the District of Columbia, and Puerto Rico, 2018. MMWR Surveill Summ. 2022:71. https://doi.org/10.15585/mmwr. ss7103a1. 11. Hagens M, Pasman HRW, Onwuteaka-Philipsen BD. Cross-sectional research into people passing away through self-ingesting self-collected lethal medication after receiving demedicalized assistance in suicide. Omega J Death Dying. 2022;84:1100–21. https://doi.org/10.1177/00302 22820926771. Acknowledgements g The authors thank Laura Hendrick and Clasine Haringsma for their help in the data collection, coding and first analysis. The authors thank Roosje van der Graaf for her help in data collection. f p g j j 4. Kõlves K, de Leo D. Suicide bereavement: piloting a longitudinal study in Australia. BMJ Open. 2018;8:e019504. https://doi.org/10.1136/bmjop en-2017-019504. 5. 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We consulted the Medical Ethics Research Committee Utre- cht (protocol number 19/596), who exempted the study from formal review because the Medical Research Involving Human Subjects Act (WMO) does not apply to the study. Each participant provided written informed consent prior to enrolling in the study. 12. Wojtkowiak J, Wild V, Egger J. Grief Experiences and Expectance of Sui- cide. Suicide Life Threat Behav. 2012;42:56–66. https://doi.org/10.1111/j. 1943-278X.2011.00070.x. 13. Boelen PA, de Keijser J, Smid GE. Cognitive-behavioral variables mediate the impact of violent loss on post-loss psychopathology. PsycholTrauma. 2015;7:382–90. https://doi.org/10.1037/tra0000018. 14. Emanuel EJ, Onwuteaka-Philipsen BD, Urwin JW, Cohen J. Attitudes and practices of euthanasia and physician-assisted suicide in the united states, canada, and europe. JAMA. 2016;316:79–90. https://doi.org/10. 1001/jama.2016.8499. of Clinical Psychology and Experimental Psychopathology, Faculty of Behav- ioral and Social Sciences, University of Groningen, Grote Kruisstraat 2/1, 9712, TS, Groningen, The Netherlands. 4 Expertisecentrum Euthanasie, P.O. Box 13480, 2501, EL, Den Haag, The Netherlands. 5 ARQ National Psychotrauma Centre, Nienoord 5, 1112 XE, Diemen, The Netherlands. 6 Department of Clinical Psychology, Faculty of Social Sciences, Utrecht University, P.O. Box 80140, 3508, TC, Utrecht, The Netherlands. 7 Department of Humanistic Chaplaincy Studies, University of Humanistic Studies, Kromme Nieuwegracht 29, 3512, HD, Utrecht, the Netherlands. Author details 1 g 17. Ganzini L, Goy ER, Dobscha SK, Prigerson H. Mental health outcomes of family members of Oregonians who request physician aid in dying. 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Levensbeëindiging op verzoek bij patiënten met een psychische stoornis 2018. https://richtlijnendatabase.nl/richtlijn/levensbeeindiging_ op_verzoek_psychiatrie/startpagina_-_levensbe_indiging_op_verzoek. html (accessed 30 Sept 2018). • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? 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https://openalex.org/W2174792007	https://escholarship.org/content/qt2j86667p/qt2j86667p.pdf?t=phrpfu	English	null	Variable responses of human and non-human primate gut microbiomes to a Western diet	Microbiome	2,015	cc-by	8,651	UC San Diego UC San Diego Previously Published Works Title Variable responses of human and non-human primate gut microbiomes to a Western diet Permalink https://escholarship.org/uc/item/2j86667p Journal Microbiome, 3(1) ISSN 2049-2618 Authors Amato, Katherine R Yeoman, Carl J Cerda, Gabriela et al. Publication Date 2015-12-01 DOI 10.1186/s40168-015-0120-7 Peer reviewed UC San Diego UC San Diego Previously Published Works Title Variable responses of human and non-human primate gut microbiomes to a Western diet Permalink https://escholarship.org/uc/item/2j86667p Journal Microbiome, 3(1) ISSN 2049-2618 Authors Amato, Katherine R Yeoman, Carl J Cerda, Gabriela et al. Publication Date 2015-12-01 DOI 10.1186/s40168-015-0120-7 Peer reviewed UC San Diego UC San Diego Previously Published Title Variable responses of human and non-human prim Permalink https://escholarship.org/uc/item/2j86667p Journal Microbiome, 3(1) ISSN 2049-2618 Authors Amato, Katherine R Yeoman, Carl J Cerda, Gabriela et al. Publication Date 2015-12-01 DOI 10.1186/s40168-015-0120-7 Peer reviewed UC San Diego Title Variable responses of human and non-human primate gut microbiomes to a Western diet Permalink h h l hi i 2j8666 Journal Microbiome, 3(1) ISSN 2049-2618 Authors Amato, Katherine R Yeoman, Carl J Cerda, Gabriela et al. Publication Date 2015-12-01 DOI 10.1186/s40168-015-0120-7 Peer reviewed © 2015 Amato et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Variable responses of human and non-human primate gut microbiomes to a Western diet Katherine R. Amato1,2,3, Carl J. Yeoman4, Gabriela Cerda5, Christopher A. Schmitt6,16, Jennifer Danzy Cramer7, Margret E. Berg Miller8, Andres Gomez8,9, Trudy R. Turner10,11, Brenda A. Wilson8,12, Rebecca M. Stumpf5,8, Karen E. Nelson13, Bryan A. White8,14, Rob Knight15 and Steven R. Leigh2,8 Abstract Background: The human gut microbiota interacts closely with human diet and physiology. To better understand the mechanisms behind this relationship, gut microbiome research relies on complementing human studies with manipulations of animal models, including non-human primates. However, due to unique aspects of human diet and physiology, it is likely that host-gut microbe interactions operate differently in humans and non-human primates Results: Here, we show that the human microbiome reacts differently to a high-protein, high-fat Western diet than that of a model primate, the African green monkey, or vervet (Chlorocebus aethiops sabaeus). Specifically, humans exhibit increased relative abundance of Firmicutes and reduced relative abundance of Prevotella on a Western diet while vervets show the opposite pattern. Predictive metagenomics demonstrate an increased relative abundance of genes associated with carbohydrate metabolism in the microbiome of only humans consuming a Western diet. Conclusions: These results suggest that the human gut microbiota has unique properties that are a result of changes in human diet and physiology across evolution or that may have contributed to the evolution of human physiology. Therefore, the role of animal models for understanding the relationship between the human gut microbiota and host metabolism must be re-focused. Keywords: Gut microbiome, Vervet, Chlorocebus aethiops, Western diet, Human evolution Correspondence: katherine.amato@northwestern.edu 1Department of Anthropology, Northwestern University, Evanston, USA 2Department of Anthropology, University of Colorado Boulder, Boulder, USA Full list of author information is available at the end of the article Powered by the California Digital Library University of California Powered by the California Digital Library University of California eScholarship.org RESEARCH Open Access Variable responses of human and non-human primate gut microbiomes to a Western diet Katherine R. Amato1,2,3*, Carl J. Yeoman4, Gabriela Cerda5, Christopher A. Schmitt6,16, Jennifer Danzy Cramer7, Margret E. Berg Miller8, Andres Gomez8,9, Trudy R. Turner10,11, Brenda A. Wilson8,12, Rebecca M. Stumpf5,8, Karen E. Nelson13, Bryan A. White8,14, Rob Knight15 and Steven R. Leigh2,8 Amato et al. Microbiome (2015) 3:53 DOI 10.1186/s40168-015-0120-7 Amato et al. Microbiome (2015) 3:53 DOI 10.1186/s40168-015-0120-7 Background in many of these conditions [8–10], indicating a link be- tween the human gut microbiota, diet, and physiology. A common theme in human gut microbiome research currently is the effect of diet on gut microbiota compos- ition, and ultimately, host physiology [1]. In particular, a number of studies focus on the impact of a “Western” diet (high in animal fat and protein and low in fiber) compared to a “non-Western” diet (low in animal fat and protein and high in fiber; [2–6]). Western and non- Western human diets are consistently associated with distinct gut microbial communities [2–6], and humans consuming a high-protein, high-fat Western diet also have higher rates of obesity and other metabolic syndromes such as diabetes [7]. Gut microbes appear to be implicated As data describing patterns in the human gut micro- biota accumulate, researchers are beginning to turn to- ward animal models to test the mechanisms driving host-gut microbe interactions and their physiological consequences. While the use of rodent models is particu- larly popular due to ease of manipulation, non-human primate models are being integrated as well as a result of their close phylogenetic relationships with humans and presumably similar physiology [1, 11–13]. For example, a recent study examined the impact of a high-fat diet on the gut microbiota of adult female macaques and their off- spring [12]. However, human evolution appears to reflect a major dietary shift from a predominantly plant-based diet to an increasingly carnivorous diet, with relatively recent in- creases in food digestibility due to tool use, cooking, and Amato et al. Microbiome (2015) 3:53 Page 2 of 9 other processing techniques [14–16]. Additionally, com- pared to non-human primates, humans are characterized by a number of unique physiological adaptations, includ- ing increased brain size, reduced gut size, increased fat deposition, and decreased muscle mass [17–19]. Differ- ences also exist between human and non-human primate Toll-like receptors in the gut as well as expression of enzymes for the production of hormones such as prosta- glandin [20–22]. Because diet can alter the taxonomic composition of the human gut microbiota during an indi- vidual’s lifetime [23, 24], and human metabolism and physiology is closely linked to the gut microbiota [25], it is likely that evolutionary shifts in human diet and physi- ology were accompanied by shifts in the gut microbiota. Background As a result, the human gut microbiota should exhibit unique properties when compared to the non-human pri- mate gut microbiota. If this is the case, non-human primate models of host-gut microbe relationships may be less ideal than assumed for addressing questions regarding human diet and physiology in the context of the gut microbiota. Physiological similarities to humans make vervets, an at- tractive model for biomedical trials [28], particularly with regards to human metabolic disorders and obesity [29, 30]. Vervets may therefore provide an excellent reference system for understanding how unique the responses of the human gut microbiota to diet are compared to other primates. Despite the utility of vervets as models for human physiology in many contexts, we hypothesized that the vervet gut microbiota would exhibit distinct responses to a Western diet when compared with the human gut microbiota. Specifically, we predicted that the relative abundances of fewer microbial taxa and genes would differ in response to diet in the vervet gut microbiota since vervets have not evolved to include large amounts of animal fat and protein in their natural diets [31]. Consequently, the vervet gut microbiota should be less able to adapt to this type of Western diet and may not be an appropriate model for understanding human gut microbe interactions in the context of diet. Recent data provide evidence that the human gut microbiota is less diverse and better adapted to a meat-rich diet compared to closely related extant pri- mates [26], but no direct comparison of host-gut mi- crobe dynamics in humans and non-human primates currently exists. Therefore, it is unclear if the relation- ship between the gut microbiota and host diet and physiology differs in humans compared to other pri- mates. To improve our understanding of how host-gut microbe interactions are similar or different in human and non-human primates, here, we use 454 titanium pyrose- quencing and predictive metagenomics (PICRUSt) [27] to compare the gut microbiota of humans and African green monkeys, or vervets (Chlorocebus aethiops sabaeus), consuming both a Western and a non-Western diet. Results Vervets Results show that although vervets fed a typical West- ern diet (TWD, see Methods for composition) had similar gut microbial richness compared to wild vervets consuming a non-Western diet (Fig. 1), the compos- ition of their gut microbiota was distinct (unweighted UniFrac distance, F1,26 = 4.8, p < 0.001, r2 = 0.16, Fig. 2; weighted UniFrac distance, F1,26 = 9.6, p < 0.001, r2 = 0.28, Additional file 1: Figure S1, Table S1-S5). Specifically, at the phylum level, TWD-fed vervets had significantly lower relative abundances of Firmicutes, Lentisphaerae, Proteo- bacteria, Tenericutes, and Verrucomicrobia than wild vervets (Additional file 1: Table S1). In contrast, TWD-fed Fig. 1 A Western diet reduces gut microbial diversity in humans but not vervets. Chao1 estimates (average ± SD) of microbial community richness at 1000 sequence reads per sample for vervets and humans consuming a non-Western vs. a Western diet. Star indicates significant differences (FDR-corrected p < 0.05) in microbial richness between diets. Western humans are from Italy (Schnorr et al. [3]) and the USA (Yatsunenko et al. [2]). Non-western humans are from Tanzania (Hadza, Schnorr et al. [3]), Venezuela (Guahibo, Yatsunenko et al. [2]), and Malawi (Yatsunenko et al. [2]). (N = 13 non-Western vervets; N = 14 Western vervets; N = 17 non-Western (Schnorr et al.) humans; N = 11 Western (Schnorr et al.) humans; N = 76 non-Western (Yatsunenko et al.) humans; N = 118 Western (Yatsunenko et al.) humans) Fig. 1 A Western diet reduces gut microbial diversity in humans but not vervets. Chao1 estimates (average ± SD) of microbial community richness at 1000 sequence reads per sample for vervets and humans consuming a non-Western vs. a Western diet. Star indicates significant differences (FDR-corrected p < 0.05) in microbial richness between diets. Western humans are from Italy (Schnorr et al. [3]) and the USA (Yatsunenko et al. [2]). Non-western humans are from Tanzania (Hadza, Schnorr et al. [3]), Venezuela (Guahibo, Yatsunenko et al. [2]), and Malawi (Yatsunenko et al. [2]). (N = 13 non-Western vervets; N = 14 Western vervets; N = 17 non-Western (Schnorr et al.) humans; N = 11 Western (Schnorr et al.) humans; N = 76 non-Western (Yatsunenko et al.) humans; N = 118 Western (Yatsunenko et al.) humans) Fig. 1 A Western diet reduces gut microbial diversity in humans but not vervets. Humans vs. vervets Fig. 2 The vervet gut microbiome resembles a non-Western human gut microbiome regardless of the vervet diet. Non-metric multi-dimensional scaling (NMDS) plot based on unweighted UniFrac distances illustrating clustering patterns in gut microbiomes across sampling groups at the OTU level Both human studies revealed differences in the gut micro- biota associated with diet both in terms of microbial taxo- nomic composition (unweighted UniFrac distance, Hadza/ Italy, F1,27 = 4.9, p < 0.001, r2 = 0.16, Malawi/Venezuela/ U.S., F1,193 = 31.6, p < 0.001, r2 = 0.14, Fig. 2; weighted Uni- Frac distance, Hadza/Italy, F1,27 = 5.2, p = 0.001, r2 = 0.17; Malawi/Venezuela/USA, F1,193 = 72.7, p < 0.001, r2 = 0.27 Additional file 1: Figure S1, Additional file 1: Table S1-S5) and gene relative abundances (Hadza/Italy, F1,42 = 6.1, p = 0.003, r2 = 0.13; Malawi/Venezuela/USA, F1,193 = 61.3, p < 0.001, r2 = 0.24), but we detected more differences in the relative abundances of microbial taxa between Western and non-Western gut microbiomes in the Malawi/ Venezuela/USA dataset, presumably as a result of larger sample sizes (Additional file 1: Tables S1-S7). In addition, several microbial taxa reacted similarly to a Western diet across both human and vervet datasets despite different storage, DNA extraction, and sequencing technologies as well as variation in Western and non-Western diet across countries and species. Like the vervets, Western diet humans from the USA and Italy exhibited lower relative abundances of Proteobacteria, Lentisphaerae, and Teneri- cutes (p = 0.06 for Italians) compared to those on a non- Western diet. Relative abundances of Spirochaetes were also lower in the humans from Malawi and Venezuela com- pared to the USA, and we detected lower relative abun- dances of Clostridium and higher relative abundances of Desulfovibrionaceae and Collinsella in humans from the USA compared to Malawi and Venezuela. Fig. 2 The vervet gut microbiome resembles a non-Western human gut microbiome regardless of the vervet diet. Non-metric multi-dimensional scaling (NMDS) plot based on unweighted UniFrac distances illustrating clustering patterns in gut microbiomes across sampling groups at the OTU level vervets had significantly higher abundances of Bacteroi- detes and TM7, with a trend for higher relative abun- dances of Spirochaetes (Additional file 1: Table S1). Results Vervets Chao1 estimates (average ± SD) of microbial community richness at 1000 sequence reads per sample for vervets and humans consuming a non-Western vs. a Western diet. Star indicates significant differences (FDR-corrected p < 0.05) in microbial richness between diets. Western humans are from Italy (Schnorr et al. [3]) and the USA (Yatsunenko et al. [2]). Non-western humans are from Tanzania (Hadza, Schnorr et al. [3]), Venezuela (Guahibo, Yatsunenko et al. [2]), and Malawi (Yatsunenko et al. [2]). (N = 13 non-Western vervets; N = 14 Western vervets; N = 17 non-Western (Schnorr et al.) humans; N = 11 Western (Schnorr et al.) humans; N = 76 non-Western (Yatsunenko et al.) humans; N = 118 Western (Yatsunenko et al.) humans) Page 3 of 9 Page 3 of 9 Page 3 of 9 Amato et al. Microbiome (2015) 3:53 Humans vs. vervets While people in the USA exhibited increased relative abun- dances of Firmicutes and reduced relative abundances of Page 4 of 9 Page 4 of 9 Amato et al. Microbiome (2015) 3:53 Bacteroidetes compared to people in Venezuela and Malawi, vervets exhibited the following opposite pattern: a reduction in Firmicutes relative abundances and an in- crease in Bacteroidetes relative abundances in response to a Western diet (Fig. 3a). Furthermore, Western human populations exhibited lower relative abundances of Prevo- tella and higher relative abundances of Bacteroides and Bifidobacterium, which contrasts sharply with the patterns observed in the vervets (Fig. 3b). Vervets also exhibited increased levels of Cantenibacterium on a Western diet, but both human populations we examined showed the opposite pattern. Finally, in both human populations, rela- tive abundances of Succinovibrio and Treponema were lower for people consuming a Western diet, but diet did not signficantly affect these taxa in vervets. in humans on a Western diet while in vervets its relative abundance increased (Additional file 1: Table S6). The same was true of K01858 (myo-inositol-1-phosphate synthase), K02548 (general secretion pathway protein I), and K02619 (4-amino-4-deoxychorismate lyase), associated with carbohydrate metabolism, cofactor and vitamin metabolism/biosynthesis, and folate biosynthesis, respect- ively (Additional file 1: Table S6). Additionally, vervets showed shifts in the relative abundances of eight genes that did not shift in response to diet in humans in either study. At the pathway level, humans also showed an increased rela- tive abundance of genes associated with carbohydrate me- tabolism when consuming a Western diet, a difference that was not observed in vervets (Additional file 1: Table S7). Humans vs. vervets At the genus level, we also detected lower relative abundances of Clostridium and a tendency for lower relative abundances of Bifidobacterium in TWD-fed vervets as well as higher relative abundances of Desulfovibrio, Prevotella, Canteni- bacterium, and Collinsella (Additional file 1: Table S5). Despite the similarities noted above, we also observed key differences between the gut microbiota of humans and vervets associated with each type of diet. First, exam- ination of the presence or absence of microbial taxa revealed that the vervets clustered more strongly with non-Western humans, especially the Hadza, regardless of whether the vervets were consuming a wild or Western diet (Fig. 2, Additional file 1: Figure S2). Although recent bottlenecks have resulted in low genetic diversity in both vervet populations sampled [28, 32], gut microbiome data from another wild, non-human primate species (Cercoce- bus agilis, Central African Republic) also cluster with the vervets (Additional file 1: Figure S3), indicating that in- creased genetic diversity among human populations com- pared to the vervet populations is not driving the observed patterns. ( ) Predictive metagenomics also revealed differences in the vervet gut microbiome in response to diet (F1,57 = 4.94, p = 0.01, r2 = 0.08). Specifically, 22 genes differed in relative abundance between wild vervets and TWD-fed vervets. For example, K00432, a glutathione peroxidase, which is associ- ated with lipid and amino acid metabolism, was present at higher relative abundances in vervets consuming a TWD than wild vervets (Additional file 1: Table S6). The same pattern was observed in the relative abundance of K02619, a 4-amino-4-deoxychorismate lyase, which is associated with folate biosynthesis. In contrast, K00068 (sorbitol-6- phosphate 2-dehydrogenase), K00844 (hexokinase), and K05884 (L-2-hydroxycarboxylate dehydrogenase), with roles in fructose and mannose metabolism, carbohy- drate and lipid metabolism, and coenzyme M biosyn- thesis, respectively, were all predicted in lower relative abundances in TWD-fed vervets (Additional file 1: Table S6). At the pathway level, TWD-fed vervets exhib- ited increased relative abundances of genes associated with amino acid metabolism (Additional file 1: Table S7). In addition, Chao1 species diversity estimates tended to be lower in humans consuming a Western diet vs. a non-Western diet (Fig. 1), while diversity in vervets was unaffected by diet. Compared to humans, vervets also exhibited distinct changes in the relative abundances of microbial taxa in response to a Western diet. Discussion Predictive metagenomic data also showed strong differ- ences in the reactions of the vervet and human gut micro- biota to diet. Although the relative abundance of K00432 (glutathione peroxidase) changed in response to diet in both humans and vervets, its relative abundance decreased As hypothesized, our data indicate that the composition of the human and vervet gut microbiota is distinct on a Western diet. However, we did not observe differences in the relative abundance of fewer microbial taxa and Fig. 3 Human and vervet gut microbiomes react differently to a Western diet. Relative abundances of key a phyla and b genera in humans and vervets consuming non-Western and Western diets. Western humans are from Italy (Schnorr et al. [3]) and the USA (Yatsunenko et al. [2]). Non-Western humans are from Tanzania (Hadza, Schnorr et al. [3]), Venezuela (Guahibo, Yatsunenko et al. [2]), and Malawi (Yatsunenko et al. [2]). Stars indicate significant differences (FDR-corrected p < 0.05) in relative abundances between diets for both humans and vervets. However, Bacteroides relative abundances were not significantly different between diets for vervets Fig. 3 Human and vervet gut microbiomes react differently to a Western diet. Relative abundances of key a phyla and b genera in humans and vervets consuming non-Western and Western diets. Western humans are from Italy (Schnorr et al. [3]) and the USA (Yatsunenko et al. [2]). Non-Western humans are from Tanzania (Hadza, Schnorr et al. [3]), Venezuela (Guahibo, Yatsunenko et al. [2]), and Malawi (Yatsunenko et al. [2]). Stars indicate significant differences (FDR-corrected p < 0.05) in relative abundances between diets for both humans and vervets. However, Bacteroides Fig. 3 Human and vervet gut microbiomes react differently to a Western diet. Relative abundances of key a phyla and b genera in humans and vervets consuming non-Western and Western diets. Western humans are from Italy (Schnorr et al. [3]) and the USA (Yatsunenko et al. [2]). Non-Western humans are from Tanzania (Hadza, Schnorr et al. [3]), Venezuela (Guahibo, Yatsunenko et al. [2]), and Malawi (Yatsunenko et al. [2]). Stars indicate significant differences (FDR-corrected p < 0.05) in relative abundances between diets for both humans and vervets. However, Bacteroides relative abundances were not significantly different between diets for vervets Fig. 3 Human and vervet gut microbiomes react differently to a Western diet. Relative abundances of key a phyla and b genera in humans and vervets consuming non-Western and Western diets. Discussion Western humans are from Italy (Schnorr et al. [3]) and the USA (Yatsunenko et al. [2]). Non-Western humans are from Tanzania (Hadza, Schnorr et al. [3]), Venezuela (Guahibo, Yatsunenko et al. [2]), and Malawi (Yatsunenko et al. [2]). Stars indicate significant differences (FDR-corrected p < 0.05) in relative abundances between diets for both humans and vervets. However, Bacteroides relative abundances were not significantly different between diets for vervets Amato et al. Microbiome (2015) 3:53 Page 5 of 9 exposure to local microbial pools, and antibiotic use. Like- wise, future studies must control for the potential effects of non-human primate captivity such as host social contact networks and early life influences on the gut microbiota. However, it is unlikely that the results we present here are primarily driven by these potential confounds. Patterns in confounding factors such as antibiotic use across populations of the same host are likely to be similar for both humans and vervets, with Western humans and captive vervets generally having more exposure to antibiotics than non-Western humans and wild vervets. Therefore, while diet may not be the only factor causing differences between populations of the same host species, the comparisons of patterns between the two host species remains valid. Additionally, the captive population of vervets was taken from St. Kitts between 1975 and 1980. As a result, the two popula- tions are only separated by 3–8 generations, reducing the impact of host genetics as a potential confound for the vervets. genes in the vervets compared to the humans. Instead, the effect of a Western diet on the vervet gut microbiota was driven by distinct patterns in microbial taxa and genes compared to humans. Many of the patterns we detected in both vervets and humans have been reported in other studies. For ex- ample, we observed elevated microbial richness and higher relative abundances of Prevotella in non-Western humans and elevated relative abundances of Bacteroides in Western humans. These results are concordant with a study of the human gut microbiota that associates diets high in protein and animal fat with high levels of Bacter- oides and diets high in plant carbohydrates with high levels of Prevotella [23, 33]. Similarly, all published stud- ies of Western and non-Western humans to-date report higher microbial richness and higher relative abundances of Prevotella in non-Western populations [2–6, 34]. Discussion In contrast, vervets on a Western diet showed similar micro- bial richness and higher relative abundances of Prevotella, a change that is mirrored in the macaque model [12]. In addition, we detected an elevated relative abundance of Collinsella in humans on a Western diet, and Collinsella has been associated with obesity in other studies of humans [35]. Similarly, while decreased relative abundances of Bac- teroidetes coupled with increased relative abundances of Firmicutes, like those we observed in Western humans from the USA, are not reported in many studies comparing Western and non-Western humans [4, 5, 34]; they have been associated with obesity in human and mouse studies [36]. In our TWD-fed vervets, we measured the opposite patterns, and these findings are analogous to previous work contrasting a high-fat diet to a captive control (chow) diet in a macaque model (Macaca fuscata) where a shift to a high-fat diet also resulted in significant increases in Bacter- oidetes, especially Prevotella and no significant variations in Firmicutes [12]. Despite the comparison of multiple datasets in this analysis, studies generally indicate that host diet has the strongest effect on the gut microbiota compared to other factors [37], reducing the potential for confounding fac- tors associated with different host populations to drive the observed patterns. For example, when human studies broadly control for host ethnic backgrounds, they continue to illustrate strong impacts of Western and non- Western diets, suggesting that differences in gut micro- biota composition between populations of the same host species with distinct diets are unlikely to be genetically- driven [6]. Furthermore, although the vervet Western diet is distinct from a human Western diet, it is important to note that Western and non-Western human diets vary markedly across populations (e.g., USA vs. Italy and Malawi vs. Venezuela vs. Tanzania; [2, 3]). Despite differ- ences within these diet categories, Western and non- Western human populations still cluster together in terms of gut microbiota composition, and other studies compar- ing Western and non-Western humans consistently report similar patterns [2–6, 34], suggesting that differences in the human and vervet gut microbiota are not driven by subtle variation in the composition of either a Western or non- Western diet or by idiosyncrasies in the selected datasets. Discussion Likewise, while the data we present were not all generated using the same methodology, the potential effects of dis- tinct DNA extraction protocols, PCR primers, or sequen- cing platforms on gut microbiota data have been shown to be small [38, 39] compared to the effects of diet. Addition- ally, all DNA extractions utilized bead-beating step, which reduces extraction bias [40], and our use of the same closed-reference OTU picking pipeline (see methods) on rarefied data also reduces the potential effect of se- quencing error and read depth on the results [41]. These commonalities suggest that our results are not unique to the human and vervet populations that we ex- amined in this study. The human gut microbiota and its response to an easy-to-digest Western diet that is low in fiber differ fundamentally from the non-human primate gut microbiota. These findings provide the first evidence implying a specialization of the human gut microbiota. Therefore, while non-human primates may serve as comparative biomedical models for other aspects of hu- man physiology [28], we suggest that the non-human primate gut microbiota may not provide an ideal direct model for understanding the effect of the human gut microbiota on host metabolism and nutrition in the context of a Western diet. It is important to note that both the human and vervet data used in this study compare distinct populations, and therefore it is impossible to control for potential non-diet influences on the gut microbiota such as host genetics, Page 6 of 9 Amato et al. Microbiome (2015) 3:53 protein, high-fat diet can impact the gut microbiota almost immediately in humans, and changes are similar to those observed across populations with distinct diets [24], suggesting that a 6-month interval was sufficient. Although the captive vervets weighed more than the wild vervets both before and after the diet challenge, indicating an effect of captivity on body weight, this effect is likely a result of increased food availability and decreased activity, both of which would be expected to exacerbate weight gain and negative health outcomes on a Western diet, not mitigate them. While additional research is necessary to confirm and examine this pat- tern in more detail, we suggest that a side effect of the proposed unique human gut microbiota may be an increased susceptibility to obesity and metabolic disor- ders, particularly when hosts are consuming a high- protein, high-fat diet. Discussion If the non-human primate gut microbiota does, in fact, possess properties that make it resistant to obesity when subjected to a Western diet, it may open new avenues of exploration for translational metabolic therapies. What might confer this resistance and whether it can be maintained over generations of Western diet consumption remains to be investigated, but understanding these factors could help develop treatments to improve human resistance to obesity via the gut microbiota. Therefore, while we cannot completely eliminate the biases of study-specific methodology on this dataset, we are confident that the patterns presented are biologically-driven. Although data for other non-human primate taxa must be collected, we propose two explanations for the distinct responses of the human and non-human primate gut microbiota to diet. First, it is possible that the unique properties observed in the human gut microbiota are simply the result of unique human diet and physiology (namely an easy-to-digest, meat-heavy diet and increased brain size, reduced gut size, increased fat deposition, and decreased muscle mass [17–19]). Since both host diet and physiology drive gut microbiota composition [42], evolu- tionary changes in human diet and physiology could have easily led to a distinct gut microbiota. Another possible explanation is that, in addition to other factors such as diet, unique properties of the human gut microbiota con- tributed to the evolution of human physiology. For ex- ample, if over time the human gut microbiota shifted in a way that confers an increased capacity for energy produc- tion and storage, it could have promoted increased brain size during human evolution. Similarly, because the hu- man gut microbiota plays a role in regulating host energy intake and fat production [43], and an increased capacity to store energy as fat has been hypothesized to have en- abled humans to develop larger brains [18, 19], changes in the gut microbiota that affected host metabolic pathways could have contributed to the evolution of the human brain as well. To distinguish between these two alterna- tives, further studies are necessary that measure the meta- bolic potential of the human and non-human gut microbiota and more directly compare the physiological consequences of consuming a Western diet. However, a recent study suggests that large-brained primates endure seasonal periods of food limitation more successfully than small-brained primates [44]. Conclusions Although animal models such as non-human primates are commonly used to test mechanisms of human gut microbe interactions, direct comparisons of the influ- ence of commonly investigated factors on the gut micro- biota of humans and non-human primates are lacking. This study demonstrates that similar host diets differen- tially affect the human and non-human primate gut microbiota. These results indicate that non-human pri- mates are not appropriate models for directly testing the relationships between human diet, physiology and the gut microbiota. However, the observed patterns have implica- tions for human evolution since they suggest an association between the gut microbiota and unique shifts in human diet and physiology across evolutionary time. A more de- tailed examination of this association has the potential to transform our understanding of the role of gut microbes in human biology. Finally, our data indicated that, in addition to the gut microbiota, the physiological responses of humans and non-human primates to a Western diet may be distinct. Although vervets have an adverse metabolic reaction to a Western diet and can become obese in captivity [46, 47], most of the TWD-fed vervets did not gain weight during the study (Additional file 1: Table S8). It is possible that two factors restricted vervet weight gain: (1) finite food availability and (2) study duration (6 months). We regard these factors as unlikely to be limiting because the ver- vets were provided with enough food to result in a 10 % daily surplus, and no obvious reduction in food intake was observed [47]. Moreover, a sufficiently high- Discussion Together with evidence that gut microbes compensate for periodically reduced energy intake in some wild primates [45], these data could indicate a role for the gut microbiota in buffering hosts nutritionally against energetically expensive physiological adaptations. Sample collection and processing l b d l DNA was extracted from rectal swabs using a MoBio UltraClean Soil DNA Isolation kit (MoBio, Carlsbad, CA, USA) per manufacturer instruc- tions. The V1–V3 region of the bacterial 16S rRNA gene was amplified using pyrotagged primers (MID 1-15) 27f and 534r. Amplicons were checked for specificity with gel electrophoresis and cleaned using the AMPure XP system (Beckman Coulter, Danvers, MA, USA). Amplicons were sequenced using 454 pyrosequencing technology at the J. Craig Venter Institute and/or the University of Illinois KECK Center. No differences were seen in the microbial taxonomic distributions of samples sequenced at both sequencing centers. There- fore, sequencing center was eliminated as a possible effect. Chao1 diversity estimates were produced for each sample using QIIME. We also predicted the metagen- ome associated with each sample using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt 1.0.0) [27] after normal- izing for 16S copy number. The average Nearest Se- quenced Taxon Index was 0.11 ± 0.03, with Western humans falling at the lower end of the range and non- Western humans and vervets falling at the higher end. We tested for significant differences between wild and TWD vervets as well as Western and non-Western humans from each human study. Although inter-study effects on patterns of gut microbial community com- position have been demonstrated to be low [38, 39], we tested for the effect of diet within each individual data- set independently to eliminate the potential for con- founding factors. Principle coordinate analyses based on unweighted and weighted UniFrac distances were used to visualize differences in gut microbial commu- nity composition among populations. PERMANOVA (R software, version 3.0.2, adonis package) was used to test for the effect of diet on microbial community compos- ition and predicted metagenomes for all three datasets. Similarly, for each dataset, we tested for differences in the relative abundances of microbial taxa and genes be- tween wild and TWD vervets or between Western and non-Western humans using a series of Kruskal-Wallis tests at each taxonomic level. p values were adjusted using either a family-wide detection rate (microbial taxonomy) or a Bonferroni correction (predicted meta- genomes). All statistics were performed using QIIME’s implementation of R. Raw sequence data from non-Western and Western human populations published by Yatsunenko et al. [2] and Schnorr et al. [3] were also obtained for analysis. Non- Western human populations were sampled in Malawi, Venezuela (Guahibo Amerindians), and Tanzania (Hadza). Sample collection and processing l b d l Rectal swabs were used to sample the gut microbiota of 27 wild and captive vervets. In January 2010, 15 wild vervet monkeys were captured, sampled, and released on the island of St. Kitts. Wild vervets on St. Kitts are not provisioned and consume mainly ripe fruits, flowers, and seeds [48]. We considered this a non-Western diet. In Page 7 of 9 Page 7 of 9 Amato et al. Microbiome (2015) 3:53 database with OTUs defined as sharing ≥97 % identity. This strategy made it possible to compare patterns across studies despite the use of different primers and/ or sequencing platforms and reduced the impact of se- quencing error on the data. Due to the use of 454 tech- nology in two of the datasets, we rarefied the number of sequences from each sample to 1000 for all studies. Previ- ous studies suggest that this level allows for meaningful comparison [50]. Additionally, nine TWD vervet samples were removed from analysis as a result of unusually high relative abundances of Brachyspira. Several Brachyspira species are known gut pathogens, and loose stools were observed in some of the enclosures during the diet trial. Removing these samples did not affect the overall patterns observed in beta diversity within and across studies (Additional file 1: Figure S4, S5) but did lead to Brachy- spira relative abundances in a more typical range. September 2009, 23 vervets at the Wake Forest University Primate Center were sampled in a similar manner. These vervets are directly descended from the St. Kitts popula- tion (with an initial group obtained in 1975) and were fed a typical Western human diet (TWD; LabDiet 5L0P, Purina, St. Louis, MO; 18 % protein, 37 % fat, 45 % carbohydrates, 9 % fiber) for 6 months before sampling. Sampling of and care for the captive TWD vervets was approved by the Institutional Animal Care and Use Committee at Wake Forest University (IACUC #A10-091). Sampling of the wild monkeys was approved by the University of Illinois Institu- tional Animal Care and Use Committee (IACUCs #08044, #11046) as well as the University of California Los Angeles IACUC (#2009-053-13). All sampling was carried out in ac- cordance with the approved guidelines at each institution. All rectal swabs were immediately placed in RNAlater (Qiagen Inc., Valencia, CA, USA) and stored at −80 °C until processing. Data analysis All All sequence data were quality filtered so that se- quences shorter than 200 nt, longer than 1000 nt, con- taining incorrect primer sequences, more than six ambiguous base calls and/or homopolymers longer than 7 nt were discarded, resulting in 387,785,923 sequences across all studies. All subsequent data ana- lyses were performed using QIIME (version 1.8.0) [49]. Operational taxonomic units (OTUs) were picked closed-reference against the Green Genes 13_8 Sample collection and processing l b d l Western human populations were sampled in the USA and Italy. Only individuals between the ages of 18 and 50 were included from the Yatsunenko et al. [2] dataset (Malawi/Venezuela N = 76 people, USA N = 118 people), while all samples from Schnorr et al. 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Competing interests Competing interests 11. Faith JJ, McNulty NP, Rey FE, Gordon JI. Predicting a human gut microbiota’s response to diet in gnotobiotic mice. Science. 2011;333:101–4. The authors declare that they have no competing interests. 12. Ma J, Prince AL, Bader D, Hu M, Ganu R, Baquero K, et al. High-fat maternal diet during pregnancy persistently alters the offspring microbiome in a primate model. Nat Comm. 2014;5:3889. doi:10.1038/ncomms4889. 34. Martinez I, Stegen JC, Maldonado-Gomez MX, Eren AM, Siba PM, Greenhill AR, et al. The gut microbiota of rural Papua New Guineans: Composition, diversity patterns, and ecological processes. Cell Rep. 2015;11:527–38. Additional file 1: Supplementary tables and figures. (DOC 2591 KB) 10. Hosseini E, Grootaert C, Verstraete W, Van de Wiele T. Propionate as a health-promoting microbial metabolite in the human gut. Nutr Rev. 2011;69(5):245–58. doi:10.1111/j.1753-4887.2011.00388.x. Author details 1D f 1Department of Anthropology, Northwestern University, Evanston, USA. 2Department of Anthropology, University of Colorado Boulder, Boulder, USA. 3BioFrontiers Institute, University of Colorado Boulder, Boulder, USA. 4Department of Range Sciences, Montana State University, Bozeman, USA. 5Department of Anthropology, University of Illinois, Urbana, USA. 6Department of Anthropology, Boston University, Boston, USA. 7Department of Sociology, Anthropology, and Women’s Studies, American Military University and American Public University, Charles Town, USA. 8The Institute for Genomic Biology, University of Illinois, Urbana, IL 61801, USA. 9Department of Ecology, Evolution, and Behavior, University of Minnesota, Minneapolis, USA. 10Department of Anthropology, University of Wisconsin, Milwaukee, USA. 11Department of Genetics, University of the Free State, Bloemfontein, South Africa. 12Department of Microbiology, University of Illinois, Urbana, USA. 13The J. Craig Venter Institute, Rockville, USA. 1Department of Anthropology, Northwestern University, Evanston, USA. 2Department of Anthropology, University of Colorado Boulder, Boulder, USA. 3BioFrontiers Institute, University of Colorado Boulder, Boulder, USA. 4Department of Range Sciences, Montana State University, Bozeman, USA. 5Department of Anthropology, University of Illinois, Urbana, USA. 6Department of Anthropology, Boston University, Boston, USA. 7Department of Sociology, Anthropology, and Women’s Studies, American Military University and American Public University, Charles Town, USA. 8The Institute for Genomic Biology, University of Illinois, Urbana, IL 61801, USA. 9Department of Ecology, Evolution, and Behavior, University of Minnesota, Minneapolis, USA. 10Department of Anthropology, University of Wisconsin, Milwaukee, USA. 11Department of Genetics, University of the Free State, Bloemfontein, South Africa. 12Department of Microbiology, University of Illinois, Urbana, USA. 13The J. Craig Venter Institute, Rockville, USA. 14 15 21. Casanova JL, Abel L, Quintana-Murci L. Human TLRs and IL-1Rs in host defense: Natural insights from evolutionary, epidemiological, and clinical genetics. Immunology. 2011;29:447–91. 22. Kargman S, Charleson S, Cartwright M, Frank J, Riendeau D, Mancini J, et al. Characterization of prostaglandin G/H synthase 1 and 2 in rat, dog, monkey, and human gastrointestinal tracts. Gastroenterol. 1996;111(2):445–54. 23. Wu GD, Chen J, Hoffmann C, Bittinger K, Chen YY, Keilbaugh SA, et al. Linking long-term dietary patterns with gut microbial enterotypes. Science. 2011;334:105–8. 24. David LA, Maurice CF, Carmody RN, Gootenberg DB, Button JE, Wolfe BE, et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature. 2014;505:559–66. doi:10.1038/nature12820. 25. Tremaroli V, Backhed F. Functional interactions between the gut microbiota and host metabolism. Nature. 2012;489:242–9. 14Department of Animal Sciences, University of Illinois, Urbana, USA. 15School of Medicine, University of California San Diego, La Jolla, USA. Author details 1D f 16Center for Neurobehavioral Genetics, University of California, Los Angeles, CA, USA. 14Department of Animal Sciences, University of Illinois, Urbana, USA. 15School of Medicine, University of California San Diego, La Jolla, USA. 16Center for Neurobehavioral Genetics, University of California, Los Angeles, CA, USA. 26. Moeller AH, Li Y, Ngole EM, Ahuka-Mendeke S, Lonsdorf EV, Pusey AE, et al. Rapid changes in the gut microbiome during human evolution. PNAS. 2014;111(46):16431–5. doi:10.1073/pnas.1419136111. 27. Langille MGI, Zaneveld J, Caporaso JG, McDonald D, Knights D, Reyes JA, et al. Predictive functional profiling of microbial communitiesusing 16S rRNA marker gene sequences. Nat Biotech. 2013;8:1–10. Received: 20 May 2015 Accepted: 29 September 2015 References Characterization and heritability of obesity and associated risk factors in vervet monkeys. Obesity. 2007;15:1666–74. 3. Schnorr SL, Candela M, Rampelli S, Centanni M, Consolandi C, Basaglia G, et al. Gut microbiome of Hadza hunter-gatherers. Nat Comm. 2014;5:3654. doi:10.1038/ncomms4654. 3. Schnorr SL, Candela M, Rampelli S, Centanni M, Consolandi C, Basaglia G, et al. Gut microbiome of Hadza hunter-gatherers. Nat Comm. 2014;5:3654. doi:10.1038/ncomms4654. 31. Jaffe KE, Isbell LA. The guenons: polyspecific associations in socioecological perspective. In: Campbell C, Fuentes A, MacKinnon KC, Bearder SK, Stumpf RM, editors. Primates in perspective. Second Editionth ed. New York: Oxford University Press; 2011. p. 277–99. 4. Obregon-Tito AJ, Tito RY, Metcalf JL, Sankaranarayanan K, Clemente JC, Ursell LK, et al. Subsistence strategies in traditional societies distinguish gut microbiomes. Nat Comm. 2015;6:6505. 4. Obregon-Tito AJ, Tito RY, Metcalf JL, Sankaranarayanan K, Clemente JC, Ursell LK, et al. Subsistence strategies in traditional societies distinguish gut microbiomes. Nat Comm. 2015;6:6505. 32. Aarnink A, Jacquelin B, Dauba A, Hebrard S, Moureaux E, Muller-Trutwin M, et al. MHC polymorphism in Caribbean African green monkeys. Immunogen. 2014;66(6):353–60. 5. Clemente JC, Pehrsson EC, Blaser MJ, Sandhu K, Gao Z, Wang B, et al. The microbiome of uncontacted Amerindians. Sci Adv. 2015;1(3):e1500183. 5. Clemente JC, Pehrsson EC, Blaser MJ, Sandhu K, Gao Z, Wang B, et al. The microbiome of uncontacted Amerindians. Sci Adv. 2015;1(3):e1500183. 6. Ou J, Carbonero F, Zoetendal EG, DeLaney JP, Wang M, Newton K, et al. Diet, microbiota, and microbial metabolitesin colon cancer risk in rural Africans and African-Americans. Am J Clin Nutr. 2013;98(1):111–20. 33. De Filippo C, Cavalieri D, Di Paola M, Ramazzotti M, Poullet JB, Massart S, et al. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. PNAS. 2010;107(33):14691–6. 7. Hu F, van Dam R, Liu S. Diet and risk of type II diabetes: the role of types of fat and carbohydrate. Diabetol. 2001;44:805–17. 34. Martinez I, Stegen JC, Maldonado-Gomez MX, Eren AM, Siba PM, Greenhill AR, et al. The gut microbiota of rural Papua New Guineans: Composition, diversity patterns, and ecological processes. Cell Rep. 2015;11:527–38. 8. Ley RE. Obesity and the human microbiome. Curr Opin Gastroenterol. 2010;26(1):5–11. Page 9 of 9 Page 9 of 9 Amato et al. Microbiome (2015) 3:53 Amato et al. Microbiome (2015) 3:53 35. Turnbaugh PJ, Hamady M, Yatsunenko T, Cantarel BL, Duncan A, Ley RE, et al. References A core gut microbiome in obese and lean twins. Nature. 2009;457:480–4. 36. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature. 2006;444(21):1027–31. harvest. Nature. 2006;444(21):1027–31. 37. Xu ZZ, Knight R. Dietary effects on human gut microbiome diversity. Br J Nutr. 2015;113(S1):S1–5. 38. Lozupone C, Stombaugh J, Gonzalez A, Ackermann G, Wendel D, Vasquez- Baeza Y, et al. Meta-analysis studies of the human microbiota. Genome Res. 2013;23:1704–14. doi:10.1101/gr.151803.112. 39. Wesolowski-Andersen A, Bahl MI, Carvalho V, Kristiansen K, Sicheritz-Ponten T, Gupta R, et al. Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis. Microbiome. 2014;5(2):19. 40. Walker AW, Martin J, Scott P, Parkhill J, Flint HJ, Scott KP. 16s rRNA gene- based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice. Microbiome. 2015;3:26. 41. Schloss PD, Gevers D, Westcott SL. Reducing the effects of PCR amplification and sequencing artifacts on rRNA-based studies. PLoS One. 2011;6(12):e27310. 42. Dethlefsen L, Eckburg PB, Bik EM, Relman DA. Assembly of the human intestinal microbiota. Trends Ecol Evol. 2006;21(9):517–23. 43. Backhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A, et al. The gut microbiota as an environmental factor that regulates fat storage. PNAS. 2004;101:15718–23. 44. van Woerden JT, Willems EP, Van Schaik CP, Isler K. Large brains buffer energetic effects of seasonal habitats in catarrhine primates. Evol. 2012;66(1):191–9. 45. Amato KR, Leigh SR, Kent A, Mackie RI, Yeoman CJ, Stumpf RM, et al. The gut microbiota appears to compensate for seasonal diet variation in the wild black howler monkey (Alouatta pigra). Microb Ecol. 2014;69(2):434–43. doi:10.1007/s00248-014-0554-7. 46. Voruganti VS, Jorgensen MJ, Kaplan JR, Kavanagh K, Rudel LL, Temel R, et al. Significant genotype by diet (GxD) interaction effects on cardiometabolic responses to a pedigree-wide, dietary challenge in vervet monkeys (Chlorocebus aethiops sabaeus). 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission
https://openalex.org/W3186694034	https://www.researchsquare.com/article/rs-11319/v1.pdf	English	null	Imputation benchmark of β-value and M-value from DNA methylation data under different missing data mechanisms.	Research Square (Research Square)	2,020	cc-by	567	Preprint: Please note that this article has not completed peer review. Preprint: Please note that this article has not completed peer review. CURRENT STATUS: UNDER REVISION CURRENT STATUS: UNDER REVISION methylation, M-value, β-value, Missing data mechanisms, MCAR, 1 1 Abstract Background: High-throughput technologies enable the cost-effective collection and analysis of DNA methylation data throughout the human genome. This naturally entails missing values management that can complicate the analysis of the data. Several general and specific imputation methods are suitable for DNA methylation data. However, there are no detailed studies of their performances under different missing data mechanisms -(completely) at random or not- and different representations of DNA methylation levels (β and M-value). Results: We make an extensive analysis of the imputation performances of seven imputation methods on simulated missing completely at random (MCAR), missing at random (MAR) and missing not at random (MNAR) methylation data. We further consider imputation performances on the β- and M- value popular representations of methylation levels. Overall, β -values enable better imputation performances than M-values. Imputation accuracy is lower for mid-range β -values, while it is generally more accurate for values at the extremes of the β -value range. The MAR values distribution is on the average more dense in the mid-range in comparison to the expected β -value distribution. As a consequence, MAR values are on average harder to impute. Conclusions: The results of the analysis provide guidelines for the most suitable imputation approaches for DNA methylation data under different representations of DNA methylation levels and different missing data mechanisms. Full Text Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF. Figures 2 Figure 1 β-value distributions of different missingness mechanisms. Comparison of the β-value distribution against the distribution of simulated MCAR, MAR and MNAR missing values. Figure 1 β-value distributions of different missingness mechanisms. Comparison of the β-value β-value distributions of different missingness mechanisms. Comparison of the β-value distribution against the distribution of simulated MCAR, MAR and MNAR missing values. 3 3 Figure 2 Figure 2 β-value distributions of CpGs with frequently missing values. Comparison of the β-value distribution against the β-value distribution of CpGs with missing values on > 20%;> 25%;> 30% samples. MAR simulated distribution included. β-value distributions of CpGs with frequently missing values. Comparison of the β-value distribution against the β-value distribution of CpGs with missing values on > 20%;> 25%;> 30% samples. MAR simulated distribution included. 4 5 Figure 3 Healthy datasets. Average RMSE with respect to β-value range. Average RMSE for M-value and β-value imputation with respect to different - value ranges and with respect to the MCAR, MAR, MNAR (low, mid, high) missing data mechanisms. Figure 3 Healthy datasets. Average RMSE with respect to β-value range. Average RMSE for M-value and β-value imputation with respect to different - value ranges and with respect to the MCAR, MAR, MNAR (low, mid, high) missing data mechanisms. Healthy datasets. Average RMSE with respect to β-value range. Average RMSE for M-value and β-value imputation with respect to different - value ranges and with respect to the MCAR, MAR, MNAR (low, mid, high) missing data mechanisms. 5 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. AdditionalFile2.pdf AdditionalFile1.pdf AdditionalFile2.pdf AdditionalFile1.pdf 6 6
W4387276480.txt	https://amu.hal.science/hal-03938731/document	en		Homeland in the world.	HAL (Le Centre pour la Communication Scientifique Directe)	2,022	cc-by	3,959	La patria nel mondo. Il mondo in una Patria. Claudio Milanesi To cite this version: Claudio Milanesi. La patria nel mondo. Il mondo in una Patria.: Effetti di transculturalità e modi di rappresentazione delle culture straniere nell’Italia del tempo presente. Enrico Deaglio, Patria (1978-2000).. TRANSCULTURALITÀ E PLURILINGUISMI NELLA LETTERATURA ITALIANA DEGLI ANNI DUEMILA, A cura di Alessandro Benucci, Silvia Contarini e Giuliana Pias, Franco Cesati Editore, 2022, 978-88-7667-993-3. �hal-03938731� HAL Id: hal-03938731 https://amu.hal.science/hal-03938731 Submitted on 23 Jan 2023 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Distributed under a Creative Commons Attribution 4.0 International License Claudio Milanesi, Aix Marseille Université, CAER, Aix-en-Provence, France La patria nel mondo. Il mondo in una Patria. Effetti di transculturalità e modi di rappresentazione delle culture straniere nell’Italia del tempo presente. Enrico Deaglio, Patria (1978-2000). Alla base di questo lavoro vi è la volontà di verificare in che modo il buon giornalismo italiano renda conto dei fenomeni transazionali, intendendo in questo caso le rappresentazioni dei paesi stranieri, le rappresentazioni degli italiani nel mondo, le rappresentazioni delle influenze di paesi e fenomeni culturali, sociali e politici stranieri nella politica, nella cultura e nella società italiana. Il campione di osservazione prescelto è costituito dai tre volumi Patria di Enrico Deaglio che coprono un periodo che va dal 1968 al 2020, il tutto per complessive 2250 pagine1. Del primo volume che va dal 1978 al 2010 avevo già parlato in un convegno a Varsavia2, in un dialogo con il compianto Remo Ceserani. Avevamo fatto una lettura incrociata del libro di Deaglio, e Cesarani ne parlava come del “grande romanzo italiano”, mutuando l’espressione dalla critica americana. Intendeva, per “grande romanzo italiano”, che Patria avesse i numeri per rappresentare, sia per quello che raccontava che per come rendeva conto di vent’anni anni di vita italiana, IL romanzo che si candidava a meglio raccontare lo spirito del paese e di un’epoca, che diventava fra l’altro metonimia di tutta la sua storia e delle continuità che l’avevano attraversata. Solo che non era un romanzo. Ma una non fiction: Deaglio non inventava niente (o meglio, stringeva un patto col lettore secondo il quale non avrebbe inventato niente)3, ma inanellava fatti di cronaca, personaggi, citazioni di libri, evocazioni di canzoni, di film, di romanzi, dichiarazioni di personaggi dell’epoca… Finiva così per compilare una grande narrazione esplosa, spezzettata in mille tasselli, un grande racconto per frammenti, un patchwork in puro stile postmoderno. Una storia globale fondata però sulla sfiducia postmoderna nella storia globale delle grandi narrazioni novecentesche. Il grande romanzo italiano era quindi un non romanzo sulla storia della fine del XX secolo fondato su una sostanziale sfiducia nella capacità della Storia di inserire gli eventi in un quadro coerente. Ne risultava una sorta di storia per microstorie, per spie, senza una direzione, priva di una filosofia della storia, e per questo ricca, variegata, libera, laica, soggettiva e oggettiva al tempo stesso, e per tutte queste ragioni profondamente intrigante. 1 DEAGLIO Enrico, Patria. 1978-2000, Milano,Il Saggiatore, 2010 (prima edizione Patria. 1978-2008 Milano, Il Saggiatore, 2018), id. Patria. 1967-1977, Milano, Feltrinelli, 2017 ; id., Patria. 2011-2020, Milano, Feltrinelli, 2020. 2 Si tratta del convegno Fiction Faction Reality: incontri, scambi, intrecci nella letteratura italiana dal 1990 ad oggi tenutosi all’Università di Varsavia nel novembre 2009. 3 Infatti, nel testo troviamo alcuni dialoghi cui lo scrittore non ha potuto assistere. Per esempio: : il colloquio fra i reclutatori delle Brigate Rosse e patrizio Peci: “Come sarà la mia vita? Come mi mantengo?” […] “Devi troncare tutti i rapporti con la famiglia e con il paese, cambi nome e ti diamo 200mila lire al mese, che è la paga di un metalmeccanico, vestiti e bollette li paghiamo noi. Stai agli ordini dell’Esecutivo”, DEAGLIO Enrico, Patria. 1978-2000, cit., p. 81. Oppure: Salvo Lima risponde ai maggiorenti democristiani che lo interrogano sull’omicidio di Piersanti Mattarella, dicendo: “Sapete, i patti vanno rispettati”, ibid., p. 90. 1 Da quel convegno di Varsavia (2009) in cui discutevamo di Patria con Ceserani, i cui atti sono poi usciti nel 20114, sono passati 12 anni, ma sembra un secolo. Nel frattempo Deaglio ha aumentato l’arco temporale della sua Patria: se il primo volume andava dal 1978 al 2010, il secondo (il prequel) risale dal 1968 al 1977, il terzo (il sequel), uscito l’anno scorso, va dal 2011 al 2020. Nel quadro di un lavoro molto più ampio (un volume collettivo sui grandi libri della non fiction italiana, francese, spagnola e latinoamericana, ad opera del Network Non Fiction, un’équipe di ricercatori dedicata alla ricerca sull’emergere del genere), sto cominciando un corposo capitolo su questi tre volumi che a mio modo di vedere – e con l’autorità di Cesarani alle spalle – costituiscono il monumento della non fiction italiana, e probabilmente europea, pur non essendo tradotti in nessuna lingua del continente. Il presente articolo è una sorta di spin-off di questo lavoro. La questione affrontata riguarda la forma e la rappresentazione degli interscambi fra l’Italia e il resto del mondo in questa storia frammentata. Quali interazioni fra l’Italia e il resto del mondo vengono raccontati fra le microstorie selezionate da Deaglio? Che idea dei paesi stranieri appare nel “grande romanzo italiano”? Come vengono rappresentati i fatti e i personaggi d’oltrefrontiera? E che influenza si pensa esercitino le culture e le politiche dei Paesi Altri sulla cultura, sulla politica sulla società della penisola? Per far questo ho cominciato a isolare un decennio particolare, quello degli anni ’80, e sono andato a verificare quali fossero le modalità di apparizione del resto del mondo in questa sezione di Patria. In questo articolo prendo come esempio un triennio di storia italiana di questo decennio in modalità Patria. Si tratta di un triennio di svolta in cui sembra concentrarsi un grande numero di fenomeni che segnano tutta la storia italiana da allora a oggi. Proprio come voleva la retorica del “grande romanzo americano”, è una metonimia di tutto l’insieme. Quasi una monade che concentra il tutto nell’uno. Si tratta del triennio 1979/1981. L’oggetto della ricerca consiste nell’analisi delle rappresentazioni del flusso degli eventi e delle influenze straniere sui fatti italiani. E inversamente di come i fatti e i personaggi italiani si trovino ad agire all’estero lasciando così la loro impronta fuori dai confini. La cosa può sembrare di poco interesse. Ma non è così. In quest’epoca di mondialismo e sovranismo, ondeggiamo fra due rappresentazioni estreme, opposte e a somma zero. Una è quella della metafora del battito d’ala della farfalla, secondo la quale le interconnessioni della globalizzazione avrebbero fatto sparire le differenze fra i luoghi e le culture, l’altra quella dell’esaltazione del confine e della frontiera, del ritorno nostalgico e fantasmatico di sovranità nazionali che si nutrirebbero da sé. Ma questi sono, appunto, solo fantasmi: da un lato, nonostante la globalizzazione, le differenze fra le culture e i modi di vivere continuano ad esistere, e dall’altro le frontiere sono ancora– per fortuna – permeabili. Permeabili alle 4 SERKOWSKA Hanna (a cura di), Finzione cronaca realtà. Scambi intrecci e prospettive nella narrativa italiana contemporanea, Massa, Transeuropa, 2011. Negli atti del convegno, i due articoli in cui si discute di Patria sono: MILANESI Claudio, « Enrico Deaglio, Bella ciao, Besame mucho, Patria: dalle storie minime alla Storia per frammenti », pp. 69-79 e CESERANI Remo, « Il caso di un montaggio di cronache, ricordi, documenti e interpretazioni della realtà che sembrano costruire un romanzo italiano: Patria di Enrico Deaglio », pp. 81-94. 2 merci, certo, ma permeabili anche alle idee, ai sogni e a volte anche agli uomini e alle donne, e persino ai bambini, quando non li si fa morire di freddo al confine. Ma è da qui che voglio partire. Mi sono chiesto che tipo di rappresentazione fornissero a questi moti dello spirito e dei corpi le pagine di Patria. Mi sono chiesto se non ci fosse il modo, invece di bloccarsi davanti ai fantasmi, di vedere cosa succede davvero agli albori della nostra epoca glocal. Certo giornalismo italiano è gretto e becero. E a guardare la televisione italiana certe volte vien de credere che l’Italia sia il centro del mondo e possa ancora “fare da sé”, appartata dal resto, e che possa, per usare l’espressione dei proto sovranisti di inizio secolo, “tornare alla vecchia lira”. Rileggendo Patria, ho voluto vedere se l’oltrefrontiera nel giornalismo italiano possa esistere non solo come proiezione dei fantasmi, ma come realtà concreta, specifica, coi suoi eventi e personaggi particolari, unici, singolari e irripetibili. Ho voluto vedere se il buon giornalismo italiano, che esiste eccome, espanso fino a diventare non fiction, possa essere un modo per reimmettere il mondo in Italia e l’Italia nel mondo. Ho voluto verificare se ne emerga un‘idea transnazionale del Paese che faccia da contrasto alle deliranti posizioni sovraniste. Il 1980 è un anno cerniera, il passaggio fra l’impegno e il riflusso, fra l’epoca dei movimenti e il decennio dell’edonismo reaganiano. È l’anno di Ustica e della strage di Bologna, della marcia dei Quarantamila, degli omicidi di Carlo Casalegno e di Walter Tobagi. Ho quindi preso in considerazione il triennio di cui il 1980 è al cuore. Il 1979, l’anno del 7 aprile e degli omicidi di Guido Rossa e di Guido Ambrosoli. E il 1981, l’anno della P2, l’anno in cui le BR uccidono Roberto Peci e in cui Alì Agcà non riesce a uccidere Papa Woytila. Ce n’era abbastanza per riempire le pagine dei giornali e i telegiornali. Eppure… Quello che mi interessava era vedere se in Patria la preminenza di eventi così sconvolgenti finiva per inghiottire, annullandolo, lo sguardo verso l’esterno o se invece la selezione dei frammenti significativi della cronaca teneva comunque viva la coscienza del legame necessario fra il Paese e il resto del mondo. Così ho fatto un primo sondaggio dei frammenti in questione per rilevare la presenza dell’estero nelle cronache italiane, il tipo di rappresentazioni e il loro peso nell’economia della narrazione. Non mi soffermo sull’analisi quantitativa delle presenze di queste interazioni nel testo. L’unico dato che riporto è che nel breve spazio di tre anni (per un totale di 77 pagine), i paesi citati perché in un qualche modo interagiscono con l’Italia (e/o perché l’Italia interagisce con loro) sono 25. Il grosso delle citazioni è costituito dai paesi dell’Unione europea più la Svizzera. Seguiti dalla coppia USA/Canada. L’altro grosso insieme è costituito dai paesi dell’America Latina. Seguono i Paesi del Medio Oriente. Vengono poi la Russia e la Somalia, che è l’unico paese africano ad essere evocato, anche se di riflesso. Non c’è traccia dell’Estremo Oriente (Cina, Giappone, Vietnam…) né di Australia e Nuova Zelanda. Forse il fatto che la guerra del Vietnam fosse finita, e che in Cina, morto Mao e liquidata la Banda dei Quattro, le riforme di Deng fossero ancora agli albori, spiega questa assenza. Nessuna sorpresa che l’Unione Europea, con Francia e Germania in testa, e il Nordamerica facciano la parte del leone. Più sorprendente la presenza preminente del Medio Oriente, nonostante lo scontro di civiltà non fosse ancora iniziato, e dell’America Latina, che oggi ci appare più lontana di quanto apparentemente non fosse allora. 3 Il mondo è cambiato da allora, inutile negarlo. Tuttavia, certe continuità “italiane” sono quasi scioccanti. Faccio un inciso prima di tornare ai Paesi stranieri. Leggendo le cronache di quei tre anni è come se assistessimo a una sorta di onda lunga del caso Italia: diversi fenomeni d'attualità di allora sono realtà di lungo periodo, e certi casi raccontati in Patria e relativi all’inizio degli anni ’80 sono ancora i nostri di oggi. Appare stupefacente che personaggi e fenomeni del triennio rivelino una continuità che resiste e ritorna nei quarant’anni successivi: vi sono già l’immigrazione e il razzismo (un cittadino somalo viene bruciato vivo da una banda vicino Piazza Navona5), ci sono i fuoriusciti della lotta armata in Francia (allora accolti dalla dottrina Mitterrand oggi sotto la minaccia di estradizione dalla controdottrina Darmanin), c’è Berlusconi , allora occupato a stringere un patto coi siciliani perché non gli rapiscano i figli, oggi alla ricerca di un’elezione a Presidente della Repubblica, e c’è persino il progetto del ponte sullo stretto, allora elaborato in Svizzera in gran segreto da Licio Gelli della P2 e Carlo Pesenti, patron dell'Italcementi. Il ponte poi non si è fatto, ma il progetto ritorna fuori a ritmo costante come un’araba fenice, rilanciato ancora di recente dal ministro delle Infrastrutture Giovannini6. Ultimo degli elementi di questa onda lunga dell’attualità italiana: la violenza sulle donne, fenomeno già diffuso allora, e rimasto di tragica attualità fino a noi. È nel 1979 che viene trasmesso dalla RAI il Processo per stupro, il processo per la violenza perpetrata su una ragazza da un gruppo di maschi in una villetta di Nettuno nel 1977. Durante il dibattimento, la madre di uno degli imputati giustifica lo stupro subita dalla minorenne perché il ragazzo “s’è andato a diverti`”; e un avvocato usa questo argomento: “Se questa ragazza fosse stata a casa, se l’avessero tenuta presso il caminetto non le sarebbe successo tutto questo”7. Argomenti analoghi saranno utilizzati nel 2021 da un noto comico diventato uomo politico per minimizzare le violenze di gruppo perpetrate da suo figlio e da un gruppo di amici nei confronti di una studentessa diciannovenne in una villa in Costa Smeralda nel 20198. Di fronte a queste ossessioni che scavalcano il tempo, mi sono chiesto se sia lo sguardo di Deaglio a istituire queste continuità, se sia cioè lo scrittore a selezionare, del passato, le linee di eventi che si riverberano sul presente della scrittura. O se invece queste continuità possiedono una loro oggettività, e in quale misura. La domanda è legittima, e riguarda l’equilibro fra l’obiettività della conoscenza storica e il suo aspetto soggettivo, insito nella necessità del ricorso alla narrazione. È un dibattito epistemologico di fondo che si protrae da diversi anni. Ma nel caso di Patria non ho ancora una risposta sull’equilibrio fra conoscenza documentata e soggettività della narrazione, e la questione resta in questa fase della ricerca ancora aperta. 5 Il caso dell’omicidio di Ahmed Alì Giama, ingegnere e dissidente somalo, verrà evocato anche in un’altra opera di non fiction, la cui prima edizione risale al 1983. Nel resoconto sul suo soggiorno nel carcere romano di Rebibbia, Goliarda Sapienza racconta di avervi incontrato, fra le altre, « uno degli imputati dell’omicidio del somalo bruciato vivo vicino a Piazza Navona » : SAPIENZA Goliarda, L’Università di Rebibbia, [1983] Torino, Einaudi, 2012, p. 77. 6 Il Sole 24 Ore, 4 agosto 2021 7 DEAGLIO Enrico, Patria 1978-2008, cit., p. 63. 8 “Beppe Grillo difende il figlio accusato di stupro : ‘Non ha fatto niente, arestate me’”, Il Messaggero, s.d. https://www.ilmessaggero.it/video/politica/beppe_grillo_figlio_stupro_cosa_e_successo_ultime_notizie5908927.html (consultato l ’11 dicembre 2021). 4 Torniamo ai fatti transazionali. Per quali motivi i vari Paesi vengono evocati? La prima grande impresa transnazionale che appare in Patria è – non ce ne stupiremo – la mafia. Onnipresente: a New York Miami Chicago Detroit, in Canada, in Belgio Germania Francia Svizzera e Venezuela. Qui i siciliani sono emigrati, da almeno un secolo a questa parte, qui alcuni di loro hanno fatto fortuna, chi con il lavoro chi con il crimine, da qui mandano le loro rimesse a casa. Deaglio ricostruisce un giro vorticoso di denaro, perlopiù sotterraneo, che a volte emerge in superficie come nel caso Sindona, il banchiere della mafia e del Vaticano, che scende al Pierre o al Regency Hotel a Manhattan, dove convoca il presidente di Mediobanca Enrico Cuccia e gli annuncia che il curatore fallimentare del Banco Ambrosiano che ha osato andargli contro ha le ore contate. Ambrosoli, il curatore, che Cuccia non avvertirà, verrà ammazzato, davanti casa, a Milano, l’11 luglio del 1979. E nessuna autorità si farà vedere al suo funerale. Intanto, il banco Ambrosiano, prima di fallire, di investimenti e finanziamenti all’estero ne aveva fatti: i suoi beneficiari stanno in tutto il mondo, dal sindacato Solidarnosc nella Polonia di Lech Walesa alle dittature di Massera in Argentina e dei Gorillas in Brasile. E in Argentina fa affari Licio Gelli della P2, che dalla ESMA, la scuola di polizia di Buenos Aires, si faceva fare uno dei suoi tanti passaporti falsi, stampato proprio là, dove si facevano sparire gli oppositori prima di buttarli nell’oceano dagli elicotteri dell’esercito. Una brutta storia, il peggio dell’Italia fuori d’Italia. Più incoraggiante è il ritratto che Deaglio fa del consiglio di fabbrica della FIAT e delle sue aperture al mondo. Certo, nel “consiglione” si discuteva di quanto succedeva in fabbrica (siamo poco prima del grande sciopero che finirà con la marcia dei Quarantamila) ma si guardava anche al di là della frontiera: gli scioperi in Polonia appunto (i membri del consiglio di fabbrica ignoravano all’epoca che fossero finanziati dal Banco Ambrosiano), i colpi di Stato in America Latina (ignoravano che ci fosse lo zampino della P2), la rivoluzione sandinista in Nicaragua (che è poi finita com’è finita). Grandi ideali, grandi speranze, volontà di capire non solo il quotidiano presente, ma il quadro internazionale. E più tardi, e a Deaglio basta evocare i fatti senza orientarne la lettura per far risaltare la loro inattualità, grandi disillusioni della sinistra. La FIAT: l’altra grande impresa italiana che va oltre confine è la Fiat, la sola “America d’Italia”9. Deaglio evoca l’aspetto transnazionale della società, allora ben impiantata in Italia, senza però evocare gli stabilimenti impiantati nel 1966 a Togliattigrad in Russia; si sofferma piuttosto su un caso che, della grande impresa torinese, fornisce una luce particolare: chi salvò la Fiat dal fallimento a metà degli anni ‘70 ? I fondi di investimento americani? No. Le case concorrenti francesi o tedesche? Nemmeno. Il grande investitore straniero che aveva salvato la prima casa automobilistica italiana era il colonnello libico Gheddafi, che comprò il 10% delle azioni della società. Proprio quel colonnello Gheddafi che secondo alcune versioni mai acclarate sarà, il 27 giugno 1980, il bersaglio (mancato) di un missile che colpì invece un aereo Itavia che attraversava il cielo sopra Ustica, facendo 81 vittime. Un intrigo internazionale mai risolto. Quella della FIAT e dell’America è un’altra lunga ossessione che culminerà nel nostro secolo con l’arrivo di Sergio Marchionne alla direzione del gruppo e l’acquisizione di Chrysler nel 2014. Giovanni Agnelli, quando vi entra come Amministratore Delegato, dice che il Lingotto è come “la General Motors di Detroit”, Deaglio la chiama “l’unica America d’Italia”10. E qui cambiamo registro, e dai misteri internazionali siamo passati a un altro aspetto, quello delle 9 DEAGLIO Enrico, Patria 1978-2008, cit., p. 82. Ibid. 10 5 mitologie esterofile dell’Italia degli anni ‘80. Certo, fra queste ci troviamo l’ammirazione sconfinata per l’America dell’industria e del successo che leggiamo nelle parole di Agnelli. Ma ci troviamo anche altro: per Gianna Nannini, che ha scritto un pezzo chiamato America pubblicato in un LP intitolato California, l’America è la libertà, e l’essenza di questa libertà celebrata nella canzone è la libertà sessuale: “fammi l’amore forte sempre più forte e io sono l’America”11. Sempre per stare in tema di cultura giovanile, qual è il romanzo che segna la letteratura italiana del 1981? Per Deaglio è Treno di Panna dell’esordiente Andrea De Carlo che “narra le vicende felici di una ragazzo italiano in California, all’epoca uno dei miti della felicità giovanile”12. Eh sì, l’America non è solo quella della mafia, della CIA e della General Motors, ma anche quella di Andrea De Carlo e Gianna Nannini, le voci della giovane generazione di allora che la celebrano come il mito della libertà e della felicità. E fra l’altro, l’America sarà anche quella degli aiuti alla città di Napoli colpita dal terremoto dell’Irpinia del 23 novembre del 1980. Per restare in tema di riferimenti culturali transnazionali, il più controverso riguarda però Franco Piperno, l’allora leader dell’Autonomia Operaia, che parlando della strage di via Fani in cui persero la vita i cinque uomini della scorta di Aldo Moro, parlò della “terribile bellezza”13 dell’agguato. Citava William Butler Yeats, Piperno. Yeats aveva cantato la ”terrible beauty” dell’assalto al Post Office di Dublino della Pasqua del 1916: un gruppo di patrioti irlandesi aveva assalito l’ufficio postale, la rivolta era fallita e poi era finita in un bagno di sangue14. Certo, a sentir parlare di terribile bellezza della violenza politica a noi italiani vien più in mente lo “zang zang tumb tumb” del Marinetti filofascista che il linguaggio della sinistra. Ma tant’è. Piuttosto, vien da ricordare, ma Deaglio questa volta trascura il riferimento, che l’assalto al Post Office era già stato decostruito e trasformato in una sorta di commedia sprizzante sensualità e comicità da un altro scrittore francese, Raymond Quéneau in Troppo buoni con le donne (1947), pubblicato in Italia solo nel 199215. Ma al giovane fisico Piperno l’ironica decostruzione delle mitologie rivoluzionarie a quanto pare non piaceva molto. Preferiva evidentemente la retorica del sangue e della morte. O forse non leggeva il francese. Arrivo a una prima e parziale conclusione, molto generica, in attesa di un lavoro più sistematico: la Patria di Deaglio è un colabrodo, un passoir. Non c'è fenomeno italiano di peso che non sia collegato a un paese "di fuori". Che sia un fenomeno criminale (l'esportazione della mafia in mezzo mondo), un fenomeno culturale (le mitologie giovanili rivolte alla parte migliore degli Stati Uniti), un fenomeno politico (il ruolo di Mitterrand negli affari italiani, la protezione degli esuli degli anni di piombo, e anche il suo modello vincente di unione delle sinistre): sempre e comunque, fra le determinanti dei fenomeni che segnano l’attualità italiana, c'è una variabile straniera. I mafiosi emigrano in Venezuela e in USA e di là mandano soldi in Sicilia; e i riferimenti obbligati sono i politici stranieri: Mitterrand per la sinistra e Margaret Thatcher per la Destra; le mitologie giovanili (il sesso libero) e industriali (la fabbrica) si nutrono di California, Detroit e New York. 11 ID., Patria 1978-2008, cit., a p. 77 cita L’America di Mauro Paoluzzi e Gianna Nannini, inclusa nell’LP di NANNINI Gianna, California, Milano, Ricordi, 1979. 12 DEAGLIO Enrico, Patria 1978-2000, cit. p. 126. 13 ID., p. 124. 14 YEATS William Butler, Easter, 1916, composta nel settembre 1916 e pubblicata inizialmente su The New Statesman e su The Dial nell’autunno 1920; trad. italiana in Poesie, Milano, Mondadori, 1974, p. 185. 15 QUENEAU Raymond, On est toujours trop bons avec les femmes [1947]. Traduzione italiana, Troppo buoni con le donne, Torino, Einaudi, 1992. 6 E verso l'estero vanno, certo, gli emigrati italiani, di allora come adesso (in Svizzera, Francia, USA), ma anche gli esuli (Francia) e gli uomini d'affari, buoni o cattivi che siano (Ferruzzi in Argentina e USA, Sindona a New York, tornando poi a Palermo via Patrasso). Leggere Patria è il miglior antidoto ai sovranismi attuali. Non perché plauda a un ingenuo mondialismo, e nemmeno perché trasudi esterofilia di maniera, ma perché mostra nei fatti, nelle circostanze, nel modo di vivere delle persone reali, quanto sia sterile e vuoto il chiacchiericcio sulle presunte virtù di una comunità che si vorrebbe chiusa su se stessa, e quanto invece siano intensi e inevitabili - e il più delle volte produttivi di senso oltre che di esiti concreti - gli scambi fra la Patria e li mondo. Per concludere. La storia, fra le tante, che mi piaciuta di più, sia per l’affettuosa ironia della scena riportata che per il suo carattere simbolico di passaggio da un’epoca all’altra, è quella che riguarda gli operai Fiat e le sigarette: reduci da un ciclo di lotte operaie durato un decennio, nei primi anni ‘80 ormai fumano Marlboro, però le nascondono nei pacchetti di MS. 7
https://openalex.org/W3116427975	http://revistascientificas.filo.uba.ar/index.php/RPS/article/download/9695/8506, http://repositorio.filo.uba.ar/bitstream/filodigital/11895/1/uba_ffyl_a_punto%20sur_3_1-8.pdf	es		Geografía, política y resistencias: perspectivas, casos y estrategias de abordaje	Punto Sur	2,020	cc-by	3,692	DOSSIER ISSN 2683-7404 Punto Su.r3 (julio-diciembre, 2020): [1-8] doi: 10.34096/ps.n3.9695 Presentación al Dossier Geografía, política y resistencias: perspectivas, casos y estrategias de abordaje " Mariana Arzeno CONICET - Universidad de Buenos Aires. Facultad de Filosofía y Letras. Instituto de Geografía “Romualdo Ardissone”. Buenos Aires, Argentina. Mónica Farías CONICET - Universidad de Buenos Aires. Facultad de Filosofía y Letras. Instituto de Geografía “Romualdo Ardissone”. Buenos Aires, Argentina. Sam Halvorsen School of Geography. Queen Mary University of London. Reino Unido. Este trabajo está bajo una Licencia Creative Commons Atribución 4.0 Internacional Introducción El presente dossier es resultado de una serie de colaboraciones e intercambios entre investigadores del Grupo de Estudios Geografías Emergentes del Instituto de Geografía de la UBA (GEm-IIGEO) y de la red de trabajo Latin American Geographies in the United Kingdom (LAG-UK), que tuvieron un hito importante en 2018 a partir de la organización de la Mesa Redonda “Geografía y Política en América Latina: espacialidad de y en los procesos políticos”. Dicha Mesa reunió a investigadores de distintas unidades académicas de Latinoamérica y también del Reino Unido, así como a militantes de organizaciones sociales, interesados en debatir respecto de dos interrogantes centrales: ¿qué papel le compete a la geografía en la política de América Latina en la actualidad? ¿Cómo contribuye la disciplina a pensar lo político tanto en su dimensión teórica como en la materialidad de las prácticas cotidianas? Las discusiones giraron en torno a los procesos políticos de resistencias y conflictos que vienen cobrando visibilidad en las últimas décadas en América Latina, en particular aquellos que surgen en torno a tensiones entre actores por la apropiación del espacio y organización de la vida socioespacial. La organización del dossier constituyó una instancia para materializar algunas de las discusiones que se plantearon durante este evento –así como para sumar otras nuevas– con el objetivo de profundizar la discusión teórica y epistemológica relativa a la espacialidad de esos procesos políticos, en cuanto elemento indisociable de los mismos. Otro de los objetivos ha sido el de promover el diálogo entre geógrafxs y otras disciplinas en lo que respecta a su estudio. En las últimas décadas en Argentina, tal como en otros países de la región, hubo un notorio giro espacial en torno al análisis de los procesos de resistencias frente al avance y la consolidación de políticas y prácticas –por parte de privados y también del Estado– excluyentes, desiguales y hasta violentas. Esto se evidencia, por ejemplo, en 1 DOSSIER ISSN 2683-7404 Punto Su.r3 (julio-diciembre, 2020): [1-8] Presentación al Dossier Geografía, política y resistencias: perspectivas, casos y estrategias de abordaje Mariana Arzeno, Mónica Farías, Sam Halvorsen el rol central que ha ocupado el territorio en múltiples trabajos sobre la movilización colectiva, en donde se subraya ya sea la importancia de las identidades, los vínculos sociales y los valores producidos en los barrios urbanos (Grimson, Ferraudi y Segura, 2009; Manzano, 2013; Merklen, 2005; Rodríguez, 2018; Rodríguez y Di Virgilio, 2016; Svampa y Pereyra, 2009, Rossi, 2017), así como las experiencias comunitarias en zonas extractivas y del agronegocio (Domínguez, 2009, 2016; Svampa, 2011; Wahren, 2012). Desde el campo de la geografía algunos trabajos se han centrado específicamente en la dimensión espacial de las movilizaciones colectivas. Por ejemplo, los trabajos de Tobío (2011; 2014) sobre la territorialidad piquetera, los de Sznol (2007) sobre la espacialidad de la protesta social en ámbitos urbanos y Pintos (2004) sobre la espacialidad de las resistencias de movimientos sociales. En este dossier nos proponemos, en primer lugar, subrayar la importancia de seguir pensando el giro espacial en clave geográfica, es decir, desde las orientaciones epistemológicas y con las herramientas conceptuales y metodológicas del campo disciplinar geográfico. En segundo lugar, buscamos llamar la atención acerca de la relevancia de conceptos como el de “lugar” o ideas como “ordenamiento espacial” que no son utilizados con tanta frecuencia como el de “territorio” para dar cuenta de los procesos políticos. Por último, consideramos necesario destacar la variedad de actores sociales en los procesos políticos de resistencia, actores a los que frecuentemente no se los ve actuando e incidiendo en el plano de lo político. Por ello, este dossier busca interrogar y problematizar el recurso del espacio y la constelación de conceptos asociados a él (Haesbaert, 2014) que abundan en el lenguaje cotidiano de las ciencias sociales en la actualidad. Consideramos necesario fomentar un diálogo que examine la importancia de la geografía y el de un enfoque geográfico sobre los fenómenos políticos, que también avance en la discusión teórica sobre el espacio, el territorio y el lugar como conceptos nodales para problematizar la dimensión espacial de los procesos políticos de resistencias. Los seis artículos que conforman este dossier presentan discusiones que no sólo buscan problematizar la dimensión espacial implicada en los procesos que analizan, sino que además proponen, en algunos casos, temas poco trabajados en general desde el campo disciplinar. Cada uno de ellos aporta elementos teóricos y empíricos sobre procesos de resistencia, algunos de los cuales son protagonizados por movimientos sociales/ movimientos socioterritoriales (en el caso de Torres y Halvorsen) y, en otros casos, por actores que no se definen como movimiento, pero que sin embargo presentan grados diversos de organización y/o articulación entre sí y con el Estado (son los casos de Fernández et al., Jurado, Cardozo y Palladino). En estos trabajos el espacio y otras categorías espaciales se vuelven relevantes para comprender la diversidad y complejidad de los procesos de resistencia en el contexto actual, así como para pensar estrategias de lucha y de cambio social. De este modo, lxs autores contribuyen a profundizar la discusión teórica relativa a la espacialidad de (su expresión espacial), así como también la espacialidad en los procesos (en tanto elemento co-constitutivo y por lo tanto productor de estos). Para su abordaje recurren a diversas herramientas analíticas –espacio, orden espacial, territorio, territorialización, lugar– que ponen en juego en el análisis empírico a través de distintas formas de operacionalización. El dossier comienza con el artículo de Fernández Romero, Muñecas, Zanotti y Piccinali, Mapeando el (des)orden espacial: cartografía social en Cabure-í, Misiones. Allí lxs doi: 10.34096/ps.n3.9695 2 DOSSIER ISSN 2683-7404 Punto Su.r3 (julio-diciembre, 2020): [1-8] Presentación al Dossier Geografía, política y resistencias: perspectivas, casos y estrategias de abordaje Mariana Arzeno, Mónica Farías, Sam Halvorsen autores exploran los modos en que la cartografía social puede contribuir a poner de relieve la dimensión espacial en las situaciones donde existen tensiones en torno al uso, ocupación o apropiación del espacio. El artículo analiza una práctica de mapeo colectivo que llevaron a cabo con una cooperativa campesina del norte de la provincia de Misiones en conflicto con los titulares registrales de las tierras que habitan. Recuperando discusiones recientes del campo de las cartografías críticas, esta práctica de mapeo les permitió problematizar las ideas de orden/desorden espacial, así como el rol de las cartografías oficiales en la producción/contestación del ordenamiento espacial. El mapeo colectivo resultó en un mapa en donde la distribución de las tierras ocupadas por las familias respeta los límites perimetrales del loteo del catastro, aunque los usos y apropiaciones son diferentes a los prescriptos. Asumiendo al ordenamiento como una práctica intrínseca a la producción del espacio, en donde la definición de “orden” está siempre en disputa, este caso demuestra que las cartografías oficiales pueden ser apropiadas, interpretadas y usadas creativamente de múltiples formas como parte de órdenes espaciales alternativos. De alguna manera, este artículo deja planteado el interrogante sobre qué sujetos se constituyen como actores políticos y cómo la politicidad de las acciones queda en evidencia cuando recurrimos a un análisis espacial. Un tema que se repite en los tres siguientes artículos. En este sentido, los dos primeros se adentran en discusiones sobre la economía social y solidaria (ESS), un campo de estudios académicos y de debate político que desde hace dos décadas viene cobrando relevancia en Argentina. En su artículo Territorio y lugar: la espacialidad en debate en la Economía Social y Solidaria, Jurado recupera algunas de las principales discusiones teóricas sobre los conceptos “territorio” y “lugar”, y hace un ejercicio analítico para abordar con cada concepto el caso de una organización campesina de la ESS en Mendoza. El autor reafirma la pertinencia del concepto de territorio en el abordaje de las luchas contra los cercamientos y la integración a la lógica del capital de espacios comunitarios por parte de productores campesinos. Sin embargo, también encuentra limitaciones en la capacidad de dar cuenta de otras dimensiones. Así, recurre al concepto de lugar para analizar el entramado de acciones que desarrollan estas experiencias una vez que lograron territorializarse (es decir, controlar un área geográfica que deviene espacio apropiado por la organización, donde llevan adelante sus proyectos productivos). Según el autor, la concepción de lugar que articula las dimensiones de localización o emplazamiento (y su relación con otros lugares), locale (o escenas donde las actividades diarias se desarrollan) y sentido de lugar, resulta muy productiva para explicar desde una perspectiva geográfica el origen y despliegue de circuitos alternativos en contextos de resistencia campesina. Por su parte, Cardozo, en su artículo Políticas de promoción de la Economía Social y Solidaria en la comunidad mocoví Com-Caia de Recreo (Santa Fe, Argentina). La construcción de circuitos cortos de comercialización en el período 2012-2017, focaliza en la territorialización de las políticas para ESS en esa comunidad. El autor recupera algunas discusiones que permiten conceptualizar la dimensión territorial de la implementación de las políticas públicas. Para ello, retomando el planteo de Haesbaert (2018), pone en diálogo la idea de territorio como categoría normativa que enfatiza la mirada estatal sobre el mismo, con la idea del territorio como categoría de la práctica política de los sectores subalternos que son objeto de esas políticas. Este cruce le permite analizar el “encuentro” entre las formas de territorialización de ciertos procesos espaciales que atraviesa la comunidad y las intervenciones de distintos doi: 10.34096/ps.n3.9695 3 DOSSIER ISSN 2683-7404 Punto Su.r3 (julio-diciembre, 2020): [1-8] Presentación al Dossier Geografía, política y resistencias: perspectivas, casos y estrategias de abordaje Mariana Arzeno, Mónica Farías, Sam Halvorsen actores con proyectos que involucran a esta población. Así, la territorialización de la política se manifiesta en nuevas estrategias de organización socioespacial que emergen de la negociación entre los actores. Esto implica negociar las condiciones de posibilidad para la puesta en funcionamiento de circuitos cortos de comercialización campo-ciudad de productos hortícolas. Cardozo, además, se detiene y reflexiona en algunos de los procesos por los que atraviesa una comunidad indígena que logra el acceso a la tierra y que además avanza en el desarrollo de procesos productivos para su reproducción. Se trata, en definitiva, de comunidades cuya adscripción territorial es producto de un complejo proceso de desplazamientos y de luchas, algo que es retomado en el siguiente artículo. El trabajo de Palladino, Territorio(s) e indigeneidad(es): aportes a partir del trabajo de campo con comunidades comechingonas cordobesas, focaliza su análisis en la forma en que los procesos de re-emergencia comunal de los comechingones en la provincia de Córdoba construye la adscripción territorial apelando a distintas formas de identificación con el territorio. Para ello el autor toma dos casos de estudio en donde discute el papel del territorio en los procesos de comunalización desde la perspectiva relacional e integradora propuesta por el geógrafo Rogerio Haesbaert, lo que le permite abordar los procesos de territorialización indígena desde la movilidad e identificación territorial con distintos sitios. Así, el autor se distancia de los enfoques que identifican las reivindicaciones indígenas con territorialidades sustentadas en la continuidad espacial, es decir,en la fijación y la rigidez en lo que respecta al vínculo identidad-territorio. Esto da lugar a la posibilidad de problematizar dichas territorialidades como móviles y fluidas, y asociadas a los procesos de desagregación y fractura a las que fueron sometidas durante la colonia. El autor muestra cómo los procesos de des-territorialización y re-territorialización se vuelven elementos clave en la reconstrucción de narrativas y memoria territorial en la que se basan esos procesos de reemergencias y comunalización. Finalmente, cierran el dossier dos artículos que ponen el foco en los movimientos socioterritoriales, aportando distintas discusiones que permiten, por un lado, operacionalizar el concepto de manera tal de abordar la dimensión espacial intrínseca a ellos y, por otro, complejizar el análisis relativo a sus vínculos con el Estado en la producción del territorio de los movimientos. En el artículo Movimientos sociales e institucionalización: la especificidad de los movimientos socioterritoriales, Torres presenta un método analítico para entender las relaciones entre movimientos sociales e instituciones, recurriendo a la categoría de movimiento socioterritorial y recuperando las discusiones teóricas planteadas por Fernandes en torno a sus particularidades (2005). La autora se centra en la dimensión de la institucionalización de las formas organizacionales, en y a través del territorio, característica de los movimientos (Halvorsen, Fernandes y Torres, 2019); es decir, la producción de instituciones que garanticen las relaciones de poder que sustentan esa apropiación del espacio convertido en el territorio del movimiento. Para ello utiliza el caso de la Organización Barrial Túpac Amaru de Jujuy, que por medio de la ocupación de tierras logró construir un barrio y producir instituciones laborales, educativas, de salud y culturales, entre otras. El control del territorio habilita relaciones sociales, reglas y prácticas que regulan la vida cotidiana desde lógicas distintas a la mercantil. De este modo, para la autora, los movimientos socioterritoriales dejan en evidencia la importancia de la dimensión espacial, específicamente la producción y control de territorios para la creación de instituciones y nuevas ciudadanías territorializadas. doi: 10.34096/ps.n3.9695 4 DOSSIER Presentación al Dossier Geografía, política y resistencias: perspectivas, casos y estrategias de abordaje Mariana Arzeno, Mónica Farías, Sam Halvorsen Halvorsen también recurre al concepto de movimiento socioterritorial en su artículo El Territorio en disputa: estrategias políticas y movimientos socioterritoriales, para discutir el concepto “territorio”. El autor pone en diálogo las discusiones anglosajonas sobre el territorio –como el ámbito de ejercicio del poder estatal– y las latinoamericanas –en donde abundan las referencias al territorio como ámbito de poder emancipatorio para los movimientos–. Apoyándose en tres tipos de movimiento de América Latina cuyo objetivo es el de la apropiación de un espacio determinado, Halvorsen sugiere que lejos de ser algo estable, el territorio es un proceso de apropiación siempre aconteciendo. Propone entonces una definición abierta de territorio que resulta de la apropiación del espacio a través de estrategias políticas intrincadas y superpuestas. Esto supone considerar al menos tres dimensiones: las configuraciones territoriales hegemónicas, pasadas y presentes, en donde tiene un rol predominante el Estado; la coexistencia de diversas apropiaciones que generan múltiples territorios; y el cruce de diversas prácticas e ideas que materializan relaciones de poder en el espacio. El autor recupera estas dimensiones en el análisis de los casos para detenerse en las distintas negociaciones y puntos de tensión entre éstos y las estrategias de control del Estado. Esto le permite alejarse de las miradas romantizadas sobre los movimientos que enfatizan mayormente su poder emancipatorio, al tiempo que deja en evidencia la potencialidad de los cruces epistemológicos provenientes de distintas tradiciones académicas. En su conjunto el dossier sugiere algunas posibles líneas de trabajo para continuar con el debate sobre la espacialidad de los procesos políticos y el potencial de la geografía para dar cuenta de ellos. Creemos necesario continuar y extender las discusiones acerca de la/s espacialidad/es de las resistencias presentes en la región. Dado el fuerte interés que el concepto de territorio ha suscitado en las ciencias sociales en América Latina en los últimos años –al punto de plantearse un “giro territorial” más que espacial en la región (Haesbaert, 2009; Souza, 2009)–, consideramos particularmente importante ahondar en otras espacialidades de la política y en los modos en los que las mismas se relacionan. Pensar los procesos políticos a partir de los conceptos de lugar, escala, red, paisaje (ej. Jessop et al., 2008) no sólo permite enriquecer su discusión teórica, sino que además habilita nuevos campos de acción para la práctica política. Además, creemos necesario dar lugar a más y mayores diálogos interdisciplinarios en lo que respecta a la producción de saberes sobre la espacialidad de y en los procesos políticos. Para ello será necesario tener presente la construcción de los bordes disciplinares y las “regiones ontológicas” (Lander, 2000) de las disciplinas implicadas en el “giro espacial”, de modo de alentar su constante revisión y enriquecer así el intercambio. Por último, resultaría interesante considerar la importancia de los lugares y los ámbitos donde estos saberes emergen, se manifiestan y circulan. Tener en cuenta la situacionalidad de los saberes anclados en contextos geohistóricos específicos –Argentina, América Latina– necesariamente requeriría repensar los modos en los que se traducen y comunican esos saberes. La foto de la portada fue tomada en el año 2016 en la localidad de Tilcara, situada en la Quebrada de Humahuaca, provincia de Jujuy. Se trata de un graffiti que expresa una serie de resistencias que se vienen dando en ese lugar y en la Quebrada en general: resistencias a la minería a cielo abierto, de uranio y otros minerales, resistencias a la valorización inmobiliaria que se deriva del turismo. Como dijo Rodolfo Walsh, las paredes son la imprenta de los pueblos, de aquellxs que, sin tener el poder para llegar a los grandes medios de comunicación, buscan modos de comunicarse, de hacer oír su voz ISSN 2683-7404 Punto Su.r3 (julio-diciembre, 2020): [1-8] doi: 10.34096/ps.n3.9695 5 DOSSIER Presentación al Dossier Geografía, política y resistencias: perspectivas, casos y estrategias de abordaje Mariana Arzeno, Mónica Farías, Sam Halvorsen y visibilizar sus reclamos. Podemos pensar a este graffiti como una práctica espacial de resistencia (Souza, 2013), un modo de visibilizar y rectificar una situación de injusticia socioespacial. Nos pareció entonces apropiado, sumar esta imagen a las geografías de las resistencias presentes en este dossier. ISSN 2683-7404 Punto Su.r3 (julio-diciembre, 2020): [1-8] doi: 10.34096/ps.n3.9695 6 DOSSIER Presentación al Dossier Geografía, política y resistencias: perspectivas, casos y estrategias de abordaje Mariana Arzeno, Mónica Farías, Sam Halvorsen # Bibliografía » Domínguez, D. (2009). La lucha por la tierra en Argentina en los albores del Siglo XXI. La recreación del campesinado y de los pueblos originarios. Tesis de doctorado, Facultad de Ciencias Sociales, Universidad de Buenos Aires. » Domínguez, D. (2016). Territorialidades campesinas entre lo heterónomo y lo disidente: formas de gestión de la producción y tenencia de la tierra en el campo argentino. Política & Trabalho. Revista de Ciências Sociais, 45, 67-84. » Fernandes, B. M. (2005). Movimentos Socioterritoriais e Movimentos Socioespaciais: Contribuição Teorica Para Uma Leitura Geografica Dos Movimentos Sociais. 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Pobres ciudadanos: Las clases populares en la era democrática (Argentina 1983–2003). Buenos Aires: Editorial Gorla. » Pintos, P. (2004). La espacialidad de la resistencia social: entre la visibilidad en las calles y la acción en el territorio. Notas sobre la espacialidad piquetera en la Argentina reciente. Reflexiones geográficas, 11, 45-67. ISSN 2683-7404 Punto Su.r3 (julio-diciembre, 2020): [1-8] doi: 10.34096/ps.n3.9695 7 DOSSIER ISSN 2683-7404 Punto Su.r3 (julio-diciembre, 2020): [1-8] Presentación al Dossier Geografía, política y resistencias: perspectivas, casos y estrategias de abordaje Mariana Arzeno, Mónica Farías, Sam Halvorsen » Rodríguez, M. C. (2018). Género, espacialidad y urbanismo autogestionario. Algunas claves para su comprensión y debate. Revista Vivienda y Ciudad, 5, 67-79. » Rodríguez, M. C. y Di Virgilio, M. (2016). A city for all? Public policy and resistance to gentrification in the southern neighborhoods of Buenos Aires. 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https://openalex.org/W3022202828	https://periodicos.uem.br/ojs/index.php/ActaSciEduc/article/download/44870/751375149883	Portuguese	null	Teachers’ learning at schools: developing pedagogical capacity for curriculum design trhough {co-teaching \| cogenerative dialogue}	Acta Scientiarum. Education	2,020	cc-by	10,472	Aprendizagens docentes na escola: desenvolvendo a capacidade pedagógica para desenvolvimento curricular por meio do (co-ensino\| diálogo cogerativo) RESUMO. Este artigo objetiva discutir o desenvolvimento de uma experiência em que professores e alunos se engajam no design do currículo escolar em uma escola pública. Está baseado no referencial teórico da perspectiva sócio-historico-cultural. Os dados foram coletados em um projeto durante dois anos por meio da gravação das interações dos participantes e escrita de diários e analisados por meio da análise crítica do discurso (transitividade) e análise da conversação. Os resultados desse estudo demonstram que no processo de ter a oportunidade de interagir em encontros de co-planejamento na construção do currículo novos atitudes surgiram. Além disso, demonstra que participar em espaços mais democráticos e serem capazes de contribuir para desenvolver o currículo durante sua prática de ensino tem efeitos no modo como participantes percebem os alunos do ensino médio. Conclui-se eu o desenvolvimento da capacidade pedagógica dos alunos para o currículo influencia o grau no qual professores se engajam na transformação da escola para criar oportunidades para seus alunos. Palavras-chave: capacidade pedagógica; aprendizagem; escolas; formação de professores Michele Salles El Kadri Michele Salles El Kadri Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Pr-445 Km 380, Cx. Postal 10.011, 86057-970, Londrina, Paraná, Brasil. E-mail: mielkadri@hotmail.com ABSTRACT. This article aims at discussing development in an experience of engaging teachers and students in the design of the curriculum at a public school. It is grounded on a socio-historical perspective of learning and in (coteaching \| cogenerative dialogue) as a theorethical-methodological approach. Data was gathered in a two-year project through interactions meetings at schools and analyzed through critical discourse analysis (transitivity) and conversational analysis. The results of the study point out that having had the opportunity to interact in co-plannings and redesigning the curriculum, new attitudes emerged. Furthermore, participating in more democratic spaces and being able to redesign their own curricula during teaching practicum had effects on how the participants perceive the participation of high school students. It is concluded that developing teachers' pedagogical capacity for curriculum design may influence the degree to which teachers engage in a principled, reform-oriented analysis when creating learning opportunities for students. Keywords: pedagogical capacity; learning; schools; teacher education. Acta Scientiarum http://periodicos.uem.br/ojs/acta ISSN on-line: 2178-5201 Doi: 10.4025/actascieduc.v42i1.44870 Acta Scientiarum http://periodicos.uem.br/ojs/acta ISSN on-line: 2178-5201 Doi: 10.4025/actascieduc.v42i1.44870 http://periodicos.uem.br/ojs/acta ISSN on-line: 2178-5201 Doi: 10.4025/actascieduc.v42i1.44870 TEACHERS' FORMATION AND PUBLIC POLICY Introduction Acting as ‘curriculum makers’ is particularly important for novice teachers because it (a) influences the degree to which they engage in a principled, reform-oriented analysis when creating learning opportunities for students (Beyer & Davis, 2012); (b) emphasizes the agency of teachers in curricular processes and positions teachers as agents of change (Craig & Ross, 2008) and (c) allows a deeper understanding of the ways in which teachers exercise control and agency in their curricular work (Harris-Hart, 2009). More importantly, wherein teachers act as curriculum makers, there are close connections with leadership and this implies a strong relationship between the structures, norms, and patterns of interactions in the working conditions of schools (Grimett & Chinnery, 2009). While in the literature of teacher education the discourse of involving teachers in the production of the curriculum is widely recognized through the ‘image of teachers as curriculum makers’ (Clandinin & Conelly, 1992; Fullan, 2003; Craig & Ross, 2008), in practice, it goes far beyond this recognition. Teachers generally have very few opportunities to engage in the redesign of the curriculum. In the teaching practicum, it rarely occurs as most teacher education programs fail to provide enough resources (like context, time and conditions for schoolteacher participation) for such experiences and continue to emphasize technical rationalist conceptions of the curriculum. Some studies have acknowledged the importance of developing teachers' pedagogical capacity for curriculum design by affirming that this is an essential aspect in teaching practice and it is common for novice teachers to encounter many difficulties when attempting to develop this capacity (Davis, 2006; Nicol & Crespo, 2006; Grossman & Thompson, 2008; Beyer & Davis, 2012). Despite this, few studies have focused on the attempt to develop this pedagogical capacity when novice teachers are in the field, more specifically, when they are involved in their teaching practicum. Developing teachers' pedagogical capacity for curriculum design may influence the degree to which teachers engage in a principled, reform-oriented analysis when creating learning opportunities for students (Beyer & Davis, 2012). In an experience of engaging teachers in the design of the curriculum in a teaching methods course, Beyer and Davis (2012), for example, highlight that that most of the novice teachers also experienced an increase in confidence by the end of the course, with regard to the designing of lessons and saw themselves as curriculum designers during their first year of teaching. El Kadri Received on October 5, 2018. Accepted on May 10, 2019 Received on October 5, 2018. Accepted on May 10, 2019 Introduction They concluded that it is paramount that novice teachers are afforded opportunities to develop their pedagogical design capacity. Beyer and Davis (2009) also note that developing teachers' pedagogical design capacity includes developing their ability to act upon these personal resources while interacting with particular material resources when creating powerful learning opportunities for students (Ball & Cohen, 1999; Remillard, 2005). For them, in fostering their pedagogical design capacity, teachers must learn how to negotiate the affordances and constraints of particular curricular features while taking into consideration their own understandings, instructional goals, and classroom needs. It is, therefore, an important area in which novice teachers need support (Beyer & Davis, 2012). Acknowledging the importance of developing teachers' pedagogical capacity for curriculum design, this article reports an experience of the development of pedagogical capacity through a co-planning. I demonstrate that taking part in collaborative environments, such as co-planning, is one of the ways of acting that allows stakeholders to engage in curriculum production. Participating in more democratic spaces and being able to redesign their own curricula during teaching practicum has effects on how novice teachers perceive the participation of high school students. Thus, I argue that having had the opportunity to interact in co-plannings and redesigning the curriculum, new attitudes emerges at school. Aprendizaje de profesores en la escuela: desarrollando la capacidad pedagógica para el diseño del currículo escolar por el (co-ensino\| diálogo cogerativo) RESUMEN. Este artículo objetiva discutir el desarrollo de una experiencia en que profesores y alumnos se comprometen en el diseño del currículo escolar en una escuela pública. Está basado en el referencial teórico de la perspectiva sociocultural-cultural. Los datos fueron colectados en un proyecto durante dos años por medio de la grabación de las interacciones de los participantes y escritura de diarios y analizados por medio del análisis crítico del discurso (transitividad) y análisis de la conversación. Los resultados de este estudio demuestran que en el proceso de tener la oportunidad de interactuar en encuentros de co-planificación en la construcción del currículo nuevas actitudes fueron creadas. Además, demuestra que participar en espacios más democráticos y ser capaces de contribuir a desarrollar el currículo durante su práctica de enseñanza tiene efectos en el modo como participantes perciben a los alunos. Se concluye que el desarrollo de la capacidad pedagógica de los alumnos para el currículo influye en el grado en que los profesores se comprometen en la transformación de la escuela para crear oportunidades para sus alumnos. Palabras-clave: capacidad pedagógica; aprendizaje; escuelas; formación de profesores. Acta Sci. Educ., v. 42, e44870, 2020 Page 2 of 13 The context This article is part of a doctoral dissertation which aimed at understanding the transformation of teacher identities during their teaching practicum organized within PIBID through (coteaching \| cogenerative dialogue) as a methodological approach (El Kadri, 2014). Teacher identities were investigated through new teachers’ ways of acting, interacting, representing and being and data sources include audio-records of (coteaching \| cogenerative dialogue) meetings, lectures by novice teachers given in academic seminars, papers, journals and reports. The analysis carried out was a longitudinal one focusing on two particular new Acta Sci. Educ., v. 42, e44870, 2020 Desenvolvendo capacidade pedagógica para design curricular Page 3 of 13 Page 3 of 13 teachers during two years. Thus, this study is also part of the Brazilian Institutional Bursary Program for the Initiation to Teaching (henceforth PIBID1). PIBID is a program designed by CAPES to enhance teacher education and teaching in public schools by offering bursary to teachers. teachers during two years. Thus, this study is also part of the Brazilian Institutional Bursary Program for the Initiation to Teaching (henceforth PIBID1). PIBID is a program designed by CAPES to enhance teacher education and teaching in public schools by offering bursary to teachers. Our group was comprised of 1 teacher educator and researcher (me), one schoolteacher (Alice) and 12 novice teachers. In the beginning, all of the novice teachers were the third and fourth year of the undergraduate course, which means they were all required to engage in the teaching practicum. In this article, I exemplify the findings by drawing on the data involving this group. The research praxis lasted from August 2009 to December 2011. We met regularly during this time in co- planning, (coteaching \| cogenerative dialogue) classes, cogenerative dialogues, schools meetings and supervisions. Data, in this article is analyzed through (coteaching \| cogenerative dialogue) heuristic (Roth, 2002). During the two-years of working in the project, I recorded all forms of engagement in teaching-learning tasks on the part of the PIBID (18 new teachers during the two-year program). All the encounters were recorded and fully transcribed. The new teachers’ written assignments were assembled into portfolios that entered the database. The data sources also include talks in seminars, reports, transcribed cogenerative dialoguing session, and recordings of individual supervision sessions. The database now consists of more than 8,000 pages of transcripts produced over the two years. 1 The program aims at promoting innovative teaching practices in public schools and the integration of theory and practice by inserting novice teachers in public schools and by positioning the schoolteachers as co-supervisors it provides practitioners with incentives to take a central role in teacher education. 2 The ‘representation of social actors’, according to Fairclough (2003) involves a number of choices. For example, it is important to know if (a) social actors are included or excluded, (b) they are realized as a pronoun or as a noun, (c) they are realized as a Participant in the clause (e.g. actor, affected), or if (d) they are the Actors in processes or the affects or beneficiaries of the processes, for example. And it is precisely all these distinctions available in the theory that matter. 3 The ‘Transitivity systems’ are significant categories for analyzing representation and identity, that is, for analyzing how social actors position themselves and others in discourse because it generally refers to how meaning is represented in the clause. ‘Transitivity’ is part of the ideational function of language and is a fundamental and powerful semantic concept thus making it an essential tool in the analysis of representation (Matu, 2008). The context Our reflection on this data and other analysis can be seen in several other papers (El Kadri et al., 2018; El Kadri, 2018). In this paper, however, my analysis is based on some interactions and some extracts from novice teachers’ journals and conference presentations. The transcripts of cogenerative dialogues are analyzed through categories extracted from the Critical Discourse Analysis (Fairclough, 2003; Van Leeuwen, 2008), the heuristic of (coteaching \| cogenerative dialogue) and interaction conversational studies (Cheyne & Tarulli, 1999). The extracts of journals are analyzed through the Representation of social actors theory2 (Fairclough, 2003; Van Leeuween, 2008) and the transitivity systems3 (Halliday & Matthiessen, 2004). The project proposed by our Language teaching education program entitled, ‘Collaborative Teacher Education and curriculum innovation in English teaching’, aimed at addressing two main issues: the need to increase the quality of teacher education courses and to innovate the English teaching curriculum. This proposal was based on contemporary trends that the relationship between schools and universities is the core of teacher education models in which theory and practice are reconstructed in a dialectical way through collaboration. Based on these tenets, the PIBID-English project proposed to develop actions to place the novice teachers in the school context and to construct (collectively) innovative pedagogical practices to then be implemented collaboratively. In this article, I report the experience of redesigning the curriculum and developing teachers’ pedagogical capacity. Co-planning meeting, October 10th, 2011 Fragment 10 01 An: oh...what is the objective of the plan? Reading the ads critically? 02 Le: sometimes it it not just that... I want them to read the ad, and understand why it is like that, what the ad wants to reach… Page 4 of 13 generating an understanding of praxis (Roth, Lawless, & Tobin, 2000), which, in my view, cannot be misunderstood power-relations-free. Thus, the main theoretical underpinnings of such encounters is the belief that each participant brings unique understanding and experiences to the field of activity while experiencing and interacting with the field in different ways (Wassell & Lavan, 2009). Furthermore, the meetings conjure an emancipatory feature that works against the control of interests and ruling relations that characterize most current educational systems (Roth, 2002). Roth et al. (2000) explain that coteaching and the associated Cogenerative dialogue evolved because they found that such processes led to understandings that allowed them to generate new possibilities for action and thereby engage in expansive learning and in the transformation of praxis. They argue that they have realized that conceiving (coteaching \| cogenerative dialogue) as a dialectical unit offers far more benefits than conceiving them separately. A considerable amount of literature has been published on the results and benefits of (coteaching \| cogenerative dialogue). These studies are not solely interpreted as a methodology for the improvement of teacher education by providing new opportunities for learning to teach and for the enhancement of student learning, but also as means to overcome the theory/practice gap. The main benefits of implementing (coteaching \| cogenerative dialogue), according to the studies, are its impacts on the teaching and learning experiences of all participants, as it increases opportunities for actions that would otherwise not occur. For instance, several researchers have noted that a very important type of learning occurs from pairing up both activities: the creation of resources that provide teachers (not only novice teachers but all the participants involved) with opportunities for the development of agency (Elden & Levin, 1991; Roth et al., 2000; Stith & Roth, 2006, 2010). Eick, Ware, and Williams (2003) also argue that coteaching provides shared experiences for student teaching supervisors, methods instructors, schoolteachers and novice teachers in order to engage in theoretical discussions and improve their practice in the classroom. Others have highlighted that the process makes merging the usually separate activities of professional development, supervision, evaluation and research viable (Roth &Tobin, 2001a). In doing so, the process sets a pretense for ongoing evaluation in which the focus is placed on teaching with the intent of enhancing the learning of the students (Roth & Tobin, 2001) (Coteaching \| cogenerative dialogue) and co-planning as models for teacher education Cogenerative dialogue is characterized by encounters in which students and teachers participate in conversations regarding praxis that focuses on teaching and learning with the purpose to further develop existing understandings of the learning/teaching situation in order to build local theory and thereby increase the action potential of all participants (Roth, 2002). Cogenerative dialogue has seen increased usage among many researchers with the majority emphasizing the premise that students and teachers should be participating in conversations about praxis with a focus on teaching and learning. Although it may be argued that this is equivalent to reflective sessions, the difference is within the premise that in order to participate in cogenerative dialogue, participants are required to have shared experience in teaching (coteaching). One of the most significant features of cogenerative dialogue meetings is the ability for such meetings to provide space for all participants to utilize a more equitable approach towards making sense of and Acta Sci. Educ., v. 42, e44870, 2020 Page 4 of 13 El Kadri Page 5 of 13 03 An: yah, but is it what I wanted to say, look... strategies of knowledge…. Ok...but let’s do it right: objective: so that the students read the ads critically and understand their structure and the persuading (I don’t know how to say in English) strategies. 03 An: yah, but is it what I wanted to say, look... strategies of knowledge…. Ok...but let’s do it right: objective: so that the students read the ads critically and understand their structure and the persuading (I don’t know how to say in English) strategies. 04 Le: Ah, this is the principal, right... 05 Ma: learn the verbs in the imperative... 05 Ma: learn the verbs in the imperative... 06 Le: apprehension of vocabulary too.... 07 An: Done, it’s great… Content… 08 Ma: oh, I don’t understand 09 Le: ads and hedlines of magazines... (explaining to Mari) 10 An: Aren’t they the same? The headline is in the ad, right.... so they understand how it works…. So, we can use of all of them, right? 10 An: Aren’t they the same? The headline is in the ad, right.... so they understand how it works…. So, we can use of all of them, right? 11 Ma: no, we have to choose which one Will.... 12 Le: I liked this one here... Well, we have to think that there are boys and girls, right… if a boy ge ed this one here... Well, we have to think that there are boys and girls, right… if a boy get this one to do e, he will not get motivated… liked this one here... Well, we have to think that there are boys and girls, right… if a boy get this one to d for example, he will not get motivated… 13 An: Ah ok.... 14 Mi: yeah… This one is nice. In this excerpt, Aline initiates asking about the objective of the class they are planning (turn 01). However, she not only poses a question about it but also suggests what it should be. Leandro is the one who answers her (turn 02) and Aline elaborates on her ideas as a way to help him understand what she meant. As Aline expands (turn 03), Leandro agrees with her (turn 04). Then, Mari and Leandro add another topic to be included in the objective of the class (turn 05 and 06). Page 5 of 13 Aline then evaluates their utterance by stating, ‘done, its great’. Mari elicits explanation and Leandro provides her with this (turns 08 and 09). Aline questions them (turns 10), Mari disagrees, explaining it to her (turn 11) and Leandro offers a personal opinion (turn 12). What this episode demonstrates is that a very common way of acting in co-planning is by (a) alternating leadership and (b) alternating the position of the competent peer. Here, Aline leads the meeting in which the objective was planning classes. In these meetings, power appeared more balanced even as participants move themselves in the position of leading and listening. We can see Aline dominating the scene, making her intentions for the lesson while at the same time inviting her co-teachers to contribute to the lesson. Aline places herself in the position of someone who poses questions with the expectation of receiving answers, indicating her position as being one with power (Young & Fitzgerald, 2007). She also exerts her power even further by evaluating her coteachers (turn 07). She coordinates the discussion, elaborates on ideas, initiates dialogue and demonstrates willingness to participate. There is also an uneven distribution of questions and answers, if compared to Mari’s participation, but new ways of acting for Aline are evident when compared to the excerpts analyzed in the first section. This is important for understanding Aline’s ways of acting and interacting because the symmetrical discussion is not only visible at the level of content. What is being taught-learned are the positions and dispositions of how participants place themselves in the discussion as well (they contribute, argue, disagree, agree, exemplify, etc.). These ways of acting have a neat fit with the subjectivities created and their ways of representing and being. This episode reveals an important way of acting because as teachers redesigned a new curriculum, they also redesigned their identities as accountable actors. That is, by acting, leading, coordinating, agreeing, disagreeing, controlling and also exerting power, Aline, specifically, creates her subjectivity as an accountable actor in this context. As the novice teachers are able to assume the position of authorship of the curriculum as a result of an activity system that empowers collective action, new possibilities become available for legitimate participation at the school. Redesigning the curriculum through (coteaching \| cogenerative dialogue) Co-planning meetings is a model of (coteaching \|cogenerative dialogue). They occurred once a week on Monday morning with the purpose of planning the classes for the week; adjustments in the planning of classes were made via e-mail interactions or informal meetings among coteachers. In such meetings, coteachers (a) discussed the purpose of the lesson in accordance with national guidelines and students’ feedback, (b) reflected upon lessons and the objectives and goals for teaching English and (c) decided on resources, strategies, time and issues regarding the development of the lesson. In these meetings, the cohort planned the English teaching curriculum as a whole along with all individual lessons. Our implementation of co-planning therefore coincides with the model employed by Scatlebury and collaborators in the sense that they provided the setting for another type of co-generative dialogue (Scantlebury, Gallo-Fox, & Wassel, 2008). Distinctively, our implementation included the teacher educator in the co-planning meetings as I thought it would be an important site for sharing contribution. Based on the needs identified while coteaching, the group felt the need to produce situated materials in order to deal with the (local) needs of ‘these’ students. The schoolteacher was a substantive proponent for this idea, emphasizing that the person “[w]ho produces is the one who knows more what it is about” (Schoolteacher, oral presentation) . The excerpt below is one example of how cohort members enacted ‘shared responsibility’ for the production of the curriculum during cogenerative dialogue meetings in which they planned lessons and produced materials to be used in the class. The excerpt below is also an example of what Aline defined as ‘coming together’ when preparing classes. Acta Sci. Educ., v. 42, e44870, 2020 Desenvolvendo capacidade pedagógica para design curricular Page 5 of 13 Page 5 of 13 Page 6 of 13 05 An: Me too… say it again, Evelyn! 06 Ev: uhum….How do I write a message to share with my friends? g y (during the questions, there's a parallel explanation of how to it for Michele and Alice because they do not know how) 07 Ev: where do you click to find a friend? Where do you click to make comments on photos? We could ask that…. 08 Mi: It is the scrap? p 09 Ev: no… where do you click to see the recent updates? And there is also this option, I did not put it here,but there is private, public, friends… 09 Ev: no… where do you click to see the recent updates? And there is also this option, I did not put it here,but there is private, public, friends… 10 Mi: And can you select what each person can see…? 11 An: can we? And what is the scrap? 12 Ev: they are the messages. Here, Evelyn assumes the role of the competent peer and teaches Alice, myself (turns 02, 04, 08 and 10) and Aline (turns 05 and 11) how to use Facebook because at that time, we were not yet members. In this episode, Evelyn is in the know and guides the development of the construction of the lesson as she dominates the topic of social networks (turns 01, 02, 06, 07, 09 and 12). Although her institutional position is of a novice teacher, here, she assumes her role in the collective responsibility and contributes to the design of the curriculum in ways that neither the schoolteacher nor the teacher educator were able to in this situation. Here, Evelyn exerted leadership: she controls the turns and Aline assumes a more passive role in this interaction, accepting the shared power. What is possible to argue here is that because of the repositioning of roles – everybody teaching – there is a higher probability of symmetry in the relations, which means power is thus, shared. Therefore, if knowledge is power (Fairclough, 2003), in these situations in which knowledge is distributed as a result of the confluence of institutional positions, there is a higher probability of shared power. Page 5 of 13 These spaces were a unique locus for decision making and conceiving a sense of authorship and freedom in the process and in the constructing the self, transforming the participants into accountable actors of teaching responsible not only for their own learning but also each other's. This is so because “[…] getting your ideas through and, especially, seeing them having practical implications, give the learner a true sense of authority and agency” (Niestz, 2010, p. 816). In the episode above, she also acts as a curriculum maker. Along with this, by being involved in the redesign of the curriculum, teachers had the opportunity for development beyond their institutionalized positions, which means novice teachers, schoolteachers and teacher educators alternated the position of the competent peer, and therefore, all the participants enacted the role of teachers and learners (Roth & Radford, 2010) in praxis. The episode below – extracted from a co-planning meeting in which we were using Acta Sci. Educ., v. 42, e44870, 2020 El Kadri Page 6 of 13 social networks to teach English – exemplifies that through the process of promoting collective curriculum leadership, Alice, Aline and I also learned from novice teachers. Here, Aline displays a way of acting that differs from the previous episode in which she coordinates the discussion. Here, she alternates the position of the competent peer: she listens while Evelyn leads the discussion on how to use Facebook. social networks to teach English – exemplifies that through the process of promoting collective curriculum leadership, Alice, Aline and I also learned from novice teachers. Here, Aline displays a way of acting that differs from the previous episode in which she coordinates the discussion. Here, she alternates the position of the competent peer: she listens while Evelyn leads the discussion on how to use Facebook. Co-planning meeting, September 22nd, 2010 Fragment 10 01 Ev: then this is an specific thing so they talk about it, right…. How do I write a testimonial to a friend? Then he know… How do I download videos in my profile?? Because you can put video too… 02 Mi: but how do I do it? 03 Ev: so, it is written here, it is just look at it..(laughs) 04 Mi: (laughing) calm down, people.. I am learning too. 05 An: Me too… say it again, Evelyn! 06 Ev: uhum….How do I write a message to share with my friends? (during the questions, there's a parallel explanation of how to it for Michele and Alice because they do not know how) 07 Ev: where do you click to find a friend? Where do you click to make comments on photos? We could ask that…. 08 Mi: It is the scrap? 09 Ev: no… where do you click to see the recent updates? And there is also this option, I did not put it here,but there is private, public, friends… 10 Mi: And can you select what each person can see…? 11 An: can we? And what is the scrap? 12 Ev: they are the messages. 01 Ev: then this is an specific thing so they talk about it, right…. How do I write a testimonial to a friend? know… How do I download videos in my profile?? Because you can put video too… 3 Ev: so, it is written here, it is just look at it..(laughs) 04 Mi: (laughing) calm down, people.. I am learning too. Evaluating the enactement of the curriculum and the participation in school collective meetings: teachers attitudes towards the experience Teachers’ confidence in the redesigned curriculum steadily increased until the end of the second year. Although tensions and contradictions permeated the whole process of construction and enactment of a new curriculum, teachers involved considered it a positive experience and demonstrated a great deal of satisfaction. Paula’s writing in her final research paper exhibit this feeling of ‘getting proof’ that that they could actually transform the curriculum in a public school in ways they initially thought impossible. She writes: These doubts were only lessened after 2 years teaching, when we had the opportunity to confirm that our objectives and activities done in class were exactly what’ the national exam for high school (ENEM) asked for. (Paula, final research paper). Here, she represents through relational processes, which represent states of mind or states of being, how she evaluates the experience: exhibit that the doubts were in a relation of being diminishing as new teachers were able to confirm that the ‘objective and activities done’ were in a relation of being in accordance or similar to the requirements of National guidelines: These doubts were’ (intensive relational process) only lessened (state of being) after 2 years teaching (circumstance)” and “[…] our objectives and activities done in class were (intensive relational process) exactly (state of being) what ENEM asked for […] (Paula, final research paper) These doubts were’ (intensive relational process) only lessened (state of being) after 2 years teaching (circumstance)” and “[…] our objectives and activities done in class were (intensive relational process) exactly (state of being) what ENEM asked for […] (Paula, final research paper) Constructing together a list of criteria to analyze their own curriculum was also important to the emergent sense of achievement. The coteachers felt responsible for the curriculum and its evaluation, as Mari stated during a public discussion: ‘We are evaluating what we have done’. ‘We also discuss’ a lot our own practices, what has worked or not. ‘We implemented’ the curriculum, that was ‘ours, we created and implemented’ at school and each class had a curriculum that fit their needs (Mari, in a teaching conference) . The sense of responsibility and engagement as new ways of acting and interacting at school is visible in this excerpt of Mari’s speech in a teaching conference. Page 6 of 13 Both excerpts analyzed here exhibit how (a) different novice teachers act, assuming the role of the competent peer by developing authorship through active participation and (b) how Aline also developed shared responsibility for the curriculum production knowing how and when to participate by either leading or listening. In this section, I attempted to delineate on how teachers not only engaged in co-planning meetings when producing new curriculum and acted as accountable actors but also how institutionalized positions of power were altered and all participants became teachers and learners simultaneously. These, and similar episodes in the database, demonstrate and exemplify participants’ ways of acting. It is an example of how horizontal relations in teacher education programs can emerge (rather than hierarchical, ‘ruling’ relations of power) and how ways of acting shaped Aline’s representations and identities. The ways of acting displayed thus far appear crucial aspects with regard to Aline’s ways or representing and being. In arriving at this particular point, Aline began to express herself publicly, and, in so doing, sustained and constituted further transformation. For example, when Aline spoke in front of others during mini–conferences, she not only exhibited empowerment but also increased her sense of empowerment by her engagement in the activity. Acting as accountable actors and curriculum makers thus appears to be important ways of acting for the crafting of teachers' identities. It ultimately seems to be the case here that by acting as curricular makers, teachers were able to develop new forms of representing and being. Jordão and Buhrer (2013) discussing the teaching practicum in general, stress that when teaching practicum is organized as a ‘enunciation locus’ which positions novice teachers in a place of ‘hybridization’, it offers opportunities for subjects to exercise agency in this stage of their education. Acta Sci. Educ., v. 42, e44870, 2020 Desenvolvendo capacidade pedagógica para design curricular Page 7 of 13 Evaluating the enactement of the curriculum and the participation in school collective meetings: teachers attitudes towards the experience The great amount of repetition of the social actor ‘we’, activated through material processes (evaluate, do, implement, create) and verbalization process (discuss) positions teachers as the Actors of the actions in which the Goal is the curriculum, demonstrating she believes in their power to enact and transform the curriculum. The sense of being evaluating the curriculum produced constructed by the group is explicit in the utterance “We (sayer) also discuss (verbalization process) a lot (circumstance) our own practices, what has worked or not” (Mari, in a teaching conference). The circumstance ‘a lot’ and the lexical choice ‘our own’ and ‘has worked or not’ contribute to this interpretation. Despite obvious engagement, however, the group did not indulge in feelings of success. The group members identified existing contradictions and insufficiencies that our curriculum was not addressing. For example, there was evidence that the high school students were not developing oral skills in English to the desired levels. They were uncertain about whether they sufficiently encouraged the amount of English spoken in the class. This characterized another way of acting: generally, teachers were reflecting and criticizing the very work they were doing. However, the same acknowledge of the gap found in the curriculum (lack of oral skills) contributed to new teachers understanding the power teachers have in redesign the curriculum: teachers have the power to choose and redesign curriculum according to students needs, national guidelines and their own believes. By doing that, new teachers become aware of the responsibility of making locally and situated decisions to teach English. Evidence of transformation regarding the relation was available in the wider school community. In my journal, an episode described exemplifies the new kind of relations that arose from the 2-year teaching practicum: Today, in staff room, the school supervisor came to talk to me and said that the new teachers are great. She said they were responsible and committed with school, that they distinguished themselves from the other groups of new teachers […] they felt to be “the teachers” of the class because [students] even go after them to solve issues, besides participating actively on the Student Evaluation Board and other schools meetings. She said she was surprised because they even know the students and call them by their names (Author’s journal, April 7th 2011). Today, in staff room, the school supervisor came to talk to me and said that the new teachers are great. Page 8 of 13 El Kadri this excerpt, it is visible my representation on the school coordinator. She is the social actor included in my representation through material and verbal processes: “Today, in staff room, (circunstances) the school supervisor (actor) came (material process) to talk (verbalization process) to me and said (verbalization process) that […]” . The fact that I reported the episode in the journal and represented the coordinator as the Actor and the Sayer of the processes positions her as someone with power in the context to act and to say. My utterance is then constructed through an indirect speech (‘She said’ – used three times) in which she is positioned as the one who has the power to evaluate new teachers relations to school and with the students. This interpretation is possible because representations are ways to attribute meaning, and such as one, it is linked to power relations because who has the power to represent has the power to define identity (Silva, 2004). The circumstances ‘today’ and in the ‘staff room’ - although it is a linguistic feature typical of journals - also allow us to imply this was something considered by me as noteworthy in the journal. The circumstance ‘Today’ here, means something ‘new’ or ‘noteworthy’ occurred, something that was not usual. The fact that in the sentence ‘she was (intensive relational process) surprised (attribute)’ I represent the coordinator, in relation to the intensive relational process, as the ‘carrier’ of the attribute ‘surprised’ contributes to this interpretation that the ways of being and acting at school was seen as not usual: being surprised means “[…] to strike or occur to with a sudden feeling of wonder or astonishment, as through unexpectedness […]4”, that is, she did not expect new teachers to act and be like she was describing. The importance of the role of the coordinator in my representation is constructed through the indirect speech in which is possible to depict the representation of the school coordinator regarding the new teachers. The transitivity analysis demonstrates that New teachers are social actors included through funcionalization (new teachers) and also by the pronoun ‘they’. They are first represented as being the ‘carrier’ of the attributes of the intensive relational process: “new teachers (carrier) are (intensive relational attribute) great (depicted attribute)” and “they (carrier) were (intensive relational attribute) responsible (depicted attribute) and committed (depicted attribute) with school” . Page 8 of 13 As the use of the relational process establishes a relation between two entities (Halliday & Matthiessen, 2004) that is, between the carrier and the attribute related to it,and the conceitualization of this type of process is irreversible, either entities can be used to identify the other (Halliday & Matthiessen, 2004). In this sense, new teachers are represented through positive attributes: they are ‘great’, ‘responsible’ and ‘commited’. Attributing qualities to new teachers through relational process, that is, making strong truth claims about the state of being of others as has done the coordinator also ascribes to her a position which displays a kind of power which has an uneven social distribution (Fairclough, 2003). That is, she is the one with the power to evaluate and attribute new teachers with such qualities, which is seen as noteworthy by me as an example that the school staff started to value the presence of the new teachers as they recognized the engagement of the latter with and commitment to the school. New Teachers’ ways of being were also constructed through the representation of themselves as Actors. Besides being represented through relational process, in this excerpt, new teachers are also activated through material processes: “[…] besides [they] participating (material process) actively on the Student Evaluation Board and other schools meetings […]” and “[…] they (actor) even know (material) the students and call them [the students] by their names” . Their identities are represented by me based on the coordinators evaluation. Through the use of the discourse markers ‘besides’ new teachers’ identities are represented as more than being responsible and commitment: they ‘participate actively’. Such identity is reinforced by the use of the discoursive marker ‘even’ “[…] to suggest that something mentioned as a possibility constitutes an extreme case or an unlikely instance […]”5: “[…] they even call students by their name […]” and “[…] students (actor) even go after (material process) them (goal) to solve issues” . Calling students by their names seem to be an indicator of commitment to the students and/or an act of personalizing students, implying the recognition that new teachers were responsible for students’ development. Interesting to notice is that the new teachers identities crafted in this representation is constructed through material process associated to experienced professionals. 4 Dictionary.com. (2014). Surprise. Retrieved from http://dictionary.reference.com/browse/surprised?s=t 5 Dictionary.com. (2014). Ever. Retrieved from http://dictionary.reference.com/browse/even?s=t Evaluating the enactement of the curriculum and the participation in school collective meetings: teachers attitudes towards the experience She said they were responsible and committed with school, that they distinguished themselves from the other groups of new teachers […] they felt to be “the teachers” of the class because [students] even go after them to solve issues, besides participating actively on the Student Evaluation Board and other schools meetings. She said she was surprised because they even know the students and call them by their names (Author’s journal, April 7th 2011). Acta Sci. Educ., v. 42, e44870, 2020 Page 8 of 13 That is, it is possible to notice that the aspects mentioned by me through the indirect speech to report the coordinator’s representations related to Acta Sci. Educ., v. 42, e44870, 2020 Desenvolvendo capacidade pedagógica para design curricular Page 9 of 13 what the coordinator acknowledge as being ways of acting of ‘teachers’ : teachers-in-service are the ones who generally ‘know the students’, ‘participate actively in school meetings’ , ‘call students by their names’ and ‘solve students issues’. Such construction is reinforced through the adjectives ‘responsible, great, committed’, all attributes expected from professionals. This interpretation is in keeping with the representation that new teachers ‘felt to be ‘the teachers’’. The reframing of new teacher’s ways of acting and beings seem to occur in the representation of the coordinator’s saying in my journal. It is possible to notice the valorization and acknowledge of the school in the proposal performed by the group. Through her representation, tough, is also possible to notice how the activities which has permeated the relationship between university-school, in fact, has been characterized by episodical moments (Ortenzi, 2007) and how surprised she was with new ways of acting. Such new ways of acting represents new ways of being in the representation. However, these new ways of being are also being crafted by explicitly differentiating the new teacher from another similar group, creating a difference between ‘us’ and ‘them’ (Van Leeuwen, 2008) Woodward, 2011). I report in the journal that one of the representations of the new teachers reported by the coordinator is that they distinguished themselves from the other groups of new teachers. The linguistically materialization of ‘they’ and the ‘other groups’ allow us to argue that new teachers’ identities are crafter here through ‘border demarcation’ (Silva, 2004), a representational process of classification and elaboration of similarities and differences (Fairclough, 2003). This is significant for new teachers ways of being because the way social actors are represented in texts indicate positions regarding them and their activities (Rezende, 2012). That is, by positioning6 someone, as the coordinator did with new teachers, affects the repertoire of acts one has access to and the parameters of identity positions assigned define what is expected of and socially possible for an actor (Reeves, 2009). In another words, this discoursive construction is only possible through the ongoing engagement with others as individuals participate in their day-to-day activities. Page 8 of 13 New teachers, therefore, are represented as being the Actors of the process and the carrier of positive attributes due to their new ways of acting and daily engagement with students and school staff. Such ways of representing position new teachers with emphasized agency and allow us to discuss the process of identifications and social relations in these interactions. The reframing of the ways of representing the school staff was also product of the new relations. Mariana also reports in her diary how she changed her mind and constructed new relations to the school as she participated on the school Student Evaluation Board. She commented on the professional development week in her journal: I did not imagine that the school (supervisor, coordinator, director) really got involved so much in student’s development. They even know their names. […] I really liked the freedom teachers have to expose their opinion. It shows the school is open to changes (Mari’s journal). I did not imagine that the school (supervisor, coordinator, director) really got involved so much in student’s development. They even know their names. […] I really liked the freedom teachers have to expose their opinion. It shows the school is open to changes (Mari’s journal). In this excerpt, Mari represents herself as a social actor included and activated through the cognitive Mental process (imagine) to exhibit her representation of the ‘school community: “I (Senser) did not imagine (cognitive mental process) that the school (supervisor, coordinator, director) (Actors) really got involved (material process) so much (circumstance) in student’s development” . The school community is here represented through ‘functionalization’, that is, when social actors are referred to in terms of an activity or in terms of something they do, like an occupation or a role (Van Leeuween, 2008): ‘supervisor, coordinator, director’. They are the actor of the material process ‘got involved’. The same sense of surprised exhibited in the coordinator’s representation is exhibited here, in Mari’s representation of the school staff: the linguistic realization ‘I did not imagine’ and the use of ‘even’ in the sentence ‘They even know (mental process) their names’ also suggest she represents the involvement as an unlikely instance. ‘Know their names’ seem to appear in the analysis as being a criteria for both school staff and new teachers to indicate engagement and responsibility towards students’ development. 6 “Positioning can be understood as the discursive construction of personal stories that make a person’s actions intelligible and relatively determinate as social acts and within which the members of the conversation have specific location” (Harre & Van Langenhove, 1999, p. 395). Final considerations This paper reported an experience of the development of pedagogical capacity through a co-planning. Having had the opportunity to interact in co-plannings and redesigning the curriculum, new attitudes emerged. Participating in more democratic spaces and being able to redesign their own curricula during teaching practicum had effects on how novice teachers perceive the participation of high school students. Ways of acting and interacting, that is, participatory positions framed and constructed by and for each individual, are made visible not only through the students' power to act but also through the interactions and actions between members. This also occurs through the kinds of opportunities to which he or she has access, their orientations to which they hold, what each other is accountable for, how they respond to each other, and how they engage in their ongoing interactions (Niestz, 2010). Based on this experience, I strongly believe that preparation periods concurrent to those of their schoolteachers and teacher educators are crucial for more horizontal relationships in classes and, as a result, more effectively cotaught classes. Teachers’ confidence in the redesigned curriculum steadily increased until the end of the second year. Although tensions and contradictions permeated the whole process of construction and enactment of a new curriculum, teachers involved considered it a positive experience and demonstrated a great deal of satisfaction. This sense of achievement is grounded in (a) the feedback of students, (b) the prove that they could actually transform the curriculum in a public school in ways they initially thought impossible, (c) evaluating and criticizing themselves the curriculum produced and (d) the ways in which this cohort started to be perceived by others. This result coincides with others regarding that fact that an accountable actor identity is fostered by the creation of interactional spaces in which novice teachers are not only positioned as contributors whose inputs are recognized and credited (Niestz, 2010) but also have the possibility to problematize and resolve noteworthy issues (Greeno, 2006). It means fostering spaces in which novice teachers are able to propose and evaluate ideas and are treated as if one can do something of one’s own volition by having experiences that exercise agency (Edwards & D’arcy, 2004). Here, the group appears to experience these ways of acting: a sense of agency and autonomy that allows them to see themselves as responsible, capable, and a relevant actors in the educational system. Page 10 of 13 El Kadri Page 10 of 13 changes […]) which positions the school as being the ‘carrier’ of the attribute ‘open’. Such representation is significant because it can relatively determinate Mari’s future actions as a teacher: representing the school as open to changes implies in the belief teachers are also agents of transformation. Final considerations Greeno (2006) and Engle and Faux, (2006) and Niestz (2010), for example, argue that one important aspect of developing agency is having the opportunity to participate and contribute in interactions where one is framed and positioned as an accountable author who is in charge of one’s actions. This is important because developing teachers' pedagogical capacity for curriculum design may influence the degree to which teachers engage in a principled, reform-oriented analysis when creating learning opportunities for students (Beyer & Davis, 2012). References Acta Sci. Educ., v. 42, e44870, 2020 Ball, D. L., & Cohen, D. K. (1999) Developing practice, developing practitioners: Toward a practice-based theory of professional education. In G. Sykes, & L. Darling-Hammond (Eds.), Teaching as the learning profession: handbook of policy and practice (p. 3-32). San Francisco, CA: Jossey Bass. Beyer, C. J., & Davis, E. A. (2009). Using educative curriculum materials to support preservice elementary teachers’ curricular planning: a comparison between two different forms of support. Curriculum Inquiry, 39 (5), 679-203. Doi: 10.1111/j.1467-873X.2009.00464.x Beyer, C. J., & Davis, E. A. (2012). Developing preservice elementary teachers’ pedagogical design capacity for reform-based curriculum design. Curriculum Inquiry, 42(3), 386-413. Doi: 10.1111/j.1467- 873X.2012.00599.x Cheyne, A., & Tarulli, D. (1999). Dialogue, difference, and the ‘third voice’ in the zone of proximal development. Theory and Psychology, 9(1), p. 5-28. Doi: 10.1177/0959354399091001 Clandinin, D. J., & Connelly, F. M. (1992). Teacher as curriculum maker. In P. W. Jackson (Ed.), Handbook of research on curriculum (p. 363-401). New York, NY: Macmillan. Acta Sci Educ v 42 e44870 2020 Ball, D. L., & Cohen, D. K. (1999) Developing practice, developing practitioners: Toward a practice-based theory of professional education. In G. Sykes, & L. Darling-Hammond (Eds.), Teaching as the learning profession: handbook of policy and practice (p. 3-32). San Francisco, CA: Jossey Bass. Beyer, C. J., & Davis, E. A. (2009). Using educative curriculum materials to support preservice elementary teachers’ curricular planning: a comparison between two different forms of support. Curriculum Inquiry, 39 (5), 679-203. Doi: 10.1111/j.1467-873X.2009.00464.x Beyer, C. J., & Davis, E. A. (2012). Developing preservice elementary teachers’ pedagogical design capacity for reform-based curriculum design. Curriculum Inquiry, 42(3), 386-413. Doi: 10.1111/j.1467- 873X.2012.00599.x Cheyne, A., & Tarulli, D. (1999). Dialogue, difference, and the ‘third voice’ in the zone of proximal development. Theory and Psychology, 9(1), p. 5-28. Doi: 10.1177/0959354399091001 Clandinin, D. J., & Connelly, F. M. (1992). Teacher as curriculum maker. In P. W. Jackson (Ed.), Handbook of research on curriculum (p. 363-401). New York, NY: Macmillan. Ball, D. L., & Cohen, D. K. (1999) Developing practice, developing practitioners: Toward a practice-based theory of professional education. In G. Sykes, & L. Darling-Hammond (Eds.), Teaching as the learning profession: handbook of policy and practice (p. 3-32). San Francisco, CA: Jossey Bass. Ball, D. L., & Cohen, D. K. (1999) Developing practice, developing practitioners: Toward a practice-based theory of professional education. In G. Sykes, & L. Page 8 of 13 Then, Mari evaluates the relations the school keep with the teachers as being desirable ‘and’ positions teachers as the Sayers of the verbalization process ‘I really liked the freedom teachers have to expose (verbalization process) their opinion’ . Such experience also allows Mari to represent the school through relational process ([…] the school (carrier) is (intensive relational process) open (depicted attributed) to Acta Sci. Educ., v. 42, e44870, 2020 Page 10 of 13 Desenvolvendo capacidade pedagógica para design curricular Page 11 of 13 Craig, C. J., & Ross, V. (2008). Cultivating the image of teachers as curriculum makers. In F. M. Connelly, M. F. HE, & J. I. Phillion (Eds.), The SAGE handbook of curriculum and instruction (p. 282-305). Thousand Oaks, CA: Sage Publications. Davis, E. A. (2006). Preservice elementary teachers’ critique of instructional materials for science. Science Education, 90(1), 348-375. Doi: 10.1002/sce.20110 ictionary.com. (2014). Ever. Retrieved from http://dictionary.reference.com/browse/even?s=t Dictionary.com. (2014). Surprise. Retrieved from http://dictionary.reference.com/browse/surpr Edwards, A., & D’arcy, C. (2004). 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Journal of Research in Science Teaching, 39(3), p. 253-282. Doi: 10.1002/tea.10018 Roth, W. M., & Radford, L. (2010). Desenvolvendo capacidade pedagógica para design curricular Page 13 of 13 Page 13 of 13 Page 13 of 13 institucionais) quanto em contextos de ambientes informais, como por exemplo, as redes sociais. A interface entre linguagens e tecnologias digitais, o papel do inglês no cenário atual e práticas democráticas de aprendizagem (deliberação, co-teaching e diálogo cogerativo, comunidades de prática) também permeiam suas práticas. Tem experiência na área de ensino de línguas estrangeiras, atuando principalmente na formação de professores em relação ao aprendizado em contexto escolar. Atualmente, interessa-se por pesquisas na área de formação do professor pela perspectiva socio-histórico-cultural, análise crítica do discurso, novas tecnologias, inglês como língua franca e formação de professores. Tem vários artigos, livros e capítulos publicados em cenário nacional e internacional. ORCID: http://orcid org/0000-0002-5836-4988 E-mail: mielkadri@hotmail.com INFORMATION ABOUT THE AUTHOR Michele Salles El Kadri: Professora Adjunta da Universidade Estadual de Londrina. Pós-Doutora em Linguística Aplicada pela Unicamp e Doutora em Estudos da Linguagem (UEL) com estágio de doutorado-sandwich na Griffith University (Australia). Mestre em Estudos da Linguagem e Especialista em língua inglesa pela mesma Universidade. Membro do GT de Linguagens e Tecnologias da Anpoll, professora no Programa de Pós Graduação em Educação da UEL e Coordenadora do Mestrado Profissional em Letras Estrangeiras Modernas da UEL. Bolsista Produtividade da Fundação Araucária. Foi coordenadora do Colegiado de Letras Estrangeiras Modernas, Presidente do FOPE (Fórum Permanente das Licenciaturas) da UEL e coordenadora Pedagógica da COPS-UEL. Desenvolve pesquisa sobre aprendizagens/transformações docentes em contexto escolar pelo viés da perspectiva sócio-histórico-cultural e dos referenciais de análise crítica da linguagem. Tem interesse em estudos que investiguem a aprendizagem de professores em contexto escolar com foco nas transformações nas relações interpessoais (modos de agir), nas práticas discursivas (modos de representar) e nas identificações (modos de ser). Também tem interesse nas formas como a escola, a comunidade e o conhecimento são representados e (re)ssignificados de acordo com as práticas experenciadas em contexto de estágio e iniciação a docência. Sua pesquisa também foca na aprendizagem de alunos, professores e comunidade no contexto de escolas e universidades mediados por diversos artefatos (tecnológicos, humanos, Acta Sci. Educ., v. 42, e44870, 2020 Acta Sci. Educ., v. 42, e44870, 2020 NOTE: Michele Salles El Kadri foi responsável pela concepção, análise e interpretação dos dados; redação e revisão crítica do conteúdo do manuscrito e ainda, aprovação da versão final a ser publicada. Acta Sci. Educ., v. 42, e44870, 2020
https://openalex.org/W3149239941	https://www.acjournal.ru/jour/article/download/1622/1367	Russian	null	Prevention of Youth Deviations in Russia and Abroad	Upravlenčeskoe konsulʹtirovanie	2,021	cc-by	4,257	ОБЩЕСТВО И РЕФОРМЫ ОБЩЕСТВО И РЕФОРМЫ DOI 10.22394/1726-1139-2021-1-97-105 РЕФЕРАТ Цель и задачи. В статье проанализированы социальные институты, входящие в си- стему профилактики девиаций среди детей и молодежи. Отмечена особая роль инсти- тутов, контролирующих деятельность людей и регулирующих их отношения в сферах общественной жизни, к которым также относится общественно опасное поведение. Рассмотрен опыт ряда зарубежных стран по профилактике девиантного поведения молодежи. Методы. В исследовании использован комплекс общенаучных методов, таких как анализ, синтез, обобщение, сравнение, системно-функциональный и комплексный подход. В рамках заявленной темы осуществлена систематизация теоретических по- ложений и современных подходов к проблеме молодежных девиаций на основе изуче- ния работ различных авторов, нормативных документов, официальной статистики. Теоретическая и практическая значимость представленного исследования со- стоит в том, что оно может стать основой для дальнейшего изучения деятельности социальных институтов по профилактике различных девиаций молодежи. Проведенный анализ российского и зарубежного опыта профилактики дает возможность выявить состояние проблем подростков и молодежи, актуализирует значимость государствен- ной политики в отношении данных категорий населения, как в процессе формирования, так и в процессе ее реализации. Полученные материалы позволяют выделить позитив- ные результаты и выявить факторы, способствующие дальнейшему развитию деятель- ности по предупреждению общественно опасного поведения молодежи. Результаты и выводы. В статье представлены результаты исследования деятель- ности социальных институтов по профилактике девиаций молодежи в России и ряде зарубежных стран. Выявлены слабые и сильные стороны в системе институтов про- филактики девиантного поведения. Обоснована недостаточная эффективность органи- зации государственной системы профилактики. Сформулированы выводы о необходи- мости объединения усилий государственных и негосударственных ресурсов в этом направлении. Ключевые слова: подростки, молодежь, социальные институты, государственная система профилактики Для цитирования: Цинченко Г. М., Орлова И. С. Профилактика молодежных девиаций в России и за рубежом // Управленческое консультирование. 2021. № 1. С. 97–105. Цинченко Г. М., Орлова И. С. Цинченко Г. М., Орлова И. С. Российская академия народного хозяйства и государственной службы при Президенте Рос- сийской Федерации (Северо-Западный институт управления РАНХиГС), Санкт-Петербург, Российская Федерация; galina_ts55@mail.ru Российская академия народного хозяйства и государственной службы при Президенте Рос- сийской Федерации (Северо-Западный институт управления РАНХиГС), Санкт-Петербург, Российская Федерация; galina_ts55@mail.ru Galina M. Tsinchenko, Inna S. Orlova Galina M. Tsinchenko, Inna S. Orlova Russian Presidential Academy of National Economy and Public Administration (North-West Institute of Management of RANEPA), Saint-Petersburg, Russian Federation; galina_ts55@mail.ru Russian Presidential Academy of National Economy and Public Administration (North-We of Management of RANEPA), Saint-Petersburg, Russian Federation; galina_ts55@mail. ABSTRACT Goals and objectives. The article analyzes the resources of support for families with disabled children living in the Leningrad region. There is substantial state support for this category of families, developing the potential of non-state support resources. The results of the govern- ment’s efforts to support families with children with disabilities, as well as the tasks set to be solved in the near and long term, are considered. Для цитирования: Цинченко Г. М., Орлова И. С. Профилактика молодежных девиаций в России и за рубежом // Управленческое консультирование. 2021. № 1. С. 97–105. Prevention of Youth Deviations in Russia and Abroad Galina M. Tsinchenko*, Inna S. Orlova ABSTRACT 97 УПРАВЛЕНЧЕСКОЕ КОНСУЛЬТИРОВАНИЕ . № 1. 2021 ОБЩЕСТВО И РЕФОРМЫ Methods. The study used a set of general scientific methods, such as analysis, synthesis, generalization, comparison, system-functional and integrated approach. Within the framework of the stated theme, theoretical provisions and modern approaches have been organized by studying the works of various authors, regulatory documents and official statistics. The theoretical and practical significance of the study is that it can be the basis for further study of social resources and life strategies of families with children with disabilities, their transformation. The analysis provides an opportunity to identify the problems of families with children with disabilities in Leningrad, actualizes the importance of state policy regarding these categories of families, both in the process of formation and in its implementation. The materials provide us with positive results achieved by the authorities of the Leningrad region and identify ways to further develop both state and non-state social support for families with children with disabilities in the region. Results and conclusions. The article presents the results of the research of the resources of state and non-state social support for families with children with disabilities in The Leningrad region. Conclusions have been drawn on the need to unite the efforts of state and non-state resources in this direction Keywords: adolescents, young people, social institutions, the state prevention system For citing: Tsinchenko G. M., Orlova I. S. Prevention of Youth Deviations in Russia and Abroad // Administrative consulting. 2021. N 1. P. 97–105. Противоречивые глобальные перемены в сферах экономики, политики, образо- вания, общественного строя Российской Федерации за последние десятилетия привели к массовому обеднению населения, повышению смертности, росту ал- коголизации и наркотизации общества, что предопределило рост подростковых девиаций. Несмотря на то, что по данным статистики в период с 2016 по 2018 г. на 31% снизилось количество несовершеннолетних, страдающих алкоголизмом, однако неизменным остается показатель детской и подростковой наркомании. В среднем в России около 30 тыс. подростков страдают от наркотической зависимости, что приводит к развитию серьезных заболеваний в этой среде и к высокой ранней смертности. Значительным также остается и показатель детской проституции. Ежегодно около 2 млн подростков женского и мужского пола в возрасте от 14 до 17 лет занимаются проституцией1. Согласно данным Росстата, Россия занимает 3-е место в мире по количеству подростковых суицидов. Показатель подростко- вого суицида в нашей стране превышает средний мировой показатель в 3 раза. На 100 тыс. 1 Единая межведомственная информационно-статистическая система (ЕМИСС) [Электрон ный ресурс]. URL: https://rosstat.gov.ru/emiss (дата обращения: 12.09.2020). 2 Федеральная служба государственной статистики [Электронный ресурс]. URL: https:// rosstat.gov.ru/0 (дата обращения: 26.09.2020). ABSTRACT детей в возрасте до 14 лет 2,5 кончают с собой, а из подростков от 15 до 19 лет — 16,3 сводят счеты с жизнью2. Отсутствие положительной дина- мики в статистике детских и молодежных девиаций свидетельствует о недоста- точной эффективности государственной системы профилактики девиантного по- ведения. В этой связи остается актуальной необходимость исследования деятельности государственных систем по профилактике девиантного поведения с целью повы- шения эффективности принятия управленческих решений и государственных мер в сфере предотвращения причин возникновения девиаций. В процессе профилактики девиантного поведения особую роль играют соци- альные институты, представляющие собой стабильные формы организации и кон- троля общественной жизни. Социальные институты контролируют деятельность УПРАВЛЕНЧЕСКОЕ КОНСУЛЬТИРОВАНИЕ . № 1 . 2021 98 людей, регулируют их отношения и поведение в различных сферах общественной жизни, в том числе и общественно опасное поведение. Особую роль берут на себя следующие социальные институты: государство, семья, политические пар- тии, молодежные общественные организации и движения, суд, культура и об- разование [1]. Они оказывают влияние на становление молодого поколения, определение его места и роли в обществе, формирование поведенческих осо- бенностей (рис.). людей, регулируют их отношения и поведение в различных сферах общественной жизни, в том числе и общественно опасное поведение. Особую роль берут на себя следующие социальные институты: государство, семья, политические пар- тии, молодежные общественные организации и движения, суд, культура и об- разование [1]. Они оказывают влияние на становление молодого поколения, определение его места и роли в обществе, формирование поведенческих осо- бенностей (рис.). ОБЩЕСТВО И РЕФОРМЫ Очевидно, что каждый социальный институт определенным образом влияет на сознание и поведение несовершеннолетних. Если устойчивые моральные ценности, сложившиеся традиции, привычки, общественное мнение воздействуют опосредо- ванно, то государство, политические партии, подростковые организации, школы, клубы по интересам действуют более активно, влияя на формирование жизненной позиции и позиционирования несовершеннолетними себя в обществе. Эти группы социальных институтов взаимосвязаны, нацелены на формирование позитивных стартовых жизненных условий для подрастающего поколения. В Российской Федерации несколько лет существует устойчивая система про- филактики безнадзорности и правонарушений несовершеннолетних. Основными задачами данной системы являются предотвращение общественно опасного по- ведения несовершеннолетних и эффективная борьба с последствиями их девиант- ного поведения. Согласно ст. 4 Федерального закона от 24.06.1999 № 120-ФЗ (ред. ABSTRACT от 23.11.2015) «Об основах системы профилактики безнадзорности и право- нарушений несовершеннолетних»1 в систему профилактики безнадзорности и пра- вонарушений несовершеннолетних входят: • органы, составляющие систему по профилактике безнадзорности и правонару- шений несовершеннолетних; • органы, составляющие систему по профилактике безнадзорности и правонару- шений несовершеннолетних; • комиссии по делам несовершеннолетних и защите их прав; • органы опеки и попечительства; • органы опеки и попечительства; • органы по делам молодежи; • органы управления социальной защитой населения; • федеральные органы государственной власти и органы государственной власти субъектов Российской Федерации, осуществляющие государственное управление в сфере образования; • федеральные органы государственной власти и органы государственной власти субъектов Российской Федерации, осуществляющие государственное управление в сфере образования; Социальные итуты инст Госуда во рст По ческие партии лити М твенн е олодежные общес ы организации Суд Семья К ьтура ул О ов ние браз а Социальные итуты инст Госуда во рст По ческие партии лити М твенн е олодежные общес ы организации Суд Семья К ьтура ул О ов ние браз а Рис. Социальные институты, контролирующие общественно опасное поведение Fig. Social institutions that control socially dangerous behaviour 1 Федеральный закон от 24.06.1999 № 120-ФЗ (с изменениями и дополнениями) «Об основах системы профилактики безнадзорности и правонарушений несовершеннолетних» [Электронный ресурс]. URL: https://base.garant.ru/12116087 (дата обращения: 26.09.2020). 99 ОБЩЕСТВО И РЕФОРМЫ ОРМЫ • органы местного самоуправления, осуществляющие управление в сфере обра- зования; ОРМЫ • органы местного самоуправления, осуществляющие управление в сфере обра- зования; ЕФО • органы управления здравоохранением; • органы службы занятости; ВО И • учреждения уголовно-исполнительной системы (следственные изоляторы, вос- питательные колонии и уголовно-исполнительные инспекции). • учреждения уголовно-исполнительной системы (следственные изоляторы, вос- питательные колонии и уголовно-исполнительные инспекции). К ф ЩЕСТВО И • учреждения уголовно-исполнительной системы (следственные изоляторы, вос- питательные колонии и уголовно-исполнительные инспекции). Контроль над деятельностью перечисленных органов осуществляют федеральные органы государственной власти и органы государственной власти субъектов Рос- Контроль над деятельностью перечисленных органов осуществляют федеральные органы государственной власти и органы государственной власти субъектов Рос- сийской Федерации. Ведущими задачами государственных органов и социальных учреждений системы профилактики безнадзорности и правонарушений совершен- нолетних являются: • предупреждение безнадзорности, беспризорности, правонарушений и антиобще- ственных действий несовершеннолетних, выявление и устранение причин и ус- ловий, способствующих этому; обеспечение защиты прав и законных интересов несовершеннолетних; б • социально-педагогическая реабилитация несовершеннолетних, находящихся в со- циально опасном положении. • социально-педагогическая реабилитация несовершеннолетних, находящихся в со- циально опасном положении. 1 Федеральный закон от 24.06.1999 № 120–ФЗ (с изменениями и дополнениями) «Об основах системы профилактики безнадзорности и правонарушений несовершеннолетних» [Электронный ресурс]. URL: https://base.garant.ru/12116087/ (дата обращения: 26.09.2020). 2 Федеральный закон «Об образовании в Российской Федерации» от 29.12.2012 № 273-ФЗ (последняя редакция) [Электронный ресурс]. URL: http://www.consultant.ru/document/cons_ doc_LAW_140174/ (дата обращения 15.10.2020). [Электронный ресурс]. URL: https://base.garant.ru/12116087/ (дата обращения: 26.09.2020). 2 Федеральный закон «Об образовании в Российской Федерации» от 29.12.2012 № 273-ФЗ (последняя редакция) [Электронный ресурс]. URL: http://www.consultant.ru/document/cons_ doc_LAW_140174/ (дата обращения 15.10.2020). ABSTRACT Деятельность по предупреждению общественно опасного поведения осущест- вляется на следующих принципах: законности, демократизма, гуманного обращения с подростками, поддержки института семьи и эффективного взаимодействия с ней, индивидуального подхода к подросткам, соблюдения конфиденциальности полу- ченных данных, обеспечения ответственности должностных лиц и граждан за на- рушение прав и законных интересов несовершеннолетних. Проведение непосредственной профилактической работы с молодежью возла- гается на социальные учреждения различной ведомственной принадлежности. Ф Согласно федеральному закону от 24.06.1999 № 120-ФЗ (ред. от 23.11.2015) «Об основах системы профилактики безнадзорности и правонарушений несовер шеннолетних»1 в систему профилактики безнадзорности и правонарушений со- вершеннолетних входят следующие органы и учреждения системы образования: • органы управления образованием; • общеобразовательные учреждения общего образования, образовательные учреждения начального профессионального, среднего профессионального образования и другие учреждения, осуществляющие образовательный про- цесс; • общеобразовательные учреждения общего образования, образовательные учреждения начального профессионального, среднего профессионального образования и другие учреждения, осуществляющие образовательный про- цесс; • образовательные учреждения для детей-сирот и детей, оставшихся без попече- ния родителей; вательные учреждения для детей-сирот и детей, оставшихся без попече- дителей; • специальные учебно-воспитательные учреждения открытого и закрыто органов управления образованием. льные учебно-воспитательные учреждения открытого и закрытого типов в управления образованием. Определение несовершеннолетних граждан, в отношении которых регулярно ведется профилактическая деятельность в образовательных учреждениях, огово- рено определением общественно опасной деятельности, согласно национальному праву. В п. 11 Закона РФ «Об образовании»2 понятия «общественно опасного» и «де- виантного» поведения тождественны. Согласно данному закону, дети, достигшие возраста 11 лет, по решению суда могут быть определены в специализированные образовательные учреждения, которые смогут обеспечить комфортные условия для Федеральный закон от 24.06.1999 № 120 ФЗ (с изменениями и дополнениями) Об основах системы профилактики безнадзорности и правонарушений несовершеннолетних» [Электронный ресурс]. URL: https://base.garant.ru/12116087/ (дата обращения: 26.09.2020). 2 Федеральный закон «Об образовании в Российской Федерации» от 29.12.2012 № 273-ФЗ (последняя редакция) [Электронный ресурс]. URL: http://www.consultant.ru/document/cons_ doc_LAW_140174/ (дата обращения 15.10.2020). 100 УПРАВЛЕНЧЕСКОЕ КОНСУЛЬТИРОВАНИЕ . № 1 . 2021 получения образования, реабилитации подростков, окажут эффективные медицин- ские услуги, обеспечат профессиональной подготовкой. получения образования, реабилитации подростков, окажут эффективные медицин- ские услуги, обеспечат профессиональной подготовкой. 1 Постановление Правительства РФ от 7 марта 1995 г. № 233 «Об утверждении Типового положения об образовательном учреждении дополнительного образования детей» (с изме- нениями и дополнениями) [Электронный ресурс]. URL: https://base.garant.ru/136913/ (дата обращения: 26.09.2020). ABSTRACT 2021 ВО И РЕФОРМЫ по предупреждению девиации среди несовершеннолетних и борьбы с ней; повы- шаются ответственность и контроль правительства за работой школьных учреж- дений по воспитанию несовершеннолетних, по их интеграции в современное японское общество, укреплению моральных ценностей в подростковой среде [4]. Эффективными мероприятиями правительства Японии по борьбе с девиацией являются: ОБЩЕСТВО И РЕФОРМЫ стимулирование активизации деятельности общественных организаций; • ответственность и контроль государства за деятельностью школы по обеспече- нию воспитания подрастающего поколения; • функционирование эффективной системы правового управления школьным об- разованием. Среди высокоразвитых стран Европы важную роль в сфере профилактики деви- антного поведения среди несовершеннолетних, а также предупреждению преступ- ности среди граждан в целом играет такая страна, как Германия [2]. Правительство Германии не применяет общее уголовное право по отношению к гражданам, не достигшим 21 года, для того чтобы помочь подросткам, еще не сформировавшим- ся в своих убеждениях, сделать грамотный выбор в сложных жизненных ситуациях, не переступая закон. В стране специфично и судопроизводство по правонаруше- ниям, совершенным несовершеннолетними. Расследование этих дел проводит специализированная прокуратура по делам несовершеннолетних граждан. Главным источником уголовного права несовершеннолетних граждан ФРГ является Закон об отправлении правосудия по делам молодежи — системный законодательный акт, который включает в себя нормы уголовного, уголовно-процессуального и уго- ловно-исполнительного права (JGG). Данный закон в качестве мер воздействия на правонарушителей предусматривает: воспитательные меры, принудительные меры, уголовное наказание. «Закон о защите молодежи» от 23.07.2002 выделяет базовые группы: дети — граждане, до наступления 14 лет, и подростки — граждане от 14 до 18 лет, от 18 до 24 лет — «молодые взрослые». Правительство Германии убеждено, что граж- данин, не достигший возраста 24 лет, находится на этапе формирования мировоз- зрения, соответственно, ему свойственно ошибаться. Находясь под эффективным влиянием социальных институтов (дом, семья, государство, образовательные уч- реждения), обучаясь в благоприятных условиях, приспособленных к росту и раз- витию, несовершеннолетний сможет грамотно определиться в своих политических и общественных взглядах. В Германии существует специальная нормативная база, регулирующая общественную профилактику девиантного поведения среди моло- дежи [2]. Эффективная политика Германии по профилактике общественно опасного по- ведения несовершеннолетних заключается во взаимодействии государственных и общественных институтов. Путем открытого диалога государство реализует пра- во подрастающего поколения на существование в комфортных условиях, положи- тельно сказывающихся на самоопределении несовершеннолетнего, как граждани- на своей страны. Государственная политика Германии в сфере предупреждения девиации несовершеннолетних направлена на эффективное образование молоде- жи, по получению которого гражданам предоставляются рабочие места, а уровень зарплат ограничивает людей от риска преступления черты закона. ABSTRACT ОБЩЕСТВО И РЕФОРМЫ Специализированные учебно-воспитательные учреждения1 создаются для не- совершеннолетних граждан, которые нуждаются в особых условиях педагогиче- ского воздействия, образовательной деятельности и требующих особого психо- логического подхода в следующих случаях: не несут уголовной ответственности, в силу того, что на момент совершения преступления не достигли совершенно- летия; достигли совершеннолетия, но не несут уголовную ответственность, так как на момент осуществления общественно опасного деяния не могли осознавать общественную опасность своих действий, в связи с отставанием в развитии, не связанным с душевно-психическим расстройством. Условия в таких учреждениях должны способствовать проведению социально-профилактической работы с не- совершеннолетними, нацеленной на их социальную реабилитацию и коррекцию поведения. Профилактика девиантного поведения является базовой предпосылкой становле- ния подрастающего поколения. Ребенок с общественно опасным поведением не становится таким случайно. Антисоциальные убеждения, противоречащие нормам морали, формируются задолго до того, как подросток перестает взаимодействовать с социальными институтами (семьей, школой, государством). Задачей системы по предупреждению девиантного поведения среди несовершеннолетних выступает его своевременная профилактика. Она достигается путем эффективного взаимодействия социальных институтов, открытого диалога с гражданами страны, совместной дея- тельности государства и общественных молодежных организаций. Проблема девиаций в молодежной среде характерна для большинства стран, в каждой стране сложился национальный опыт ее решения. Зарубежные теорети- ческие подходы и социальная профилактическая практика в работе с молодежью также могут быть полезны и для России. Япония имеет самый низкий уровень проявления девиантного поведения среди несовершеннолетних в мире. Особенностью японской политики, направленной на профилактику безнадзорности и девиантного поведения в молодежной среде, мож но считать ярко выраженный адресный характер. Он заключается в нацеленности профилактических мер как на детей и подростков, обучающихся в средних и стар- ших классах школы, так и на систему школьного образования в целом. Антидеви- антная политика Японии включает в себя систему мер, объединяющую усилия правительства, школы, семьи и общества по предотвращению общественно опас- ного поведения среди подростков. В государственной политике по отношению к подрастающему поколению эффек- тивно используется система общественного контроля, например, традиционное для граждан Японии чувство принадлежности к особой социальной группе, ассоцииро- вание личности с этой социальной группой, как ведущий основополагающий инстру- мент общественного контроля, позволяющий предотвратить проявление девиантно- го поведения среди несовершеннолетних. В числе мероприятий, реализуемых государственной властью Японии в рамках политики, направленной на предупреждение девиантного поведения, особой эф- фективностью обладает активная деятельность общественных организаций, при помощи которых налаживается взаимосвязь между социальными институтами. Создается своеобразная система, которая усиливает роль общества в действиях 101 УПРАВЛЕНЧЕСКОЕ КОНСУЛЬТИРОВАНИЕ . № 1. ABSTRACT Также государ- ство активно взаимодействует с детскими, подростковыми и молодежными объеди- нениями, в соответствии с их классификациями. ф Факторами эффективного предотвращения преступности среди несовершенно- летних в Германии через государственные институты являются [2]: Факторами эффективного предотвращения преступности среди несовершенно летних в Германии через государственные институты являются [2]: • применение комплексного подхода к образованию молодежи, а именно: соче- тание образования с обеспечением молодежи рабочими местами; • применение комплексного подхода к образованию молодежи, а именно: соче- тание образования с обеспечением молодежи рабочими местами; 102 УПРАВЛЕНЧЕСКОЕ КОНСУЛЬТИРОВАНИЕ . № 1 . 2021 • сотрудничество власти и неправительственных организаций на всех уровнях реализации молодежной политики; • сотрудничество власти и неправительственных организаций на всех уровнях реализации молодежной политики; ОБЩЕСТВО И РЕФОРМЫ • структурирование молодежной политики по уровням — общенациональным, реги- ональным и местным; • структурирование молодежной политики по уровням — общенациональным, реги- ональным и местным; • использование принципа субсидиарности, когда основная ответственность, в числе финансовая, возлагается на низовой уровень; • использование принципа субсидиарности, когда основная ответственность, в том числе финансовая, возлагается на низовой уровень; • использование принципа субсидиарности, когда основ числе финансовая, возлагается на низовой уровень; • условное деление единого массива молодежи на непосредственно молодежь и «молодых взрослых», поскольку приоритеты этих возрастных групп различны • условное деление единого массива молодежи на непосредственно молодежь и «молодых взрослых», поскольку приоритеты этих возрастных групп различны. В А В Англии акцент по предупреждению девиантного поведения молодежи делает- ся на вторичную профилактику. На лиц, нарушивших закон, распространяются такие меры государственного принуждения, как условное осуждение, ограничение свободы и штраф. Социальный работник ведет наблюдение за осужденным в те- чение всего периода условного заключения, продолжительность которого может быть сроком от 6 месяцев до 3 лет. Уголовное законодательство Англии выделяет три группы несовершеннолетних. 1. Малолетние дети в возрасте до 10 лет. Для них установлена неоспоримая пре- зумпция об уголовной недееспособности. 1. Малолетние дети в возрасте до 10 лет. Для них установлена неоспоримая пре- зумпция об уголовной недееспособности. 2. Дети в возрасте от 10 до 14 лет. Уголовная ответственность по факту возможна, но вопрос о ее применении решается в индивидуальном порядке. 2. Дети в возрасте от 10 до 14 лет. Уголовная ответственность по факту возможна, но вопрос о ее применении решается в индивидуальном порядке. 3. Подростки в возрасте от 14 до 17 лет (при назначении наказаний к ним при- равниваются молодые люди от 17 до 21 года). 3. ABSTRACT Подростки в возрасте от 14 до 17 лет (при назначении наказаний к ним при- равниваются молодые люди от 17 до 21 года). Совершеннолетие наступает в Англии в 21 год, что дает гражданину полностью сформироваться во взглядах и убеждениях. К гражданам моложе 21 года приме- няются меры, по факту не являющиеся мерой пресечения. Такие, как: приказ о надзоре социальными службами, приказ о попечительстве, приказ родителям и опекунам несовершеннолетнего создать условия для обеспечения положитель- ного поведения подростка и т. д. Гражданина, не достигшего 21 года, могут обязать оказывать предоставление социальных услуг обществу, например, выполнять обязательные не оплачиваемые общественные работы в течение времени, определенного судом (количество часов зависит от возраста подсудимого). Несовершеннолетнего могут отправить в со- циальный центр временного задержания и только в особых случаях — к не харак- терному для несовершеннолетних преступников заключению под стражу и пожиз- ненному ограничению свободы. Крайние меры по отношению к несовершеннолетним назначаются только в тех случаях, когда, по решению социальных служб и государ- ственных институтов, отсутствуют для данного нарушителя лояльные методы ис- полнения наказания [3]. В порядке сопутствующих наказаний для несовершенно- летних на территории Англии применяется лишение прав: на вождение транспорт- ного средства, на занятие указанной судом деятельностью. По примеру стран, имеющих успешный опыт профилактики девиантного пове- дения среди несовершеннолетних, правительству Российской Федерации стоит пересмотреть систему, противостоящую общественно опасному поведению моло- дежи. Очевидно, что уголовный возраст в России начинается достаточно рано, в период становления подростка как личности, в момент оценки им общественно- го и политического строя своей страны, важно не давить, а поддержать, посред- ством грамотно выстроенной социальной политики государства. Консервативные взгляды на предупреждение и профилактику девиантного поведения среди несо- вершеннолетних вряд ли помогут его предотвратить и тем более эффективно бо- роться с последствиями общественно опасных действий. Государственные органы должны более активно взаимодействовать с обще- ственными организациями на всех уровнях реализации молодежной политики. Также необходимо выстроить доверительный диалог между социальными институ- 103 УПРАВЛЕНЧЕСКОЕ КОНСУЛЬТИРОВАНИЕ . № 1. 2021 тами, для того чтобы подрастающее поколение росло в безопасной среде и раз- вивалось в комфортных условиях. тами, для того чтобы подрастающее поколение росло в безопасной среде и раз- вивалось в комфортных условиях. ОБЩЕСТВО И РЕФОРМЫ В современных условиях должны принципиально меняться подходы к органи- зации профилактической работы, с тем, чтобы от мер административно-право- вого воздействия перейти к оказанию социально-педагогической помощи семье, подростку, помощи, основанной на изучении личности несовершеннолетнего правонарушителя, условий его семейного и общественного воспитания [5]. ABSTRACT Такая работа может вестись успешно лишь в тесном взаимодействии всех социальных институтов и требует глубоких специальных психолого-педагогических знаний. Системный анализ генезиса отклоняющегося поведения выявляет различные неблагоприятные психобиологические, психолого-педагогические, социально- психологические, психологические и социальные факторы, обусловливающие отклонения в поведении детей и подростков, свидетельствует о необходимости комплексного подхода к профилактике, включающего комплекс социально-ме- дико-педагогических мер по оздоровлению условий воспитания и коррекции отклоняющегося поведения несовершеннолетних. Об авторах: Цинченко Галина Михайловна, доцент кафедры социальных технологий Северо-Западного института управления РАНХиГС (Санкт-Петербург, Российская Федерация), кандидат со- циологических наук, доцент; tsinchenko-gm@ranepa.ru Орлова Инна Степановна, доцент кафедры социальных технологий Северо-Западного ин- ститута управления РАНХиГС (Санкт-Петербург, Российская Федерация), кандидат педа- гогических наук, доцент; oca-oca@mail.ru Литература 1. Ахъядов Э. С. Отечественный и зарубежный опыт профилактики безнадзорности и право- нарушений несовершеннолетних // Молодой ученый. 2016. № 2. С. 658–662. 2. Жданова М. А. Исследование проблемы и практического опыта профилактики девиантно- го поведения подростков и молодежи в современной Германии // Проблемы педагогиче- ской инноватики в профессиональном образовании. Материалы XIX Международной на- учно-практической конференции. СПб. : Российский гос. пед. университет им. А. И. Герцена, 2018. 490 с. С. 401–408. 3. Кушнир С. И. Опыт предупреждения преступного поведения подростков в США и Вели кобритании : автореф. дис. … канд. пед. наук. Коломна, 2001. 4. Морозов Н. А. Основные классификации методов, используемых в Японии для предупреж- дения и борьбы с девиантным поведением молодежи // Alma Mater. 2013. № 3. C. 93–98. 5. Цинченко Г. М., Орлова И. С. Наркопотребление несовершеннолетних как социальная проблема // Здоровье — основа человеческого потенциала: проблемы и пути их решения. 2019. Т. 14, ч. 3. С. 1069–1077. ( ) 4. Morozov N. A. Basic classifications of methods used in Japan to prevent and combat deviant behavior of youth // Alma Mater. 2013. N 3. P. 93–98. (In rus). References 1. Akhyadov E. S. Domestic and foreign experience in the prevention of neglect and juvenile de- linquency // Young scientist [Molodoi uchenyi]. 2016. N 2. P. 658–662. (In rus). 2. Zhdanova M. A. Study of the problem and practical experience of preventing deviant behavior of adolescents and young people in modern Germany // Problems of pedagogical innovation in vocational education. Materials of the XIX International Scientific and Practical Conference. St. Petersburg: Herzen Russian State Pedagogical University, 2018. 490 p. P. 401–408. (In rus). 3. Kushnir S. I. Experience in preventing criminal behavior of adolescents in the USA and Great Britain: dissertation abstract. Kolomna, 2001. (In rus).i ( ) 4. Morozov N. A. Basic classifications of methods used in Japan to prevent and combat deviant behavior of youth // Alma Mater. 2013. N 3. P. 93–98. (In rus). 4. Morozov N. A. Basic classifications of methods used in Japan to prevent and combat deviant behavior of youth // Alma Mater. 2013. N 3. P. 93–98. (In rus). 104 УПРАВЛЕНЧЕСКОЕ КОНСУЛЬТИРОВАНИЕ . № 1 . 2021 ОБЩЕСТВО И РЕФОРМЫ 5. Tsinchenko G. M., Orlova I. S. Drug abuse of minors as a social problem // Health is the basis of human potential: problems and ways to solve them [Zdorov’e — osnova chelovecheskogo potentsiala: problemy i puti ikh resheniya]. 2019. V. 14, Part 3. P. 1069–1077. (In rus). 5. Tsinchenko G. M., Orlova I. S. Drug abuse of minors as a social problem // Health is the basis of human potential: problems and ways to solve them [Zdorov’e — osnova chelovecheskogo potentsiala: problemy i puti ikh resheniya]. 2019. V. 14, Part 3. P. 1069–1077. (In rus). About the authors:tt Galina M. Tsinchenko, Associate Professor the Chair of Social Technologies of North-West Institute of Management of RANEPA (St. Petersburg, Russian Federation), PhD in Sociology, Associate Professor; tsinchenko-gm@ranepa.ru About the authors:tt ЩЕСТВО И About the authors:tt Galina M. Tsinchenko, Associate Professor the Chair of Social Technologies of North-West Institute of Management of RANEPA (St. Petersburg, Russian Federation), PhD in Sociology, Associate Professor; tsinchenko-gm@ranepa.ru ОБЩ Inna S. Orlova, Associate Professor the Chair of Social Technologies of North-West Institute of Management of RANEPA (St. Petersburg, Russian Federation), PhD in Pedagogic, Associate Professor; oca-oca@mail.ru УПРАВЛЕНЧЕСКОЕ КОНСУЛЬТИРОВАНИЕ . № 1. 2021 105
https://openalex.org/W2750519506	https://europepmc.org/articles/pmc5575668?pdf=render	English	null	Mapping of QTLs for Seed Phorbol Esters, a Toxic Chemical in Jatropha curcas (L.)	Genes	2,017	cc-by	7,507	Kitiya Amkul 1, Kularb Laosatit 1, Prakit Somta 1,* ID , Sangrea Shim 2, Suk-Ha Lee 2, Patcharin Tanya 1 and Peerasak Srinives 1 Patcharin Tanya and Peerasak Srinives 1 Department of Agronomy, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Nakhon Pathom 73140, Thailand; heartless_gg@hotmail.com (K.A.); pigirosa@hotmail.com (K.L.); agrprt@ku.ac.th (P.T.); agrpss@ku.ac.th (P.S.) 2 Department of Plant Science and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea; kev8305@gmail.com (S.S.); sukhalee@snu.ac.kr (S.-H.L.) * Correspondence: agrpks@ku.ac.th; Tel.: +66-34-351887 Received: 5 July 2017; Accepted: 17 August 2017; Published: 18 August 2017 1 Department of Agronomy, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Nakhon Pathom 73140, Thailand; heartless_gg@hotmail.com (K.A.); pigirosa@hotmail.com (K.L.); agrprt@ku.ac.th (P.T.); agrpss@ku.ac.th (P.S.) 1 Department of Agronomy, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Nakhon Pathom 73140, Thailand; heartless_gg@hotmail.com (K.A.); pigirosa@hotmail.com (K.L.); agrprt@ku.ac.th (P.T.); agrpss@ku.ac.th (P.S.) g p ( ) g p ( ) 2 Department of Plant Science and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea; kev8305@gmail.com (S.S.); sukhalee@snu.ac.kr (S.-H.L.) * Correspondence: agrpks@ku ac th; Tel : +66-34-351887 g p g p 2 Department of Plant Science and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea; kev8305@gmail.com (S.S.); sukhalee@snu.ac.kr (S.-H.L.) 2 Department of Plant Science and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea; kev8305@gmail.com (S.S.); sukhalee@snu.ac.kr (S.-H.L.) * Correspondence: agrpks@ku.ac.th; Tel.: +66-34-351887 g ( ); ( ) * Correspondence: agrpks@ku.ac.th; Tel.: +66-34-351887 Received: 5 July 2017; Accepted: 17 August 2017; Published: 18 August 2017 Abstract: Jatropha (Jatropha curcas L.) is an oil-bearing plant that has potential to be cultivated as a biodiesel crop. The seed cake after oil extraction has 40–50% protein that can be used in animal feeds. A major limitation in utilizing the cake is the presence of phorbol esters (PE), a heat-tolerant toxic chemical. To identify the quantitative trait loci (QTLs) for PE, we constructed a genetic linkage map from an F2 population of 95 individuals from a cross “Chai Nat” × “M10” using 143 simple sequence repeat (SSR) markers. M10 is low in seed PE while Chai Nat is high. Seeds from each F2 individual were quantiﬁed for PE content by high performance liquid chromatography. A single marker analysis revealed ﬁve markers from linkage group 3 (LG3) and nine markers from LG8 associated with seed PE. Kitiya Amkul 1, Kularb Laosatit 1, Prakit Somta 1,* ID , Sangrea Shim 2, Suk-Ha Lee 2, Patcharin Tanya 1 and Peerasak Srinives 1 Inclusive composite interval mapping identiﬁed two QTLs, each on LG3 (qPE3.1) and LG8 (qPE8.1) responsible for the PE. qPE3.1 and qPE8.1 accounted for 14.10%, and 15.49% of total variation in seed PE, respectively. Alelle(s) from M10 at qPE3.1 increased seed PE, while at qPE8.1 decreased seed PE. qPE3.1 is a new loci for PE, while qPE8.1 is the same locus with that reported recently for PE. Keywords: phorbol esters; Jatropha; physic nut; quantitative trait loci; QTL; seed toxin genes G C A T T A C G G C A T genes G C A T T A C G G C A T genes Genes 2017, 8, 205; doi:10.3390/genes8080205 www.mdpi.com/journal/genes Mapping of QTLs for Seed Phorbol Esters, a Toxic Chemical in Jatropha curcas (L.) Kitiya Amkul 1, Kularb Laosatit 1, Prakit Somta 1,* ID , Sangrea Shim 2, Suk-Ha Lee 2, Patcharin Tanya 1 and Peerasak Srinives 1 Kitiya Amkul 1, Kularb Laosatit 1, Prakit Somta 1,* ID , Sangrea Shim 2, Suk-Ha Lee 2, Patcharin Tanya 1 and Peerasak Srinives 1 Keywords: phorbol esters; Jatropha; physic nut; quantitative trait loci; QTL; seed toxin 1. Introduction Jatropha or physic nut (Jatropha curcas L.) (2n = 2x = 22) is an oil-bearing plant that has potential to be cultivated as a non-edible oil crop for producing biodiesel. Although Jatropha seed has 30–44% oil with high percentage of monounsaturated oleic and polyunsaturated linoleic acid [1,2] this crop has not been fully domesticated due to several limitations including low seed yield, non-synchronous maturity, and the presence of toxins in seeds. The presence of this chemical in seed cakes after oil extraction from the seeds prevent the use of the cake which contain high protein (40–50%) as raw material for animal feed industry [3]. The principal seed toxins in Jatropha are phorbol esters (PE) and curcin [4,5]. PE, a tumor inducing substance, is tetracyclic tiglian diterpenoids. PE is found in most Jatropha accessions (called toxic Jatropha). Six forms of PE have been isolated from Jatropha in which all of them are likely to be derived from the same phorbol backbone [4,5]. Despite the interest in PE, its biosynthesis pathway is still poorly understood. Nonetheless, geranylgeranyl diphosphate (GGPP) synthase, casbene synthase, terpene synthases, terpene hydroxylase and acyltransferase are believed to implicate in PE biosynthesis [6–10]. A single knockdown of genes for GGPP synthase, terpene synthase and casbene synthases by RNA interference (RNAi) technique reduced up to 80% of PE content in the leaves of Jatropha, while a double knockdown of two of these genes reduced PE content in the leaves to less than 15% of control toxic Jatropha [10]. However, some accessions from www.mdpi.com/journal/genes Genes 2017, 8, 205 2 of 11 Mexico and Guatemala have no or low PE in seeds (non-toxic Jatropha) [11,12]. The low PE seeds are consumed as snack or sweets. The high and low PE seeds can be distinguished only by seed analysis with high performance liquid chromatography (HPLC). Mexico and Guatemala have no or low PE in seeds (non-toxic Jatropha) [11,12]. The low PE seeds are consumed as snack or sweets. The high and low PE seeds can be distinguished only by seed analysis with high performance liquid chromatography (HPLC). Breeding for non-toxic cultivar(s) is a major objective in Jatropha breeding programs, although there are a few reports on genetics of seed PE in Jatropha [12,13]. 1. Introduction Plant breeders are looking for an inexpensive and quick method to differentiate high and low PE seed Jatropha such as through molecular markers, provided that the location(s) of gene(s) controlling seed PE content is known. Recently, King [12] reported that seed PE in Jatropha is controlled by a single dominant gene without xenia effect. They also located a quantitative trait locus (QTL) controlling seed PE onto a genetic linkage map. Although the QTL is closely to several simple sequence repeat (SSR) markers, such the QTL should be validated before using for marker-assisted selection. Therefore, in this study, our objective was to we identify and validate QTLs for PE for Jatropha using different source of non-toxic cultivar. 2.2. Quantiﬁcation of Phorbol Esters Content in Seeds The mature seeds were used to analyze for PE content following the method described by Haas and Mittelbach [14] with minor modiﬁcations. Brieﬂy, kernels were grinded into ﬁne powder. A sample of four grams from each plant was extracted for PE by methanol (analytical grade) for ﬁve hours in a soxhlet apparatus (Buchi Universal Extraction System B-811, Buchi, Flawil, Switzerland). The methanol was then removed from the solvent using a rotary evaporator (Buchi Rotavapor R-205, Buchi, Flawil, Switzerland) to obtain dried extract. The dried extract was dissolved in 25 mL methanol (HPLC grade). The PE content was determined using an HPLC (Waters 600 HPLC system, Waters, Milford, MA, USA) equipped with a reverse phase C-18 column and photodiode array detector. The column temperature was maintained at 25 ◦C and the ﬂow rate was kept at 1 mL min−1. The mobile phase was acetonitrile and water in 80/20 ratio. The PE peaks appeared between 6 and 10 min were detected and integrated at 280 nm. The phorbol 12-myristate 13-acetate (TPA) (Sigma Aldrich, St. Louis, MO, USA) was used as an external standard, which appeared between 20 and 22 min (Figure 1C). The areas under the PE peaks were summed and converted to standard PE equivalent by taking its peak area and concentration. The quantiﬁcation was conducted twice for each plant. The average value of seed PE content of each plant was used for QTL analysis. 2.1. Plant Materials and DNA Extraction An F2 population of 95 individuals was developed by self-pollinating an F1 hybrid derived from a cross between “Chai Nat” (hereafter called “CN”) and “M10” as male and female parents, respectively. Homozygosity in both CN and M10 is higher than 99.0%. M10 is a non-toxic cultivar originated from Mexico possessing low seed PE, while CN is a toxic cultivar of Thailand possessing high PE. Previously, the PE content of M10 and CN was reported to be 0.05 and 1.62 mg/g, respectively [13]. During the self-pollination, ﬂowers of the F1 hybrid were covered with pollen-proof bags to prevent pollen contamination from other plants. The F2 population was grown in an experimental ﬁeld of Kasetsart University, Kamphaeng Saen Campus, Thailand during March 2014 to June 2015. The mature seeds from each plant were individually harvested. Genomic DNA of each plant was extracted from young leaves following a cetyltrimethylammonium bromide (CTAB) method described by Tanya et al. [13]. 2.4. Linkage Map Construction and QTL Analysis 2.4. Linkage Map Construction and QTL Analysis For each marker in the F2 population, plants showing homozygous DNA bands of CN and M10 parents were scored as “2” and “0”, respectively, while plants showing heterozygous DNA bands were scored as “1”. All the markers were checked for segregation distortion using chi-squared test. A genetic linkage map was constructed using software QTL IciMapping 4.0 (Chinese Academy of Agricultural Sciences, Beijing, China) [17]. The markers were grouped using a minimum log of the odds (LOD) of 4. The markers were put in order by “RECORD” and “SARF” functions. Map distance was calculated based on Kosambi’s mapping function. Linkage group was named following the linkage map reported by King et al. [12] using common marker(s). SSR markers associated with seed PE content were determined by likelihood ratio test (LRT) method implemented in the software QTL IciMapping 4.0 using signiﬁcant LOD threshold of 2.5. Locations of the QTLs controlling seed PE were determined by inclusive composite interval mapping (ICIM) [18] implemented in the QTL IciMapping 4.0. Signiﬁcant LOD score for QTLs were obtained from 3000 permutation test at p = 0.05. 2.5. Identiﬁcation of Physical Locations of Markers Flanking QTLs and Genes Associated with QTL Primer sequences of the markers locating around QTL regions detected for PE were subjected to BLASTN analysis (NCBI—National Center for Biotechnology Information, Bethesda, MD, USA) against the Jatropha curcas Database (JCDB) (http://jcdb.xtbg.ac.cn) to identify the physical location and explore potential candidate genes that may implicate in PE biosynthesis. 2.3. SSR Marker Analysis One thousand and eighty SSR markers from the reports of King et al. [12]; Laosatit et al. [8,14]; Tanya et al. [15] and Wang et al. [16] were used to screen for polymorphism between M10 and CN. Polymerase chain reaction (PCR), electrophoresis, and DNA bands visualization were carried out following Laosatit et al. [14]. Polymorphic SSR markers were used to analyze DNA of the F2 plants. 3 of 11 3 of 12 Genes 2017, 8, 205 17, 8, 205 gure 1. High Pressure Liquid Chromatography (HPLC) chromatogram of phorbol esters M10 (non-toxic Jatropha) (A) and Chai Nat (toxic Jatropha) (B) and of phorbol 12-myristate 1 TPA) (C). DHPB: 12-deoxy-16-hydroxyphorbol; PE: phorbol esters; CN: Chai Nat. ure 1. High Pressure Liquid Chromatography (HPLC) chromatogram of phorbol esters (non-toxic Jatropha) (A) and Chai Nat (toxic Jatropha) (B) and of phorbol 12-myristate A) (C). DHPB: 12-deoxy-16-hydroxyphorbol; PE: phorbol esters; CN: Chai Nat. Figure 1. High Pressure Liquid Chromatography (HPLC) chromatogram of phorbol esters in seed of M10 (non-toxic Jatropha) (A) and Chai Nat (toxic Jatropha) (B) and of phorbol 12-myristate 13-acetate (TPA) (C). DHPB: 12-deoxy-16-hydroxyphorbol; PE: phorbol esters; CN: Chai Nat. Figure 1. High Pressure Liquid Chromatography (HPLC) chromatogram of phorbol esters in seed of M10 (non-toxic Jatropha) (A) and Chai Nat (toxic Jatropha) (B) and of phorbol 12-myristate 13-acetate (TPA) (C). DHPB: 12-deoxy-16-hydroxyphorbol; PE: phorbol esters; CN: Chai Nat. 4 of 11 Genes 2017, 8, 205 3.1. Variation of Seed PE in Parents and F2 Population CN and M10 possessed a large difference in seed PE, with 2.62 and 0.05 mg/g, respectively, which is about 52-fold difference in seed PE content. In addition, M10 and CN parents exhibited different types/forms of PE. CN showed ﬁve forms (Figure 1A) while M10 showed two forms which were also present in CN (Figure 1B). Seed PE of the F2 plants distributed continuously skewing toward M10 ranging from 0.30 to 2.89 mg/g with a mean of 1.33 mg/g (Figure 2). This suggests that the seed PE content in Jatropha is a quantitative trait. Genes 2017, 8, 205 5 of 12 Figure 2. Frequency distribution of phorbol esters content in seeds (mg per gram) of the 95 F2 plants derived from the cross Chai Nat × M10. PE was determined by HPLC method. Figure 2. Frequency distribution of phorbol esters content in seeds (mg per gram) of the 95 F2 plants derived from the cross Chai Nat × M10. PE was determined by HPLC method. Figure 2. Frequency distribution of phorbol esters content in seeds (mg per gram) of the 95 F2 plants derived from the cross Chai Nat × M10. PE was determined by HPLC method. Figure 2. Frequency distribution of phorbol esters content in seeds (mg per gram) of the 95 F2 plants derived from the cross Chai Nat × M10. PE was determined by HPLC method. 3.2. SSR Polymorphism and Linkage Map Out of 1080 SSR markers screened in 3.2. SSR Polymorphism and Linkage Map 3.2. SSR Polymorphism and Linkage Map Out of 1080 SSR markers screened in 3.2. SSR Polymorphism and Linkage Map QTL mapping using inclusive composite interval mapping (ICIM) method detected two QTLs, one each on LG3 and LG8, and they were named as qPE3.1 and qPE8.1, respectively (Table 2 and Figure 4). qPE3.1 and qPE8.1 accounted for 14.10% and 15.49% of the total PE variation in the F2 population, respectively. qPE3.1 showed additive effect of −0.09 mg/g and dominant effect of −0.41 mg/g. This QTL showed overdominance effect (d/a = 4.56) (Table 2). Interestingly, allele from M10 at this QTL increased PE content. qPE8.1 showed additive effect of 0.31 mg/g and dominant effect of −0.02 mg/g. Allele from M10 at this QTL decreased the PE content. Altogether, qPE3.1 and qPE8.1 accounted 29.59% of the total PE variation in the population. Figure 3. The genetic linkage map developed from 95 F2 plants derived from the cross Chai Nat × M10 using 143 simple sequence repeat (SSR) markers. Genetic distance was shown in centimorgan unit. Linkage groups were named as per King et al. [6]. Markers showing signiﬁcant deviation from the expected segregation ratio at 0.05, 0.01, and 0.001 probability levels are marked with , , and *, respectively. 3.3. QTL Controlling Seed PE in Jatropha QTL mapping using LRT method revealed that ﬁve markers on LG3 and nine on LG8 associated with PE in the seeds. These markers accounted for 11.74% (CN_SSR199 on LG3) to 16.99% (NG286A on LG8) of the variation of seed PE in the F2 population (Table 1). QTL mapping using inclusive composite interval mapping (ICIM) method detected two QTLs, one each on LG3 and LG8, and they were named as qPE3.1 and qPE8.1, respectively (Table 2 and Figure 4). qPE3.1 and qPE8.1 accounted for 14.10% and 15.49% of the total PE variation in the F2 population, respectively. qPE3.1 showed additive effect of −0.09 mg/g and dominant effect of −0.41 mg/g. This QTL showed overdominance effect (d/a = 4.56) (Table 2). Interestingly, allele from M10 at this QTL increased PE content. qPE8.1 showed additive effect of 0.31 mg/g and dominant effect of −0.02 mg/g. Allele from M10 at this QTL Figure 3. The genetic linkage map developed from 95 F2 plants derived from the cross Chai Nat × M10 using 143 simple sequence repeat (SSR) markers. Genetic distance was shown in centimorgan unit. Figure 3. 3.2. SSR Polymorphism and Linkage Map Out of 1080 SSR markers screened in 3.2. SSR Polymorphism and Linkage Map The genetic linkage map developed from 95 F2 plants derived from the cross Chai Nat × M10 i 143 i l t (SSR) k G ti di t h i ti Figure 3. The genetic linkage map developed from 95 F2 plants derived from the cross Chai Nat × M10 using 143 simple sequence repeat (SSR) markers. Genetic distance was shown in centimorgan unit. Linkage groups were named as per King et al. [6]. Markers showing significant deviation from the expected segregation ratio at 0.05, 0.01, and 0.001 probability levels are marked with , , and , respectively. Figure 3. The genetic linkage map developed from 95 F2 plants derived from the cross Chai Nat × M10 using 143 simple sequence repeat (SSR) markers. Genetic distance was shown in centimorgan unit. Linkage groups were named as per King et al. [6]. Markers showing signiﬁcant deviation from the expected segregation ratio at 0.05, 0.01, and 0.001 probability levels are marked with , , and , respectively. Figure 3. The genetic linkage map developed from 95 F2 plants derived from the cross Chai Nat × M10 using 143 simple sequence repeat (SSR) markers. Genetic distance was shown in centimorgan unit. Linkage groups were named as per King et al. [6]. Markers showing significant deviation from the expected segregation ratio at 0.05, 0.01, and 0.001 probability levels are marked with , , and , respectively. Figure 3. The genetic linkage map developed from 95 F2 plants derived from the cross Chai Nat × M10 using 143 simple sequence repeat (SSR) markers. Genetic distance was shown in centimorgan unit. Linkage groups were named as per King et al. [6]. Markers showing signiﬁcant deviation from the expected segregation ratio at 0.05, 0.01, and 0.001 probability levels are marked with , , and , respectively. 3.2. SSR Polymorphism and Linkage Map Out of 1080 SSR markers screened in 3.2. SSR Polymorphism and Linkage Map from DNA of the CN and/or M10 (Table S1). All except a few of the amplifiable markers showed single DNA band in each parent. Out of 742 amplifiable markers, 143 markers (19.27%; 100 genic and 43 genomic SSRs) showed polymorphism. This indicated a low genetic difference between CN and M10. Out of 1080 SSR markers screened in CN and M10, only 742 markers were able to be ampliﬁed from DNA of the CN and/or M10 (Table S1). All except a few of the ampliﬁable markers showed Genes 2017, 8, 205 5 of 11 single DNA band in each parent. Out of 742 ampliﬁable markers, 143 markers (19.27%; 100 genic and 43 genomic SSRs) showed polymorphism. This indicated a low genetic difference between CN and M10. All the 143 polymorphic markers were used to analyze the F2 population. Among these markers, 14 of them showed segregation distortion. The linkage map of the F2 population developed from the 143 polymorphic SSR markers comprised 11 linkage groups (LG) spanning a total length of 1322.6 cM (Figure 3). The number of markers per LG ranged from 2 (LG7) to 27 (LG2 and LG3) with an average of 14.3. LG7 and LG3 were the shortest and the longest linkages with the length of 12.7 and 312.6 cM, respectively. The average distance between the adjacent markers was 9.3 cM. The distorted markers were distributed onto 5 LGs (Figure 3). Clusters of 4 and 3 distorted markers were found on LG11 and LG6, respectively. Genes 2017, 8, 205 6 of 12 Figure 3. The genetic linkage map developed from 95 F2 plants derived from the cross Chai Nat × M10 using 143 simple sequence repeat (SSR) markers. Genetic distance was shown in centimorgan unit. Linkage groups were named as per King et al. [6]. Markers showing significant deviation from the expected segregation ratio at 0.05, 0.01, and 0.001 probability levels are marked with , , and *, respectively. 3.3. QTL Controlling Seed PE in Jatropha QTL mapping using LRT method revealed that five markers on LG3 and nine on LG8 associated with PE in the seeds. These markers accounted for 11.74% (CN_SSR199 on LG3) to 16.99% (NG286A on LG8) of the variation of seed PE in the F2 population (Table 1). 3.3. QTL Controlling Seed PE in Jatropha QTL mapping using LRT method r 3.3. QTL Controlling Seed PE in Jatropha Log of the odds (LOD) graph of the quantitative trait loci (QTL) qPE3.1 (A) and qPE8.1 (B) controlling seed phorbol esters content detected in the F2 population of Chai Nat × M10 by inclusive composite interval mapping. Figure 4. Log of the odds (LOD) graph of the quantitative trait loci (QTL) qPE3.1 (A) and qPE8.1 (B) controlling seed phorbol esters content detected in the F2 population of Chai Nat × M10 by inclusive composite interval mapping. Figure 4. Log of the odds (LOD) graph of the quantitative trait loci (QTL) qPE3.1 (A) and qPE8.1 (B) controlling seed phorbol esters content detected in the F2 population of Chai Nat × M10 by inclusive composite interval mapping. Figure 4. Log of the odds (LOD) graph of the quantitative trait loci (QTL) qPE3.1 (A) and qPE8.1 (B) controlling seed phorbol esters content detected in the F2 population of Chai Nat × M10 by inclusive composite interval mapping. Figure 4. Log of the odds (LOD) graph of the quantitative trait loci (QTL) qPE3.1 (A) and qPE8.1 (B) controlling seed phorbol esters content detected in the F2 population of Chai Nat × M10 by inclusive composite interval mapping. Figure 4. Log of the odds (LOD) graph of the quantitative trait loci (QTL) qPE3.1 (A) and qPE8.1 (B) controlling seed phorbol esters content detected in the F2 population of Chai Nat × M10 by inclusive composite interval mapping. Table 1. Simple sequence repeat (SSR) markers associated with seed phorbol esters content in F2 population of Chai Nat × M10 detected by likelihood ratio test method. Log of the odds (LOD) value of 2.5 was used as a significant threshold. k % Table 1. Simple sequence repeat (SSR) markers associated with seed phorbol esters content in F2 population of Chai Nat × M10 detected by likelihood ratio test method. Log of the odds (LOD) value of 2.5 was used as a signiﬁcant threshold. Table 1. Simple sequence repeat (SSR) markers associated with seed phorbol esters content in F2 population of Chai Nat × M10 detected by likelihood ratio test method. Log of the odds (LOD) value of 2.5 was used as a significant threshold. Table 1. Simple sequence repeat (SSR) markers associated with seed phorbol esters content in F2 population of Chai Nat × M10 detected by likelihood ratio test method. Log of the odds (LOD) value of 2.5 was used as a signiﬁcant threshold. 3.3. QTL Controlling Seed PE in Jatropha QTL mapping using LRT method r 3.3. QTL Controlling Seed PE in Jatropha QTL mapping using LRT method revealed that five markers on LG3 and nine on LG8 associated with PE in the seeds. These markers accounted for 11.74% (CN_SSR199 on LG3) to 16.99% (NG286A on LG8) of the variation of seed PE in the F2 population (Table 1). QTL mapping using inclusive composite interval mapping (ICIM) method detected two QTLs, one each on LG3 and LG8, and they were named as qPE3.1 and qPE8.1, respectively (Table 2 and Figure 4). qPE3.1 and qPE8.1 accounted for 14.10% and 15.49% of the total PE variation in the F2 population, respectively. qPE3.1 showed additive effect of −0.09 mg/g and dominant effect of −0.41 mg/g. This QTL showed overdominance effect (d/a = 4.56) (Table 2). Interestingly, allele from M10 at this QTL increased PE content. qPE8.1 showed additive effect of 0.31 mg/g and dominant effect of −0.02 mg/g. Allele from M10 at this QTL decreased the PE content. Altogether, qPE3.1 and qPE8.1 accounted 29.59% of the total PE variation in the population QTL mapping using LRT method revealed that ﬁve markers on LG3 and nine on LG8 associated with PE in the seeds. These markers accounted for 11.74% (CN_SSR199 on LG3) to 16.99% (NG286A on LG8) of the variation of seed PE in the F2 population (Table 1). QTL mapping using inclusive composite interval mapping (ICIM) method detected two QTLs, one each on LG3 and LG8, and they were named as qPE3.1 and qPE8.1, respectively (Table 2 and Figure 4). qPE3.1 and qPE8.1 accounted for 14.10% and 15.49% of the total PE variation in the F2 population, respectively. qPE3.1 showed additive effect of −0.09 mg/g and dominant effect of −0.41 mg/g. This QTL showed overdominance effect (d/a = 4.56) (Table 2). Interestingly, allele from M10 at this QTL increased PE content. qPE8.1 showed additive effect of 0.31 mg/g and dominant effect of −0.02 mg/g. Allele from M10 at this QTL Genes 2017, 8, 205 6 of 11 ed the PE content. Altogether, qPE3.1 and qPE8.1 accounted 29.59% of the total PE variation in pulation. s 2017, 8, 205 7 of 12 decreased the PE content. Altogether, qPE3.1 and qPE8.1 accounted 29.59% of the total PE variation in the population. Genes 2017, 8, 205 7 of 12 p p Genes 2017, 8, 205 7 of 12 Figure 4. G273A 15.57 62.14 3. a Linkage group; b Coefficient of determination. a Linkage group; b Coefﬁcient of determination. 3.3. QTL Controlling Seed PE in Jatropha QTL mapping using LRT method r 3.3. QTL Controlling Seed PE in Jatropha QTL Name LG a Marker Interval Position (cM) b LOD Score Confidence Interval (cM) c PVE (%) d Additive Effect e Dominant Effect [d/a] f qPE3.1 3 CN_SSR326–CN_SSR325 153 3.82 152.5–156.5 14.10 −0.09 −0.41 4.56 qPE8.1 8 NG288C–NG286A 50 4.06 48.5–51.5 15.49 0.31 0.02 0.06 a Linkage group; b Position on the linkage group; c 1-LOD support of the QTL; d Percentage of phenotypic variance explained by the QTL; e Additive effect of alleles from Chai Nat 10; f Dominance-to-additive effects ratio. q _ _ qPE8.1 8 NG288C–NG286A 50 4.06 48.5–51.5 15.49 0.31 0.02 0.06 a Linkage group; b Position on the linkage group; c 1-LOD support of the QTL; d Percentage of phenotypic variance explained by the QTL; e Additive effect of alleles from Chai Nat 10; f Dominance-to-additive effects ratio. q _ _ qPE8.1 8 NG288C–NG286A 50 4.06 48.5–51.5 15.49 0.31 0.02 0.06 a Linkage group; b Position on the linkage group; c 1-LOD support of the QTL; d Percentage of phenotypic variance explained by the QTL; e Additive effect of alleles from Chai Nat 10; f Dominance-to-additive effects ratio. 3.4. Physical Location of the PE QTLs and Annotated Genes in the QTL Regions 3.4. Physical Location of the PE QTLs and Annotated Genes in the QTL Regions BLASTN analysis against the JCDB revealed that the markers CN_SSR326 and CN_SSR325 that ﬂank the PE QTL qPE3.1 on LG3 were at the positions 1069652 and 1074572 of the sequence NW012124225.1, respectively. There was only one annotated gene in this region, LOC105634082. LOC105634082 encodes an uncharacterized protein. The markers NG288C and G286A that ﬂank PE QTL qPE8.1 on LG8 were at the positions 242687 and 370487 of the sequence NW012130064.1, respectively (Figure 5). There were 16 annotated genes between these two markers (Table 3). BLASTN analysis against the JCDB revealed that the markers CN_SSR326 and CN_SSR325 that flank the PE QTL qPE3.1 on LG3 were at the positions 1069652 and 1074572 of the sequence NW012124225.1, respectively. There was only one annotated gene in this region, LOC105634082. LOC105634082 encodes an uncharacterized protein. The markers NG288C and G286A that flank PE QTL qPE8.1 on LG8 were at the positions 242687 and 370487 of the sequence NW012130064.1, respectively (Figure 5). There were 16 annotated genes between these two markers (Table 3). Figure 5. 3.3. QTL Controlling Seed PE in Jatropha QTL mapping using LRT method r 3.3. QTL Controlling Seed PE in Jatropha LG Marker Name R (%) Position LOD Score 3 CN_SSR199 11.74 145.32 2.58 3 CN_SSR327 12.25 150.18 2.70 3 CN_SSR326 15.88 152.84 3.57 3 CN_SSR330 11.67 165.73 2.56 3 CN_SSR336 12.88 198.11 2.84 8 NG291 14.20 47.11 3.16 8 NG288C 13.06 48.22 2.89 8 NG286A 16.99 50.41 3.84 8 MPN280 16.40 51.48 3.70 8 G262 13.55 54.15 3.00 8 G270B 15.69 55.21 3.52 8 G270D 15.64 57.34 3.51 8 G282B 13.94 59.47 3.10 8 G273A 15.57 62.14 3.49 a Linkage group; b Coefficient of determination. LG a Marker Name R2 (%) b Position LOD Score 3 CN_SSR199 11.74 145.32 2.58 3 CN_SSR327 12.25 150.18 2.70 3 CN_SSR326 15.88 152.84 3.57 3 CN_SSR330 11.67 165.73 2.56 3 CN_SSR336 12.88 198.11 2.84 8 NG291 14.20 47.11 3.16 8 NG288C 13.06 48.22 2.89 8 NG286A 16.99 50.41 3.84 8 MPN280 16.40 51.48 3.70 8 G262 13.55 54.15 3.00 8 G270B 15.69 55.21 3.52 8 G270D 15.64 57.34 3.51 8 G282B 13.94 59.47 3.10 8 G273A 15.57 62.14 3.49 a Linkage group; b Coefﬁcient of determination. 7 of 11 7 of 11 Genes 2017, 8, 205 Table 2. Locations and genetic effects of quantitative trait loci (QTLs) detected for seed phorbol esters content in the F2 population of Chai Nat × M10 by inclusive composite interval mapping. Table 2. Locations and genetic effects of quantitative trait loci (QTLs) detected for seed phorbol esters content in the F2 population of Chai Nat × M10 by inclusive composite interval mapping. QTL Name LG a Marker Interval Position (cM) b LOD Score Conﬁdence Interval (cM) c PVE (%) d Additive Effect e Dominant Effect [d/a] f qPE3.1 3 CN_SSR326–CN_SSR325 153 3.82 152.5–156.5 14.10 −0.09 −0.41 4.56 qPE8.1 8 NG288C–NG286A 50 4.06 48.5–51.5 15.49 0.31 0.02 0.06 a Linkage group; b Position on the linkage group; c 1-LOD support of the QTL; d Percentage of phenotypic variance explained by the QTL; e Additive effect of alleles from Chai Nat 10; f Dominance-to-additive effects ratio. 3.3. QTL Controlling Seed PE in Jatropha QTL mapping using LRT method r 3.3. QTL Controlling Seed PE in Jatropha Comparative map illustrating the position of qPE8.1 for seed phorbol esters content in the F2 population of Chai Nat × M10 identified in this study (right) and the location of locus controlling seed phorbol esters in the F2 population of G33 × G43 reported by King et al. [12] (left). Lines connect common markers between the two maps. Small solid bars along the maps indicate confidence intervals of the locus detected for seed phorbol esters. The QTL for PE reported by King was initially mapped between markers JCT3 and 1401433/12335072 by considering the PE as a quantitative trait. The PE locus was then fine mapped between markers NG285A and G273A by considering PE as a qualitative trait. Three solid bars along the map of Chai Nat × M10 represent Jatropha reference sequence from Jatropha curcas Database (JCDB). Figure 5. Comparative map illustrating the position of qPE8.1 for seed phorbol esters content in the F2 population of Chai Nat × M10 identiﬁed in this study (right) and the location of locus controlling seed phorbol esters in the F2 population of G33 × G43 reported by King et al. [12] (left). Lines connect common markers between the two maps. Small solid bars along the maps indicate conﬁdence intervals of the locus detected for seed phorbol esters. The QTL for PE reported by King was initially mapped between markers JCT3 and 1401433/12335072 by considering the PE as a quantitative trait. The PE locus was then ﬁne mapped between markers NG285A and G273A by considering PE as a qualitative trait. Three solid bars along the map of Chai Nat × M10 represent Jatropha reference sequence from Jatropha curcas Database (JCDB). Figure 5. Comparative map illustrating the position of qPE8.1 for seed phorbol esters content in the F o ulatio of Chai Nat × M10 ide tified i thi tudy ( i ht) a d the lo atio of lo u o t olli Figure 5. Comparative map illustrating the position of qPE8.1 for seed phorbol esters content in the Figure 5. Comparative map illustrating the position of qPE8.1 for seed phorbol esters content in the F2 population of Chai Nat × M10 identified in this study (right) and the location of locus controlling seed phorbol esters in the F2 population of G33 × G43 reported by King et al. [12] (left). Lines connect common markers between the two maps. 3.3. QTL Controlling Seed PE in Jatropha QTL mapping using LRT method r 3.3. QTL Controlling Seed PE in Jatropha Small solid bars along the maps indicate confidence intervals of the locus detected for seed phorbol esters. The QTL for PE reported by King was initially mapped between markers JCT3 and 1401433/12335072 by considering the PE as a quantitative trait. The PE locus was then fine mapped between markers NG285A and G273A by considering PE as a qualitative trait. Three solid bars along the map of Chai Nat × M10 represent Jatropha reference sequence from Jatropha curcas Database (JCDB). Figure 5. Comparative map illustrating the position of qPE8.1 for seed phorbol esters content in the F2 population of Chai Nat × M10 identiﬁed in this study (right) and the location of locus controlling seed phorbol esters in the F2 population of G33 × G43 reported by King et al. [12] (left). Lines connect common markers between the two maps. Small solid bars along the maps indicate conﬁdence intervals of the locus detected for seed phorbol esters. The QTL for PE reported by King was initially mapped between markers JCT3 and 1401433/12335072 by considering the PE as a quantitative trait. The PE locus was then ﬁne mapped between markers NG285A and G273A by considering PE as a qualitative trait. Three solid bars along the map of Chai Nat × M10 represent Jatropha reference sequence from Jatropha curcas Database (JCDB). 8 of 11 Genes 2017, 8, 205 Table 3. Annotated genes located between markers CN_SSR325 and CN_SSR326 ﬂanking qPE3.1 and between markers NG288C and NG286A ﬂanking QTL qPE8.1 that controls seed phorphol esters content detected in the Jatropha F2 population of Chai Nat × M10 by inclusive composite interval mapping. The data are from Jatropha curcas Database (JCDB). 4. Discussion King et al. [12] reported that the presence of PE is a qualitative trait controlled by a single dominant gene. They identiﬁed a single major QTL controlling the trait. On the contrary, we found in this study that the presence of PE is a quantitative trait (Figure 1). Recently, Ng [10] reported that down regulate single gene expression of GGPP synthase, terpene synthase and casbene synthases reduced up to 80% of PE content in the leaves of Jatropha, while a double down regulate of two genes reduced PE content in the leaves to 85%. This suggested that PE contents in Jatropha is a quantitative trait. Thus, our results agreed with that report of Ng [10]. The contrasting ﬁndings between our study and that of King et al. [12] may stem from the fact that the jatropha germplasm used by King et al. [12] was different from that used in this study. The non-toxic Jatropha (G43) used by them contained no PE, while the one (M10) used in this study contained very low PE. G43 was from Guatemala, while M10 was from Mexico. In addition, the extraction methods used to determine PE in the two studies were different. It is also worth noting that non-toxic (M10) and the toxic (CN) Jatropha parents used in this study exhibited different types/forms of PE (Figure 1A,B). Regardless the above argument on germplasm and quantifying methods, the location of qPE8.1 controlling seed PE detected in our study was similar to the QTL conferring seed PE reported by King et al. [12]. qPE8.1 was located between markers NG288C and NG286A which is within the genomic region reported by them (Figure 5). This suggested that the QTLs controlling seed PE for Jatropha identiﬁed in these two studies are the same locus. Thus, this QTL was validated and can be used for marker-assisted selection (MAS). qPE3.1 is a new QTL controlling seed PE content in Jatropha. In contrast to qPE8.1, the allele contributed by CN at qPE3.1 reduced PE content (Table 2). In addition, qPE3.1 showed strong dominant effect or overdominance effect (Table 2). This suggests that this QTL is possibly a cluster of linked loci for PE that resulting in pseudo-overdominance effect. Nonetheless, qPE3.1 in combination with qPE8.1 will be useful for MAS to develop new Jatropha line(s) possessing low seed PE and for map-based cloning of the gene(s) controlling PE. 3.3. QTL Controlling Seed PE in Jatropha QTL mapping using LRT method r 3.3. QTL Controlling Seed PE in Jatropha qPE3.1 Name Position Annotation GO Term KEGG LOC105634082 NW_012124225.1:1070722..1072343 Uncharacterized protein At4g22758 - - qPE8.1 Name Position Annotation GO Term KEGG LOC105650633 NW_012130064.1:251091..252362 Peptidyl-prolylcis-trans isomerase CYP26-2, chloroplastic GO:0003755 K03768 LOC105650634 NW_012130064.1:244937..251195 Uncharacterized LOC105650634 GO:0008270 K03768 LOC105650635 NW_012130064.1:253456..257226 Single-stranded DNA-binding protein, mitochondrial GO:0003697 K03111 LOC105650637 NW_012130064.1:259463..262260 Uncharacterized LOC105650637 - - LOC105650638 NW_012130064.1:262323..266102 Uncharacterized LOC105650638 - - LOC105650639 NW_012130064.1:267598..271421 Zinc ﬁnger BED domain-containing protein DAYSLEEPER GO:0003677, GO:0046983, GO:0003676 K03680 LOC105650640 NW_012130064.1:273262..278864 Translation initiation factor eIF-2B subunit delta GO:0044237 K03680 LOC105650641 NW_012130064.1:288075..288870 Oleosin 1-like GO:0016021, GO:0012511 - LOC105650642 NW_012130064.1:291937..292732 Uncharacterized LOC105650642 - K13111 LOC105650712 NW_012130064.1:279771..287264 Pseudogene - - LOC105650643 NW_012130064.1:297597..314028 Suppressor of mec-8 and unc-52 protein homolog 1 GO:0005515 K13111 LOC105650713 NW_012130064.1:317012..317392 Inactive protein FON2 SPARE1-like - - LOC105650644 NW_012130064.1:324023..347807 Transcription initiation factor TFIID subunit 2 GO:0008237, GO:0008270, GO:0005488 K03128 LOC105650714 NW_012130064.1:367045..368556 Transcription factor CYCLOIDEA - K08735 LOC105650645 NW_012130064.1:376579..386656 DNA mismatch repair protein MSH2 GO:0005524, GO:0030983, GO:0006298 K08735 LOC105650646 NW_012130064.1:392247..402394 Uncharacterized LOC105650646 GO:0005515, GO:0008270 - GO Term: Gene Ontology Term; KEGG: Kyoto Encyclopedia of Genes and Genomes. GO Term: Gene Ontology Term; KEGG: Kyoto Encyclopedia of Genes and Genomes. Genes 2017, 8, 205 9 of 11 4. Discussion In this study, two QTLs were detected explaining a total of 29.59% of PE variation in the F2 Jatropha population. The low total variance explained by the QTLs suggested that the PE is highly affected by environments. However, since the F2 population comprised only 95 plants, it is possible that QTLs with small effects controlling PE (if any exist) were not detected. In addition, effect of QTL detected under small population size is likely to be overestimated, while its position cannot be located accurately [19]. Therefore, number and effects of the QTLs for PE found in this study must be considered carefully before using in MAS and map-based cloning. Based on BLASTN analysis against the JCDB, there were 17 annotated genes between the ﬂanking markers of qPE3.1 and qPE.81 (Table 3). However, none of these genes encodes enzyme that possibly involves in PE biosynthesis such as GGPP synthase, casbene synthase, terpene synthases, terpene hydroxylase and acyltransferase [6–10]. Nevertheless, the marker ﬂanking qPE3.1 and qPE.81 will be useful for MAS and these genome regions can be used as target for ﬁne mapping to identify gene controlling PE content in Jatropha. Tanya et al. [15] studied diversity of jatropha using genic SSR markers and reported that markers MPN006, MPN007, MPN008, MPN016 and MPN046 were able to classify toxic and non-toxic Jatropha accessions. Nonetheless, in this study, none of these markers showed polymorphism between toxic and non-toxic Jatropha parents used in this study. This result indicated that these markers cannot be used for classifying high and low toxic Jatropha germplasm, at least between CN and M10. Supplementary Materials: The following are available online at www.mdpi.com/2073-4425/8/8/205/s1. Table S1: Sequences and polymorphism of 742 ampliﬁable primers to screen polymorphism between Jotropha cultivars Chai Nat and M10. Acknowledgments: This work was supported by the project on Breeding to Accelerate Domestication of Novel Jatropha for Fuel and Feeds (Grant No. P-11-00599) of the National Science and Technology Development Agency, Ministry of Science and Technology, Thailand. Part of this work was also supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ011027) of the Rural Development Administration, Korea. 10 of 11 Genes 2017, 8, 205 Author Contributions: P. So., P. Sr., S.H.L. conceived and designed the experiments; K.A., K.L., S.S., P.T. performed the experiments; K.A., K.L., P. So. analyzed the data; P. So., P. Sr. wrote the manuscript. All authors read and approved the ﬁnal manuscript. 4. Discussion Author Contributions: P. So., P. Sr., S.H.L. conceived and designed the experiments; K.A., K.L., S.S., P.T. performed the experiments; K.A., K.L., P. So. analyzed the data; P. So., P. Sr. wrote the manuscript. All authors read and approved the ﬁnal manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Gübitz, G.M.; Mittelbach, M.; Trabi, M. Exploitation of the tropical oil seed plant Jatropha curcas L. Bioresour. Technol. 1999, 67, 73–82. [CrossRef] 2. Openshaw, K. A review of Jatropha curcas: An oil plant of unfulﬁlled promise. Biomass Bioenergy 2000, 19, 1–15. [CrossRef] 3. Achten, W.M.J.; Nielsen, L.R.; Aerts, R.; Lengkeek, A.G.; Kjaer, E.D.; Trabucco, A.; Hansen, J.K.; Maes, W.H.; Graudal, L.; Akinnifesi, F.K.; et al. Towards domestication of Jatropha curcas. Biofuels 2010, 1, 91–107. [CrossRef] 4. Haas, H.S.; Sterk, H.; Mittelbach, M. 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https://openalex.org/W2955386368	https://europepmc.org/articles/pmc6630999?pdf=render	English	null	The Influence of Different Cholecalciferol Supplementation Regimes on 25(OH) Cholecalciferol, Calcium and Parathyroid Hormone after Bariatric Surgery	Medicina	2,019	cc-by	3,325	Received: 17 March 2019; Accepted: 22 May 2019; Published: 6 June 2019 Abstract: Background and objectives: Vitamin D is an essential vitamin that plays a key role in maintaining physiological calcium balance, and is also a pivotal element in the formation of bone structure. Vitamin D deﬁciency is associated with a wide array of clinical symptoms. Vitamin and mineral deﬁciencies are quite common prior to and after bariatric surgery, and therefore we have evaluated the eﬀects of two diﬀerent cholecalciferol supplementation regimes on serum calcium, 25(OH) cholecalciferol, and parathyroid hormone (PTH). Materials and Methods: In this retrospective matched cohort study, two diﬀerent cholecalciferol supplementation regimes were compared. Group A consisted of 50 patients who had 1000 mg calcium and 800 IU cholecalciferol. In Group B, 50 patients had 1000 mg calcium and 800 IU cholecalciferol with an additional 1 mL liquid cholecalciferol (50,000 IU) monthly. The primary outcome was the eﬀects on blood serum levels of calcium, 25(OH) cholecalciferol, and PTH. Results: In group A and group B, there were signiﬁcant increases in 25(OH) cholecalciferol, with a higher delta in favor of group B (for all three p < 0.001). A decrease was seen in PTH (p < 0.001), and no diﬀerences were measured in calcium levels in both groups. Conclusion: Our study suggests that an additional 1 mL cholecalciferol (50,000 IU) monthly can result in less biochemically 25(OH) cholecalciferol deﬁcient patients after bariatric surgery. No eﬀects were seen on the calcium balance. However, larger randomized clinical trials need to be done to assess the eﬀects on clinical outcomes like bone health and fracture risk. Keywords: vitamin D supplementation; cholecalciferol; parathyroid hormone; serum vitamin D; bariatric surgery medicina medicina Article Hendrika J.M. Smelt 1,2, Sjaak Pouwels 3,* and Johannes F. Smulders 2,4 Hendrika J.M. Smelt 1,2, Sjaak Pouwels 3,* and Johannes F. Smulders 2,4 Hendrika J.M. Smelt 1,2, Sjaak Pouwels 3,* and Johannes F. Smulders 2,4 1 Department of Nutrition, Catharina Hospital, 5623 EJ Eindhoven, The Netherlands; marieke.smelt@catharinaziekenhuis.nl 2 Obesity Center, Catharina Hospital, 5623 EJ Eindhoven, The Netherlands; frans.smulders@catharinaziekenhuis.nl 3 Department of Surgery, Haaglanden Medical Center, 2512 VA The Hague, The Netherlands 4 Department of Surgery, Catharina Hospital, 5623 EJ Eindhoven, The Netherlands * Correspondence: Sjaakpwls@gmail.com Hendrika J.M. Smelt 1,2, Sjaak Pouwels 3,* and Johannes F. Smulders 2,4 1 Department of Nutrition, Catharina Hospital, 5623 EJ Eindhoven, The Netherlands; marieke.smelt@catharinaziekenhuis.nl 2 Obesity Center, Catharina Hospital, 5623 EJ Eindhoven, The Netherlands; frans.smulders@catharinaziekenhuis.nl 3 Department of Surgery, Haaglanden Medical Center, 2512 VA The Hague, The Netherlands 4 Department of Surgery, Catharina Hospital, 5623 EJ Eindhoven, The Netherlands * Correspondence: Sjaakpwls@gmail.com 2 Obesity Center, Catharina Hospital, 5623 EJ Eindhoven, The Netherlands; frans.smulders@catharinaziekenhuis.nl 3 Department of Surgery, Haaglanden Medical Center, 2512 VA The Hague, The Netherlands 4 Department of Surgery, Catharina Hospital, 5623 EJ Eindhoven, The Netherlands * Correspondence: Sjaakpwls@gmail.com 1. Introduction In general, vitamin D is part of the complex physiology maintaining calcium balance and bone structure, but also aids in the absorption of calcium from the intestine and reabsorption in the distal renal tubules (in presence of parathyroid hormone (PTH)). Deﬁciencies in vitamin D can increase the risk of osteoporosis and fractures, but also have eﬀects on the immune system and muscle strength [1]. A vitamin D deﬁciency is often accompanied by a reduced dietary calcium intake, but this does not necessarily aﬀect the serum calcium [1,2]. Physiologically, when there is a ﬂuctuation in calcium levels, PTH levels will also ﬂuctuate. In case of a decreased blood calcium level, PTH will www.mdpi.com/journal/medicina www.mdpi.com/journal/medicina Medicina 2019, 55, 252; doi:10.3390/medicina55060252 Medicina 2019, 55, 252 2 of 6 increase [1,2]. Clinical symptoms of a hypocalcaemia can vary, but symptoms like leg cramping, tetany, and neuromuscular hyper excitability are often reported [2]. increase [1,2]. Clinical symptoms of a hypocalcaemia can vary, but symptoms like leg cramping, tetany, and neuromuscular hyper excitability are often reported [2]. Vitamin D deﬁciencies are common in bariatric surgical practice, and the reported prevalence prior to surgery varies between 54% and 80% [3]. In patients with obesity, this is often a combination of several problems: (1) Limited sun exposure, (2) decreased bioavailability of vitamin D due to sequestration in the excess fatty tissue, and (3) inadequate dietary intake of vitamins and minerals [3]. A secondary hyperparathyroidism can also contribute negatively because it results in increased 25(OH)D hydroxylation, therefore decreasing vitamin D [2,3]. In addition to hyperparathyroidism, several cases of osteomalacia have been described following bariatric surgery [4,5]. It needs to be taken into account that these are rare phenomena. Currently, it is advised by several guidelines that blood levels of vitamin D of ≥75–80 nmol/L are optimal, especially for patients before and after bariatric surgery [6,7]. However, if deﬁciencies occur, there is no consensus on how we should supplement these patients to achieve normal vitamin D levels. In our bariatric practice, we have used several supplementation regimes. One of them was 1000 mg oral calcium carbonate and 800 IU oral cholecalciferol daily from three weeks postoperatively. Unfortunately, too many patients remained deﬁcient after this regimen, and therefore we added additional cholecalciferol to our supplementation protocol. However, it is unclear whether this switch has eﬀects on 25(OH) cholecalciferol, PTH, and calcium. 1. Introduction We aimed to evaluate the eﬀects of these supplementation regimes on blood levels of calcium, 25(OH) cholecalciferol, and PTH. Therefore, we hypothesize that a supplementation regime of 800 IU oral and additional cholecalciferol monthly and lifelong leads to higher 25(OH) cholecalciferol levels and less deﬁciencies. 2.1. Patient Selection In this pilot study, we included 100 patients who have had bariatric surgery in the period of June 2015 until January 2016. All patients underwent a sleeve gastrectomy or Roux-en-Y gastric bypass either as a primary or a revisional procedure in the Obesity Center Catharina Hospital Eindhoven, the Netherlands. Patients with kidney disease or gastrointestinal disorders suggestive for malabsorption were excluded from this study. The included patients from group A (n = 50) were selected on the operation date in June 2015 and started with our standard supplementation regime of 1000 mg oral calcium carbonate and 800 IU oral cholecalciferol. The included patients from group B (n = 50) were selected on the operation date in January 2016 and used the previously mentioned supplementation regime and additional 50,000 IU liquid cholecalciferol monthly. Both groups were matched for age, gender, preoperative body mass index (BMI), current BMI, and surgical procedure. Due to the retrospective nature of this study, approval from the Institutional Review Board of the Catharina Hospital Eindhoven was not needed. 2.2. Blood Sampling and Deﬁnitions Baseline blood analysis of all patients was done three months preoperatively and six months postoperatively. For this study, serum calcium, vitamin D, and PTH were assessed. Vitamin D deﬁciency was deﬁned as serum vitamin D < 75 nmol/L. Vitamin D (25-hydroxy vitamin D) was determined in serum by an immunometric competition assay on Liason® using Diasorin® reagents. Reference values of calcium and PTH were 2.10–2.55 mmol/L and 1.6–6.9 pmol/L, respectively. 2.3. Correction of Deﬁciencies Preoperatively Preoperative deﬁciencies in all patients was treated with cholecalciferol. Dosage: 50,000 IU/week for the ﬁrst six weeks, and afterwards 50,000 IU monthly up to the bariatric procedure. Medicina 2019, 55, 252 3 of 6 2.4. Statistical Analysis Data were retrospectively collected, managed, and analyzed using SPSS version 22, for Windows (SPSS Inc., IBM Corporation, Armonk, NY, USA). Quantitative data are denoted as mean ± standard deviation (SD). Categorical variables were presented as frequencies with percentages. The χ-square test was used to compare categorical variables among groups. Distribution of the data was veriﬁed using the Shapiro–Wilks test. Depending on the distribution, the paired t-test was used for parametric data and the Mann–Whitney U test for non-parametric data. To analyze diﬀerences in calcium, 25(OH) cholecalciferol, and PTH levels, the deltas were calculated (follow-up levels minus baseline levels), and these values were compared between groups using the one-way analysis of variance (ANOVA) test. Deltas were determined by the following calculation: Follow-up levels minus baseline levels. A p-value ≤0.05 was considered statistically signiﬁcant. Summative ﬁgures and tables were used where necessary. 3. Results This work has been presented at the IFSO World Congress in Dubai and the meeting abstract was published in Obesity Surgery [8]. Table 1 gives an overview of the baseline characteristics of the patients included in each group. None of the included patients had symptoms of hypocalcaemia. In case of a Roux-en-Y Gastric Bypass (RYGB), a Roux limb of 150 cm was used. Table 1. Baseline characteristics of the included patients (n = 100) (mean ± standard deviation (SD)). Diﬀerent Groups Group A (n = 50) Group B (n = 50) p Value Age (years) 43.8 ± 11.6 47.5 ± 9.7 p = 0.075 Gender (n) - Male:female 9:41 10:40 p = 0.799 Preoperative body mass index (kg/m2) 42.6 ± 5.7 42.5 ± 5.2 p = 0.911 Current body mass index (kg/m2) 31.8 ± 4.6 31.7 ± 4.6 p = 0.879 Procedures (n) p = 0.359 - SG 28 29 - RYGB 18 20 - Revision surgery 4 1 SG = Sleeve gastrectomy, RYGB = Roux-en-Y gastric bypass. Table 1. Baseline characteristics of the included patients (n = 100) (mean ± standard deviation (SD)). 3.1. 25(OH) Cholecalciferol 3.4. Sub-Analysis Between Sleeve Gastrectomy and Roux-En-Y Gastric Bypass In group A, no signiﬁcance diﬀerences were found in 25(OH) cholecalciferol (p = 0.573), calcium (p = 0.341), or PTH (p = 0.995) preoperatively. No signiﬁcance diﬀerences were found in 25(OH) cholecalciferol (p = 0.851), calcium (p = 0.080), or PTH (p = 0.482) between laparoscopic sleeve gastrectomy (LSG) and RYGB patients six months postoperatively. In group B, no signiﬁcance diﬀerences were found in 25(OH) cholecalciferol (p = 0.970), calcium (p = 0.796), or PTH (p = 0.127) preoperatively. No signiﬁcance diﬀerences were found in 25(OH) cholecalciferol (p = 0.984), calcium (p = 0.615), or PTH (p = 0.992) between LSG and RYGB patients six months postoperatively. 3.1. 25(OH) Cholecalciferol 3.1. 25(OH) Cholecalciferol Both groups showed increases in 25(OH) cholecalciferol levels in the follow-up (p < 0.001 for groups A and B) (Table 2). Of all included patients, 91 patients had a 25(OH) cholecalciferol deﬁciency (<75 nmol/L) preoperatively. In group A, 47 patients had a 25(OH) cholecalciferol deﬁciency at baseline, and 35 had it in the follow-up. In group B, 44 patients had a 25(OH) cholecalciferol deﬁciency at baseline, and 10 had it in the follow-up. 4 of 6 Medicina 2019, 55, 252 Table 2. An overview of the baseline and postoperative levels of 25(OH) cholecalciferol, parathyroid hormone (PTH), and calcium levels per group (mean ± SD). Baseline Levels Levels Six Months Postoperatively p Value Group A - 25(OH) cholecalciferol 37.8 ± 20.6 66.7 ± 18.5 p < 0.001 - PTH 7.5 ± 3.1 6.5 ± 2.9 p = 0.032 - Calcium 2.37 ± 0.08 2.39 ± 0.08 p = 0.058 Group B - vitamin D 47.0 ± 21.5 94.2 ± 25.7 p < 0.001 - PTH 6.8 ± 2.7 5.2 ± 1.6 p < 0.001 - Calcium 2.38 ± 0.11 2.38 ± 0.09 p = 0.930 3 2 C l i d P th id H L l Baseline Levels Levels Six Months Postoperatively p Value 3.2. Calcium and Parathyroid Hormone Levels Calcium and Parathyroid Hormone Levels No diﬀerences were seen in serum calcium in group A (p = 0.058) and group B (p = 0.930). Decreases in PTH were seen in the follow-up in group A (p < 0.032) and group B (p < 0.001) (Table 2). No diﬀerences were seen in serum calcium in group A (p = 0.058) and group B (p = 0.930). Decreases in PTH were seen in the follow-up in group A (p < 0.032) and group B (p < 0.001) (Table 2). 3.3. Delta (∆) of 25(OH) cholecalciferol, PTH, and Calcium between Both Groups 3.3. Delta (∆) of 25(OH) cholecalciferol, PTH, and Calcium between Both Groups A signiﬁcant diﬀerence in delta 25(OH) cholecalciferol (p < 0.01) was seen between group A (28.9 ± 16.7) and group B (47.2 ± 24.3), in favour of group B. Delta PTH in group A was –1.0 ± –0.3, and in group B it was –1.6 ± –1.1, but this diﬀerence was not signiﬁcant (p = 0.336). No signiﬁcant diﬀerence was seen in ∆calcium (p = 0.185). 3.4. Sub-Analysis Between Sleeve Gastrectomy and Roux-En-Y Gastric Bypass 6. Conclusions According to this pilot study, a standard daily supplementation regime of 800 IU orally and additional liquid cholecalciferol of 50,000 IU monthly results in higher serum 25(OH) cholecalciferol and less deﬁciencies. This regimen cannot treat all vitamin D deﬁciencies, and therefore, lifelong follow-up is necessary. A randomized clinical trial is necessary to investigate diﬀerent vitamin D supplementation regimens, the inﬂuence on PTH, and the inﬂuence of calcium supplementation after bariatric surgery. Author Contributions: Conceptualization, H.J.M.S., S.P., and J.F.S.; data curation, H.J.M.S., S.P., and J.F.S.; formal analysis, H.J.M.S., S.P., and J.F.S. Author Contributions: Conceptualization, H.J.M.S., S.P., and J.F.S.; data curation, H.J.M.S., S.P., and J.F.S. analysis, H.J.M.S., S.P., and J.F.S. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Limitations Only biochemical outcomes were presented in this study. The clinical outcomes were not mentioned. A larger and better-designed study including DEXA scans is necessary to understanding the eﬀects on calcium metabolism and bone density. The eﬀect of calcium supplementation was not included in this study. However, there is no clear policy for calcium supplementation and no large randomized trials that conﬁrm the optimal regime after bariatric surgery [9]. Finally, there is a known seasonal variability regarding (metabolites of) vitamin D, and it is therefore diﬃcult to correct for this statistically. Furthermore, to determine clinical outcomes (osteoporosis risk and fracture incidence), a minimum follow-up of a few years is needed. 4. Discussion A standard supplementation regime of 800 IU oral cholecalciferol and 50,000 IU liquid cholecalciferol monthly leads to less deﬁciencies and results in a faster decline of PTH. There is no consensus about the right dose of vitamin D supplementation after bariatric surgery [9,10]. The recommended dosage of elemental calcium ranges from 1200 to 2000 mg daily [2,11,12]. Supplementation with 400–800 IU of vitamin D might not provide adequate protection for postoperative patients against an increase in PTH and bone resorption [13,14]. In the study of Flores et al., 56% of the bariatric patients had vitamin D levels of >75 nmol/L one year postoperatively by using 2000 IU vitamin D /day [15]. The study of Goldner et al. describes vitamin D levels of >75nmol/L in 44% of the patients with 800 IU vitamin D/day and in 78% of the patients with 2000 IU vitamin D/day [16,17]. p y p y In our study, the cholecalciferol supplementation regimen with 800 IU cholecalciferol was simply not enough to biochemically restore 25(OH) cholecalciferol levels in 35 of the 50 patients. The standard supplementation regimen with additional 50,000 IU cholecalciferol monthly showed that 10 of the 50 patients were still having a deﬁciency. A higher cholecalciferol dosage gives a faster decline of PTH and may result in diﬀerences in absorption. The high-dose cholecalciferol regime gives better results (in terms of a more prominent decrease in patients with a cholecalciferol deﬁciency). However, there are still a small proportion of patients that remain deﬁcient, despite the high doses of cholecalciferol. Medicina 2019, 55, 252 5 of 6 This might be related to compliance of intake of the supplements. This is the most diﬃcult point in this study because this is unfortunately diﬃcult to control. Next to the above-mentioned factors, we need to take procedure-speciﬁc inﬂuences into account, because bariatric procedures are not all the same regarding absorption of vitamins and minerals, especially calcium and vitamin D [7,9]. Calcium is mainly absorbed in the duodenum and proximal jejunum, which is facilitated by the presence of vitamin D in an acidic environment [9]. Vitamin D, on the other hand, is absorbed in the distal jejunum and the ileum. As mal- or hypo-absorptive eﬀects increase, so does the hypo-absorption of vitamins in speciﬁc parts of the small intestine [2]. 4. Discussion yp p p p However, in this study, no signiﬁcant diﬀerences in 25(OH) cholecalciferol, calcium, and PTH were found between the diﬀerent surgical procedures. . Recker, R.R. Calcium absorption and achlorhydria. N. Engl. J. Med. 1985, 313, 70–73. [CrossRef] [PubM References 1. Gumieiro, D.N.; Murino Rafacho, B.P.; Buzati Pereira, B.L.; Cavallari, K.A.; Tanni, S.E.; Azevedo, P.S.; Polegato, B.F.; Mamede Zornoﬀ, L.A.; Dinhane, D.I.; Innocenti Dinhane, K.G.; et al. Vitamin D serum levels are associated with handgrip strenght but not with muscle mass or length of hospital stay after hip fracture. Nutrition 2015, 31, 931–934. [CrossRef] [PubMed] 2. Aills, L.; Blankenship, J.; Buﬃngton, C.; Furtado, M.; Parrott, J. ASMBS allied health nutritional guidelines for the surgical weight loss patient. Surg. Obes. Relat. Dis. 2008, 4, S73–S108. [CrossRef] [PubMed] 3. Aarts, E.; Van Groningen, L.; Horst, R.; Telting, D.; Van Sorge, A.; Janssen, I.; De Boer, H. Vitamin D absorption Consequences of gastric bypass surgery. Eur. J. Endocrinol. 2011, 164, 827–832. [CrossRef] [PubMed] 4. Chakhtoura, M.T.; Nakhoul, N.N.; Shawwa, K.; Mantzoros, C.; El Hajj Fuleihan, G.A. Hypovitaminosis D in bariatric surgery: A systematic review of observational studies. Metabolism 2016, 65, 574–585. [CrossRef] [PubMed] 5. Chakhtoura, M.T.; Nakhoul, N.; Akl, E.A.; Mantzoros Ghada, A.; El Hajj Fuleihan, G.A. Guidelines on vitamin D replacement in bariatric surgery: Identiﬁcation and systematic appraisal. Metabolism 2016, 65, 586–597. [CrossRef] [PubMed] Medicina 2019, 55, 252 6 of 6 7. Kenny, A.M.; Prestwood, K.M.; Biskup, B.; Robbins, B.; Zayas, E.; Kleppinger, A.; Burleson, J.A.; Raisz, L.G. Comparison of the eﬀects of calcium loading with calcium citrate or calcium carbonate on bone turnover in postmenopausal women. Osteoporos. Int. 2004, 15, 290–294. [CrossRef] [PubMed] 8. IFSO 2018 23rd World Congress. Obes. Surg. 2018, 28, 131–1271. [CrossRef] [PubMed] 9. Smelt, H.J.; Pouwels, S.; Smulders, J.F. The clinical dilemma of calcium supplementation after bariatric surgery: Calcium citrate or calcium carbonate that is the question? Obes. Surg. 2016, 26, 2781–2782. [CrossRef] [PubMed] 10. Cole, A.J.; Beckman, L.M.; Earthman, C.P. Vitamin D status following bariatric surgery: Implications and recommendations. Nutr. Clin. Pract. 2014, 29, 751–758. [CrossRef] [PubMed] 11. Heber, D.; Greenway, F.L.; Kaplan, L.M.; Livingston, E.; Salvador, J.; Still, C.; Endocrine Society. Endocrine and nutritional management of the post-bariatric surgery patient: An endocrine society clinical practice guideline. J. Clin. Endocrinol. Metab. 2010, 95, 4823–4843. [CrossRef] [PubMed] 12. Mechanick, J.I.; Youdim, A.; Jones, D.B.; Garvey, W.T.; Hurley, D.L.; McMahon, M.; Heinberg, L.J.; Kushner, R.; Adams, T.D.; Shikora, S.; et al. Clinical practice guidelines for the perioperative nutritional, metabolic, and nonsurgical support of the bariatric surgery patient—2013 update: Cosponsored by American Association of Clinical Endocrinologists, the Obesity Society, and American Society for Metabolic & Bariatric Surgery. References Obesity 2013, 21, S1–27. [PubMed] 13. Coates, P.S.; Fernstrom, J.D.; Fernstrom, M.H.; Schauer, P.R.; Greenspan, S.L. Gastric bypass surgery for morbid obesity leads to an increase in bone turnover and a decrease in bone mass. J. Clin. Endocrinol. Metab. 2004, 89, 1061–1065. [CrossRef] [PubMed] 14. Riedt, C.S.; Brolin, R.E.; Sherrell, R.M.; Field, M.P.; Shapses, S.A. True fractional calcium absorption is decreased after Roux-en-Y gastric bypass surgery. Obesity 2006, 14, 1940–1948. [CrossRef] [PubMed] 15. Flores, L.; Moize, V.; Ortega, E.; Rodriguez, L.; Andreu, A.; Filella, X.; Vidal, J. Prospective study of individualized or high ﬁxed doses of vitamin D supplementation after bariatric surgery. Obes. Surg. 2015, 25, 470–476. [CrossRef] 16. Goldner, W.S.; Stoner, J.A.; Lyden, E.; Thompson, J.; Taylor, K.; Larson, L.; Erickson, J.; McBride, C. Finding the optimal dose of vitamin D following Roux-en-Y gastric bypass: A prospective, randomized pilot clinical trial. Obes. Surg. 2009, 19, 173–179. [CrossRef] [PubMed] g 17. Goldner, W.S.; Stoner, J.A.; Thompson, J.; Taylor, K.; Larson, L.; Erickson, J.; McBride, C. Prevalence of vitamin D insuﬃciency and deﬁciency in morbidly obese patients: A comparison with non-obese controls. Obes. Surg. 2008, 18, 145–150. [CrossRef] [PubMed] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2911568990	https://zenodo.org/records/3577698/files/141%20Design%20and%20Development%20of%20Automated%20Irrigation%20System.pdf	English	null	Design and Development of Automated Irrigation System	International journal of trend in scientific research and development	2,018	cc-by	6,182	ABSTRACT This type of technology is more efficient use of water for Key Words: Automation, Overhead structure, Microcontroller, Water spraying machine, Soil moisture sensor Automation, Overhead structure, ing machine, Soil vailable Online @ www.ijtsrd.com r, Water spraying machine, Soil r CTION creasing demand of food requires the hly specialized greenhouse vegetable ment in food production technology. In nd it is a simple, precise method for ndia, where the economy is mainly ation. The irrigation system provide amount of water to crop. This ation system allows it to be scaled up nhouses or open fields. An automated m was developed to optimize water use www.ijtsrd.com \| Volume – 3 \| Issue – 1 \| Nov-Dec 2018 ing machine, Soil creasing demand of food requires the hly specialized greenhouse vegetable ment in food production technology. In nd it is a simple, precise method for ndia, where the economy is mainly The irrigation system provide amount of water to crop. This ation system allows it to be scaled up nhouses or open fields. An automated m was developed to optimize water use gy irrigation in agriculture. Fig :- (1) Automated Irrigation System Dec 2018 Page: 1052 gy (1) Automated Irrigation System l lume – 3 \| Issue – 1 \| Nov-Dec 2018 l r irrigation in agriculture. Fig :- (1) Automated Irrigation System Dec 2018 Page: 1052 (1) Automated Irrigation System g g ABSTRACT In India, agriculture plays an important role for development in food production. In our country, agriculture are depends on the monsoons which is not sufficient source of water. So the irrigation is used in agriculture field. The greenhouse based modern agriculture industries are the recent requirement in every part of agriculture in India. In this technology, The overhead structure is created for the movement of water spraying machine. This machine unit which is programmed by the microcontroller will spray the water from start to end plant in the rows. This system provides the equal amount of water to every plant in the greenhouse unit. One of the objectives of this work is to see how human control could be removed from irrigation and also to optimize the use of water in the process. In greenhouse effect it is very difficult to provide adequate amount of water by manual watering process Hence, Automated irrigation system plays an important role. It also helps in time s removal of human agriculture and the climatic conditions. The most important factor of this system is microcontroller is used to control the movement of water spraying machine. In India, agriculture plays an important role for development in food production. In our country, agriculture are depends on the monsoons which is not sufficient source of water. So the irrigation is used in re field. The greenhouse based modern agriculture industries are the recent requirement in every part of agriculture in India. In this technology, The overhead structure is created for the movement of water spraying machine. This machine unit which is microcontroller will spray the water from start to end plant in the rows. This system amount of water to every plant in the greenhouse unit. One of the objectives of this work is to see how human control could be removed om irrigation and also to optimize the use of water in the process. In greenhouse effect it is very difficult adequate amount of water by manual process Hence, Automated irrigation system It also helps in time saving, removal of human agriculture and the climatic conditions. The most important factor of this system is microcontroller is used to control the movement of for agricultural crops. Agriculture is the main source for food production. Using science and technology we need to implement a method by which there can be limited consumption of water source is needed. International Journal of Trend in International Open Access ISSN No: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) International Open Access Journal \| www.ijtsrd.com ISSN No: 2456 - 6470 \| Volume - 3 \| Issue – 1 \| Nov Research and Development (IJTSRD) www.ijtsrd.com 1 \| Nov – Dec 2018 International Journal of Trend in International Open Access ISSN No: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) International Open Access Journal \| www.ijtsrd.com ISSN No: 2456 - 6470 \| Volume - 3 \| Issue – 1 \| Nov Research and Development (IJTSRD) www.ijtsrd.com 1 \| Nov – Dec 2018 Design and Development Arjunsing T. Rathod 1M.E. CAD/CAM, Scholar Prof. Ram Meghe Institute Development of Automated Irrigation Arjunsing T. Rathod1, Dr. A. U. Awate2 M.E. CAD/CAM, Scholar, 2Professor, Department of Mechanical Engineering Prof. Ram Meghe Institute of Technology and Research, Badnera, Maharashtra, India Irrigation System Professor, Department of Mechanical Engineering Badnera, Maharashtra, India ABSTRACT The green house based modern agriculture industries are the recent requirement in every part of agriculture in India. In this technology, the humidity and temperature of plants are precisely controlled. In greenhouse effect plants are planted very nearer to each other so it is very difficult to give adequate amount of water to all plants. Manual watering process is not so easy for every plant in the greenhouse effect, so the different types of irrigation systems are developed like Surface irrigation, Drip irrigation, Sprinkler irrigation, Centre pivot irrigation, Lateral move irrigation, Sub irrigation. In this project overhead structure is provided for the plant watering process. One water spraying device is located on the overhead struct which will operate by the use of microcontroller. The water spraying element is programmed in such a way that it will follow the instruction given by the microcontroller. It will spray the adequate amount of water to each plant in the greenhouse effect of technology is more efficient use of water for irrigation in agriculture. for agricultural crops. Agriculture is the main source for food production. Using science and technology we need to implement a method by which there can be limited consumption of water source is needed. The green house based modern agriculture industries are the recent requirement in every part of agriculture in India. In this technology, the humidity and temperature of plants are precisely controlled. In greenhouse effect plants are planted very nearer to ach other so it is very difficult to give adequate amount of water to all plants. Manual watering process is not so easy for every plant in the greenhouse effect, so the different types of irrigation systems are developed like Surface irrigation, Drip gation, Sprinkler irrigation, Centre pivot irrigation, Lateral move irrigation, Sub-irrigation and manual irrigation. In this project overhead structure is provided for the plant watering process. One water spraying device is located on the overhead structure which will operate by the use of microcontroller. The water spraying element is programmed in such a way that it will follow the instruction given by the microcontroller. It will spray the adequate amount of water to each plant in the greenhouse effect. 2. SOIL MOISTURE SENSOR Soil moisture content e determined via its effect on dielectric constant measuring the capacitance between two electrodes Microcontroller based automated irrigation em consists of moisture sensor, microcontroller, motors, overhead moving system The moisture sensor is buried in the field at uired depth. If the moisture content in the field gets uced to lower threshold limit, the signal is duced from the microcontroller to turn ON relay. d overhead moving system to reach r to each plant and spray water over it from the rce is supplied to the field. Moisture level sensed m the sensor will be displayed in the LCD display. er reaching upper threshold limit, the sensor gives ponding signal to the microcontroller and the Fig :- (3) Block Diagram of Microcontroller Based Automated Irrigation System Fig :- (3) Electronic Circuit This circuit includes Relay, Micro chip, LED display screen, Transformers, Soil moisture sensor circuit, Water spraying circuit 1. Relay: This unit takes signal from the microcontroller and provides output signal to the water spraying motor. 2. Microcontroller: This unit provides movement of the overhead system forward and backward motion is done by the signal of microcontroller. (3) Block Diagram of Microcontroller Based Automated Irrigation System (3) Electronic Circuit This circuit includes Relay, Micro chip, LED display screen, Transformers, Soil moisture sensor circuit, Relay: This unit takes signal from the microcontroller and provides output signal to the This unit provides movement of the overhead system forward and backward motion is done by the signal of microcontroller. Fig :- (3) Block Diagram of Microcontroller Based Automated Irrigation System Fig :- (3) Electronic Circuit This circuit includes Relay, Micro chip, LED display screen, Transformers, Soil moisture sensor circuit, Water spraying circuit 1. Relay: This unit takes signal from the microcontroller and provides output signal to the water spraying motor. 2. Microcontroller: This unit provides movement of the overhead system forward and backward (3) Block Diagram of Microcontroller Based Automated Irrigation System (3) Electronic Circuit This circuit includes Relay, Micro chip, LED display screen, Transformers, Soil moisture sensor circuit, Relay: This unit takes signal from the microcontroller and provides output signal to the This unit provides movement of the overhead system forward and backward Fig :- (2) Soil Moisture Sensor Soil Moisture Sensor Fig :- (2) Soil Moisture Sensor Soil Moisture Sensor Soil moisture sensors measure the water content in soil. 1. INTRODUCTION Continuous increasing demand of food requires the control in highly specialized greenhouse vegetable rapid improvement in food production technology. In a production and it is a simple, precise method for country like India, where the economy is mainly based on irrigation. The irrigation system provide only required amount of water to crop. This automated irrigation system allows it to be scaled up for larger greenhouses or open fields. An automated irrigation system was developed to optimize water use Continuous increasing demand of food requires the control in highly specialized greenhouse vegetable rapid improvement in food production technology. In a production and it is a simple, precise method for country like India, where the economy is mainly The irrigation system provide only required amount of water to crop. This automated irrigation system allows it to be scaled up for larger greenhouses or open fields. An automated ion system was developed to optimize water use Fig :- (1) Automated Irrigation System (1) Automated Irrigation System Fig :- (1) Automated Irrigation System (1) Automated Irrigation System @ IJTSRD \| Available Online @ www.ijtsrd.com www.ijtsrd.com \| Volume – 3 \| Issue – 1 \| Nov-Dec 2018 Dec 2018 Page: 1052 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 search and Development (IJTSRD) ISSN: 2456 search and Development (IJTSRD) ISSN: 2456 search and Development (IJTSRD) ISSN: 2456-6470 BLOCK DIAGRAM BLOCK DIAGRAM 2. SOIL MOISTURE SENSOR A soil moisture probe is made up of multiple soil moisture sensors. One common type of soil moisture sensors in commercial use is a Frequency domain sensor such as a capacitance sensor. Another sensor, the neutron moisture gauge, utilize the moderator properties of water for neutrons. Soil moisture content may be determined via its effect on dielectric constant by measuring the capacitance between two electrodes implanted in the soil. Soil moisture sensors measure the water content in soil. A soil moisture probe is made up of multiple soil e sensors. One common type of soil moisture sensors in commercial use is a Frequency domain sensor such as a capacitance sensor. Another sensor, the neutron moisture gauge, utilize the moderator properties of water for neutrons. Soil moisture content e determined via its effect on dielectric constant by measuring the capacitance between two electrodes Fig :- (3) Block Diagram of Microcontroller Based Automated Irrigation System (3) Block Diagram of Microcontroller Based Automated Irrigation System Fig :- (3) Block Diagram of Microcontroller Based Automated Irrigation System (3) Block Diagram of Microcontroller Based Automated Irrigation System Fig :- (3) Electronic Circuit (3) Electronic Circuit 3. METHODOLOGY The Microcontroller based automated irrigation system consists of moisture sensor, microcontroller, Relay driver, DC motors, overhead moving system etc. The moisture sensor is buried in the field at required depth. If the moisture content in the field gets reduced to lower threshold limit, the signal is produced from the microcontroller to turn ON relay. The relay allowed overhead moving system to reach over to each plant and spray water over it from the source is supplied to the field. Moisture level sensed from the sensor will be displayed in the LCD display. After reaching upper threshold limit, the sensor gives corresponding signal to the microcontroller and the relay is turned OFF. The Microcontroller based automated irrigation system consists of moisture sensor, microcontroller, motors, overhead moving system etc. The moisture sensor is buried in the field at required depth. If the moisture content in the field gets reduced to lower threshold limit, the signal is produced from the microcontroller to turn ON relay. d overhead moving system to reach over to each plant and spray water over it from the source is supplied to the field. Moisture level sensed from the sensor will be displayed in the LCD display. After reaching upper threshold limit, the sensor gives ponding signal to the microcontroller and the Fig :- (3) Electronic Circuit (3) Electronic Circuit Fig :- (3) Electronic Circuit (3) Electronic Circuit g This circuit includes Relay, Micro chip, LED display screen, Transformers, Soil moisture sensor circuit, Water spraying circuit This circuit includes Relay, Micro chip, LED display screen, Transformers, Soil moisture sensor circuit, g This circuit includes Relay, Micro chip, LED display screen, Transformers, Soil moisture sensor circuit, Water spraying circuit This circuit includes Relay, Micro chip, LED display screen, Transformers, Soil moisture sensor circuit, g This circuit includes Relay, Micro chip, LED display screen, Transformers, Soil moisture sensor circuit, Water spraying circuit This circuit includes Relay, Micro chip, LED display screen, Transformers, Soil moisture sensor circuit, 1. Relay: This unit takes signal from the microcontroller and provides output signal to the water spraying motor. Relay: This unit takes signal from the microcontroller and provides output signal to the 2. Microcontroller: This unit provides movement of the overhead system forward and backward motion is done by the signal of microcontroller. This unit provides movement of the overhead system forward and backward motion is done by the signal of microcontroller. 2. SOIL MOISTURE SENSOR Soil moisture content e determined via its effect on dielectric constant measuring the capacitance between two electrodes e Microcontroller based automated irrigation stem consists of moisture sensor, microcontroller, motors, overhead moving system BLOCK DIAGRAM Fig :- (3) Block Diagram of Microcontroller Based Automated Irrigation System (3) Block Diagram of Microcontroller Based Automated Irrigation System Water is a very precious resource and a driving force in irrigation. Adequate amount of water is needed t every plant in the field, getting better plant yields, reduce dependency on fertilizers and improve crop quality. Various methods are available to measure soil moisture content, but the quickest and better one is with the use of soil moisture sensor electronic devices. For successful irrigation, it is necessary to monitor soil moisture content continuously in the irrigation fields. This sensor can be used to test the moisture of soil, When the soil is having water sensor will show the moisture content on LED display and microcontroller will perform the operation. Water is a very precious resource and a driving force in irrigation. Adequate amount of water is needed to every plant in the field, getting better plant yields, reduce dependency on fertilizers and improve crop quality. Various methods are available to measure soil moisture content, but the quickest and better one is tronic devices. For successful irrigation, it is necessary to monitor soil moisture content continuously in the irrigation fields. This sensor can be used to test the moisture of soil, When the soil is having water shortage, the sensor will show the moisture content on LED display and microcontroller will perform the operation. Water is a very precious resource and a driving force in irrigation. Adequate amount of water is needed t every plant in the field, getting better plant yields, reduce dependency on fertilizers and improve crop quality. Various methods are available to measure soil moisture content, but the quickest and better one is with the use of soil moisture sensor electronic devices. Water is a very precious resource and a driving force in irrigation. Adequate amount of water is needed to every plant in the field, getting better plant yields, reduce dependency on fertilizers and improve crop quality. Various methods are available to measure soil moisture content, but the quickest and better one is tronic devices. ry plant in the field, getting better plant yields, uce dependency on fertilizers and improve crop lity. 2. SOIL MOISTURE SENSOR Various methods are available to measure soil sture content, but the quickest and better one is h the use of soil moisture sensor electronic devices. successful irrigation, it is necessary to monitor moisture content continuously in the irrigation ds. This sensor can be used to test the moisture of When the soil is having water sor will show the moisture content on LED display microcontroller will perform the operation. Fig :- (2) Soil Moisture Sensor moisture sensors measure the water content in A soil moisture probe is made up of multiple soil sture sensors. One common type of soil moisture sors in commercial use is a Frequency domain sor such as a capacitance sensor. Another sensor, neutron moisture gauge, utilize the moderator perties of water for neutrons. Soil moisture content y be determined via its effect on dielectric constant measuring the capacitance between two electrodes lanted in the soil. METHODOLOGY Microcontroller based automated irrigation em consists of moisture sensor, microcontroller, ay driver, DC motors, overhead moving system The moisture sensor is buried in the field at uired depth. If the moisture content in the field gets uced to lower threshold limit, the signal is duced from the microcontroller to turn ON relay. relay allowed overhead moving system to reach r to each plant and spray water over it from the rce is supplied to the field. Moisture level sensed m the sensor will be displayed in the LCD display. er reaching upper threshold limit, the sensor gives esponding signal to the microcontroller and the y is turned OFF. ry plant in the field, getting better plant yields, uce dependency on fertilizers and improve crop lity. Various methods are available to measure soil sture content, but the quickest and better one is tronic devices. successful irrigation, it is necessary to monitor moisture content continuously in the irrigation ds. This sensor can be used to test the moisture of When the soil is having water shortage, the sor will show the moisture content on LED display microcontroller will perform the operation. Soil Moisture Sensor moisture sensors measure the water content in A soil moisture probe is made up of multiple soil e sensors. One common type of soil moisture sors in commercial use is a Frequency domain sor such as a capacitance sensor. Another sensor, neutron moisture gauge, utilize the moderator perties of water for neutrons. 2. SOIL MOISTURE SENSOR SOIL MOISTURE SENSOR ater is a very precious resource and a driving force irrigation. Adequate amount of water is needed t ery plant in the field, getting better plant yields, duce dependency on fertilizers and improve crop ality. Various methods are available to measure soil oisture content, but the quickest and better one is th the use of soil moisture sensor electronic devices. r successful irrigation, it is necessary to monitor l moisture content continuously in the irrigation lds. This sensor can be used to test the moisture of l, When the soil is having water nsor will show the moisture content on LED display d microcontroller will perform the operation. Fig :- (2) Soil Moisture Sensor il moisture sensors measure the water content in l. A soil moisture probe is made up of multiple soil oisture sensors. One common type of soil moisture nsors in commercial use is a Frequency domain nsor such as a capacitance sensor. Another sensor, e neutron moisture gauge, utilize the moderator operties of water for neutrons. Soil moisture content ay be determined via its effect on dielectric constant measuring the capacitance between two electrodes planted in the soil. METHODOLOGY e Microcontroller based automated irrigation stem consists of moisture sensor, microcontroller, lay driver, DC motors, overhead moving system ater is a very precious resource and a driving force irrigation. Adequate amount of water is needed to ery plant in the field, getting better plant yields, duce dependency on fertilizers and improve crop ality. Various methods are available to measure soil oisture content, but the quickest and better one is tronic devices. r successful irrigation, it is necessary to monitor l moisture content continuously in the irrigation lds. This sensor can be used to test the moisture of l, When the soil is having water shortage, the nsor will show the moisture content on LED display d microcontroller will perform the operation. Soil Moisture Sensor il moisture sensors measure the water content in l. A soil moisture probe is made up of multiple soil e sensors. One common type of soil moisture nsors in commercial use is a Frequency domain nsor such as a capacitance sensor. Another sensor, e neutron moisture gauge, utilize the moderator operties of water for neutrons. 3. METHODOLOGY On that nozzles are provided for spraying unit. Two limit switches are provide at both sides for the slider so that it will not cross the limit when it perform forward and backward motion. 4. Soil Moisture Sensor Circuit: This circuit detects the soil moisture level and send signal to the motor via relay. Soil Moisture Sensor Circuit: This circuit detects the soil moisture level and send signal to the 4. Soil Moisture Sensor Circuit: This circuit detects the soil moisture level and send signal to the motor via relay. Soil Moisture Sensor Circuit: This circuit detects the soil moisture level and send signal to the 5. Water Spraying Circuit: This motor run when soil moisture level below 45 until it comes to the 45 till that limit water will be continuously sprayed. Water Spraying Circuit: This motor run when soil oisture level below 45 until it comes to the 45 till that limit water will be continuously sprayed. 5. Water Spraying Circuit: This motor run when soil moisture level below 45 until it comes to the 45 till that limit water will be continuously sprayed. Water Spraying Circuit: This motor run when soil oisture level below 45 until it comes to the 45 till that limit water will be continuously sprayed. 6. Transformer: Transformers are provided for the change of voltage in the circuit. This system requires 12V DC supply which we can provide from the battery but the battery cannot same supply for long period of time so we need to charge it again and again instead of that we have used AC supply which is converted in to DC by Transformers. Transformer: Transformers are provided for the change of voltage in the circuit. This system requires 12V DC supply which we can provide t the battery cannot provide same supply for long period of time so we need to charge it again and again instead of that we have used AC supply which is converted in to DC by IJTSRD \| Available Online @ www ijtsrd com charge it again and again instead of that we have used AC supply which is converted in to DC by Transformers. Fig :- (4) Automated Irrigation System this model we have used one plywood called base on that base we have mounted four tical pvc pipes for support structure. 3. METHODOLOGY @ IJTSRD \| Available Online @ www.ijtsrd.com www.ijtsrd.com \| Volume – 3 \| Issue – 1 \| Nov-Dec 2018 Dec 2018 Page: 1053 @ IJTSRD \| Available Online @ www.ijtsrd.com www.ijtsrd.com \| Volume – 3 \| Issue – 1 \| Nov-DD International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 3. LED Display: LED display shows the soil moisture contents, If soil moisture is low then water spraying motor will start and water will come from the nozzle until the soil moisture level come up to 45 after that system stops. LED Display: LED display shows the soil moisture contents, If soil moisture is low then spraying motor will start and water will come from the nozzle until the soil moisture level come up to 45 after that system stops. plants are implanted for better look. Two channels are provided for the movement of horizontal slider which perform forward and backward movement of spraying nozzles. One DC motor of 200 RPM used for the movement of slider with the help of long threaded rod of 10mm diameter. Two medium density fiber board (MDF) which are used for supportive structure of dimension 100mm100mm , At fixed with threaded stud and fixed at the another end. Two threaded studs are fixed at both MDF sheet with the help of nuts. One horizontal pvc pipe is fixed in between two vertical slide of MDF sheet. On that horizontal pvc pipe two nozzles are provided for spraying unit. Two limit switches are provide at both sides for the slider so that it will not cross the limit when it perform forward and backward motion. plants are implanted for better look. Two channels are provided for the movement of horizontal slider which d backward movement of spraying nozzles. One DC motor of 200 RPM used for the movement of slider with the help of long threaded rod Two medium density fiber board (MDF) which are used for supportive structure of dimension 100mm100mm , At one side DC motor is fixed with threaded stud and fixed at the another end. Two threaded studs are fixed at both MDF sheet with the help of nuts. One horizontal pvc pipe is fixed in between two vertical slide of MDF sheet. 5. ADVANTAGES Fig :- (8) 3 Way Upper Joint (8) 3 Way Upper Joint Fig :- (8) 3 Way Upper Joint (8) 3 Way Upper Joint The smart irrigation system will help us to provide significant amount of water to every plant in the greenhouse effect. It helps us to reduce human efforts for manual watering process. It improves the yields of crops which mean more income for the farmer people prosperous. Soil moisture sensors are more accurate than rain sensors because they can detect moisture the level of the root system. They are more exact in measuring how much water your plants are receiving and thus offer greater water savings. The smart irrigation system will help us to provide significant amount of water to every plant in the use effect. It helps us to reduce human efforts for manual watering process. It improves the yields of crops which mean more income for the farmer people prosperous. Soil moisture sensors are more accurate than rain sensors because they can detect moisture at the level of the root system. They are more exact in measuring how much water your plants are receiving and thus offer greater water savings. Fig :- (8) 3 Way Upper Joint Fig :- (9) Slider Fig :- (10) Nozzle (8) 3 Way Upper Joint Fig :- (8) 3 Way Upper Joint (8) 3 Way Upper Joint Fig :- (8) 3 Way Upper Joint (8) 3 Way Upper Joint International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 7. FUTURE SCOPE Fig :- (10) Nozzle Fig :- (10) Nozzle In this system we can provide reverse watering process by programming method. Overhead system works on the basis of microcontroller device. We can implant outside temperature and humidity sensor. Whenever there is a change in temperature and humidity of the surroundings these sensors sense the change in temperature and humidity of the surroundings and gives an interrupt signal to the microcontroller. If different kinds of sensors (that is, temperature, humidity, and etc.) are involved in such irrigation in future work, it can be said that an internet based remote control of irrigation automation will be possible. In this system we can provide reverse watering process by programming method. Overhead system works on the basis of microcontroller device. We can plant outside temperature and humidity sensor. Whenever there is a change in temperature and humidity of the surroundings these sensors sense the change in temperature and humidity of the surroundings and gives an interrupt signal to the f different kinds of sensors (that is, temperature, humidity, and etc.) are involved in such irrigation in future work, it can be said that an internet based remote control of irrigation automation will be Fig :- (10) Nozzle Fig :- (11) Automated Irrigation System (11) Automated Irrigation System 6. APPLICATIONS An overhead watering method is a practical choice to cover all the pots evenly with moisture. Sprinkler heads connected to overhead water pipes emit a misty spray across the entire greenhouse. Larger greenhouses benefit from this irrigation method because the sprinklers cover a significant amount of square footage in a short amount of time. This system is designed to apply water by moving over the plants. Using boom irrigation facilitates uniform water application which in turn results in healthy growth of the plants. Due to easy operation, uniformity and efficiency, boom irrigation system are estimated to register strong growth over the forecast period. An overhead watering method is a practical choice to all the pots evenly with moisture. Sprinkler heads connected to overhead water pipes emit a misty spray across the entire greenhouse. Larger greenhouses benefit from this irrigation method because the sprinklers cover a significant amount of e in a short amount of time. This system is designed to apply water by moving over the plants. Using boom irrigation facilitates uniform water application which in turn results in healthy growth of the plants. Due to easy operation, uniformity and cy, boom irrigation system are estimated to register strong growth over the forecast period. Fig :- (9) Slider Fig :- (9) Slider 3. METHODOLOGY Artificial www ijtsrd com \| Volume 3 \| Issue 1 \| Nov Dec 2018 charge it again and again instead of that we have used AC supply which is converted in to DC by Automated Irrigation System sed one plywood sheet which called base on that base we have mounted four rtical pvc pipes for support structure. Artificial 4. CATIA DESIGN Fig :- (5) Plywood Base Fig :- (6) T PVC Pipe Fig :- (7) Vertical Pipes Dec 2018 Page: 1054 (5) Plywood Base (6) T PVC Pipe (7) Vertical Pipes 4. Fig :- (5) Plywood Base (5) Plywood Base Fig :- (6) T PVC Pipe (6) T PVC Pipe Fig :- (7) Vertical Pipes (7) Vertical Pipes Fig :- (4) Automated Irrigation System Automated Irrigation System In this model we have used one plywood is called base on that base we have mounted four vertical pvc pipes for support structure. Artificial sed one plywood sheet which is called base on that base we have mounted four vertical pvc pipes for support structure. Artificial @ IJTSRD \| Available Online @ www.ijtsrd.com www.ijtsrd.com \| Volume – 3 \| Issue – 1 \| Nov-Dec 2018 Dec 2018 Page: 1054 International Journal of Trend in Scientific Resea Fig :- (8) 3 Way Upper Joint Fig :- (9) Slider International Journal of Trend in Scientific Resea (8) 3 Way Upper Joint International Journal of Trend in Scientific Resea International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 International Journal of Trend in Scientific Research and Development (IJTSR International Journal of Trend in Scientific Research and Development (IJTSR International Journal of Trend in Scientific Research and Development (IJTSR International Journal of Trend in Scientific Research and Development (IJT International Journal of Trend in Scientific Research and Development (IJT International Journal of Trend in Scientific Research and Development (IJT monitors and controls all the activities of overhead irrigation system efficiently. monitors and controls all the activities of overhead 5. Shagun Agrawal, Suyash Pandey, Shravan Kuma Kunal Chaudhery, “ Automatic Irrigation Syst Shagun Agrawal, Suyash Pandey, Shravan Kuma Kunal Chaudhery, “ Automatic Irrigation System 5. Shagun Agrawal, Suyash Pandey, Shravan Kumar, Kunal Chaudhery, “ Automatic Irrigation Syst International Journal of Advance Researchand Innovation, Volume 2, Issue 1(2014) 92 2347-3258 Shagun Agrawal, Suyash Pandey, Shravan Kumar, Kunal Chaudhery, “ Automatic Irrigation System” International Journal of Advance Researchand Innovation, Volume 2, Issue 1(2014) 92-94 ISSN 9. REFRENCES 1. R. Vagulabranam, M. Karthikeyan “Automatic Irrigation System On Sensing Soil Moisture Content” International Research Journal Of Engineering & Technology (IRJET) ISSN: 2395-0056 R. Vagulabranam, M. Karthikeyan, V. Sasikala, “Automatic Irrigation System On Sensing Soil Moisture Content” International Research Journal Of Engineering & Technology (IRJET) ISSN: 6. Abhishek Kumar, Mangesh. S, “ Automated Irrigation System Based On Soil Moisture Using Arduino” International Journal of Pure and Applied Mathematics Volume 116 No.21 319-323 Abhishek Kumar, Mangesh. S, “ Automated Irrigation System Based On Soil Moisture Using Arduino” International Journal of Pure and Volume 116 No.21 2017, 2. Prof. Rashmi Jain, Shaunak Kulkarni, Ahtesham Shaikh, Akash Sood, “ Automatic Irrigation System For Agriculture Field Using Wireless Sensor Network”, International Research Journal Of Engineering & Technology (IRJET) ISSN: 2395-0056 Prof. Rashmi Jain, Shaunak Kulkarni, Ahtesham Shaikh, Akash Sood, “ Automatic Irrigation For Agriculture Field Using Wireless Sensor Network”, International Research Journal Of Engineering & Technology (IRJET) ISSN: 7. Priyamitra Munoth, Rohit Goyal, Kuldeep Tiwari, “Sensor based Irrigation System”, International Journal of Engineering Research & Technology( IJERT) NCACE- 2016 Priyamitra Munoth, Rohit Goyal, Kuldeep Tiwari, “Sensor based Irrigation System”, International Journal of Engineering Research & Technology( 8. Ms. Deweshvree Rane , Prof. P. R. Indurkar, Prof. D. M. Khatri, “ REVIEW PAPER BASED ON AUTOMATIC IRRIGATION SYSTEM BASED ON RF MODULE”, IJAICT Volume 1, Issue9, January 2015 Ms. Deweshvree Rane , Prof. P. R. Indurkar, Prof. “ REVIEW PAPER BASED ON AUTOMATIC IRRIGATION SYSTEM BASED ON RF MODULE”, IJAICT Volume 1, Issue9, 3. Anita K [3], “ Automatic Irrigation System” 2 International Conference on “ Innovative Trends in Science, Engineering and Management” ISBN: 978-93-86171-10-8 Anita K [3], “ Automatic Irrigation System” 2nd International Conference on “ Innovative Trends g and Management” ISBN: International Conference on “ Innovative Trends in Science, Engineering and Management” ISBN: 978-93-86171-10-8 4. Bikash Chandra Saha, Avinash Kumar, Naiyer Mumtaz, [4] “ Microcontroller Based Irrigation System Of Sensing Soil Moisture” International Conference on “ Innovative Trends g and Management” ISBN: Bikash Chandra Saha, Avinash Kumar, Naiyer Mumtaz, [4] “ Microcontroller Based Irrigation , AUTOMATIC IRRIGATION SYSTEM BASED ON RF MODULE”, IJAICT Volume 1, Issue9, January 2015 AUTOMATIC IRRIGATION SYSTEM BASED ON RF MODULE”, IJAICT Volume 1, Issue9, 4. Bikash Chandra Saha, Avinash Kumar, Naiyer Mumtaz, [4] “ Microcontroller Based Irrigation System Of Sensing Soil Moisture” Bikash Chandra Saha, Avinash Kumar, Naiyer Mumtaz, [4] “ Microcontroller Based Irrigation 4. 8. CONCLUSION The primary application of this project is for farmers and gardeners who do not have enough time to water their Crops/plants. The system improves the efficiency of water use to save water, relying on modern science and technology to achieve high quality and crop yield with the little The Microcontroller based overhead irrigation system proves to be real time feedback control system which his project is for farmers and gardeners who do not have enough time to water their Crops/plants. The system improves the efficiency of water use to save water, relying on modern science and technology to achieve high quality and crop yield with the littlest water source. The Microcontroller based overhead irrigation system proves to be real time feedback control system which Fig :- (11) Automated Irrigation System (11) Automated Irrigation System @ IJTSRD \| Available Online @ www.ijtsrd.com www.ijtsrd.com \| Volume – 3 \| Issue – 1 \| Nov-Dec 2018 Dec 2018 Page: 1055 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 9. REFRENCES Bikash Chandra Saha, Avinash Kumar, Naiyer Mumtaz, [4] “ Microcontroller Based Irrigation System Of Sensing Soil Moisture” Bikash Chandra Saha, Avinash Kumar, Naiyer Mumtaz, [4] “ Microcontroller Based Irrigation @ IJTSRD \| Available Online @ www.ijtsrd.com www.ijtsrd.com \| Volume – 3 \| Issue – 1 \| Nov-Dec 2018 Dec 2018 Page: 1056
https://openalex.org/W2800405401	https://journal.ipb.ac.id/index.php/jtpk/article/download/21086/14415	Indonesian	null	PEMETAAN ZONA GEOMORFOLOGI DAN HABITAT BENTIK DI PULAU KOTOK BESAR MENGGUNAKAN KLASIFIKASI BERBASIS OBJEK	Jurnal Teknologi Perikanan dan Kelautan/Jurnal teknologi perikanan dan kelautan	2,018	cc-by-sa	4,417	_ISSN 2087-4871 _ISSN 2087-4871 Jurnal Teknologi Perikanan dan Kelautan Vol. 8 No. 2 November 2017: 209-219__ ABSTRACT This study used high-resolution satellite imagery of worldview-2 acquisition 5 October 2013. The purpose of this study was to explore the capability of high-resolution satellite imagery of worldview-2 to map the geomorphic zones of shallow water in Kotok Besar island. The method used is object-based image analysis. This method is able to define classes of objects based on spectral and spatial aspects. Image segmentation algorithm using multiresolution segmentation with different scala parameters for each level, level 1, level 2 and level 3. Shape and compactness are also customized for each level. Assign class at level 1 generates three classes, namely land, shallow water and deep water. Assign class at level 2 for geomorphic zone generates three class classes of reef flat, reef crest and reef slope. benthic habitat classification at level 3 produces 7 classes with an overall accuracy was 66.40%. Keyword: benthic habitat, geomorphic zones, OBIA, satellite imagery of worldview-2 PEMETAAN ZONA GEOMORFOLOGI DAN HABITAT BENTIK DI PULAU KOTOK BESAR MENGGUNAKAN KLASIFIKASI BERBASIS OBJEK GEOMORPHIC ZONE AND BENTHIC HABITAT MAPPING IN KOTOK BESAR ISLAND USING OBJECT-BASED CLASSIFICATION Ike Dori Candra1, Vincentius P. Siregar2, Syamsul B. Agus3 1Program Studi Teknologi Kelautan, Sekolah Pascasarjana 2Departemen Ilmu dan Teknologi Kelautan, Fakultas Perikanan dan Ilmu Kelautan, Institut Pertanian Bogor Korespondensi: i.doricandra@gmail.com, vingar56@yahoo.com, mycacul@gmail.com Kata kunci: citra satelit worldview-2, habitat bentik, OBIA, zona geomorfologi Jurnal Teknologi Perikanan dan Kelautan, IPB__ _ E-mail: jurnalfpik.ipb@gmail.com PENDAHULUAN dengan kemajuan teknologi satelit dan komputer seperti sensor dan wahana serta peningkatan resolusinya meliputi resolusi spasial, resolusi temporal, resolusi spektral dan resolusi radiometrik. Metode klasifikasi dari data citra yang dikembangkan selama ini juga berkembang dari interpretasi visual menuju interpretasi citra secara otomatis dengan bantuan komputer. Kehadiran citra resolusi tinggi menuntut para praktisi untuk mengekstrak informasi data citra dari metode berbasis piksel menjadi klasifikasi berbasis objek untuk mendapatkan hasil yang tepat dan akurat, sehingga menuju pemanfaatan yang optimal.i Pemetaan terumbu karang memerlukan data yang dapat menggambarkan distribusi spasial terumbu. Metode penginderaan jauh dapat memberikan informasi penyebaran terumbu karang yang dapat digunakan sebagai bahan di dalam menentukan rencana sampling di lapangan. Penginderaan jauh untuk terumbu karang memanfaatkan sinar radiasi elektromagnetik pada daerah spektrum sinar tampak. Energi pada spektrum ini dapat menembus kolom air sehingga dapat mendeteksi terumbu karang yang berada di bawah permukaan air. Secara umum, spektrum sinar tampak dapat dibagi menjadi tiga bagian, yaitu spektrum sinar biru, sinar hijau dan merah. Makin kecil panjang gelombangnya, maka energi pada spektrum tersebut lebih dalam dapat menembus air. Klasifikasi citra pada perkembangan baru-baru ini menyebutkan bahwa terdapat dua basis klasifikasi, klasifikasi berbasis piksel dan klasifikasi berbasis obyek (Nuvulur 2007). Klasifikasi citra yaitu proses mengelompokkan piksel ke dalam kelas- kelas yang telah ditentukan berdasarkan nilai kecerahan piksel pada citra. Klasifikasi citra berbasis piksel dibagi dua metode yaitu klasifikasi terbimbing (supervised classification) dan klasifikasi tidak terbimbing (unsupervised classification). Metode klasifikasi pada citra resolusi tinggi juga mengalami perkembangan yaitu klasifikasi citra berbasis obyek (OBIA : Object Based Image Analysis). Klasifikasi ini terbukti mampu meningkatkan akurasi untuk pemetaan geomorfologi dan ekologi ekosistem terumbu karang pada tiga perairan yang berbeda (Phinn et al. 2011). Peneliti menerapkan OBIA dalam mengklasifikasi habitat perairan dangkal. Proses klasifikasi metode ini menggunakan prosedur segmentasi dengan sistem hierarki, sehingga suatu karakteristik objek dapat ditambahkan dengan kumpulan informasi tambahan dari objek yang diklasifikasikan seperti bentuk, tekstur, konteks dan informasi yang lain terkait dengan objek yang diklasifikasikan (Blaschke 2010). Tambahan informasi untuk setiap objek akan memperkaya informasi dalam klasifikasi, sehingga dapat menghasilkan klasifikasi yang jelas dan akurat. Perbedaan mendasar pada pendekatan ini dibandingkan dengan klasifikasi berbasis piksel terletak pada unit dasar proses analisis citra berupa objek citra atau segmen, tidak pada piksel tunggal. Keberhasilan pendekatan klasifikasi berbasis objek sangat tergantung pada kualitas segmentasi citra. Kim et al. (2008) menyatakan belum ada metode objektif dalam menentukan skala segmentasi, umumnya menggunakan metode uji coba. ABSTRAK Penelitian ini menggunakan citra satelit resolusi tinggi worldview-2 akuisisi 5 Oktober 2013. Tujuan dari penelitian ini adalah untuk mengkaji kemampuan citra satelit resolusi tinggi worldview-2 dalam memetakan zona geomorfologi dan habitat bentik perairan dangkal di Pulau Kotok Besar. Metode yang digunakan adalah metode klasifikasi Object Based Image Analysis (OBIA). Metode ini mampu mendefinisikan kelas-kelas objek berdasarkan aspek spektral dan spasial. Segmentasi citra menggunakan algoritma multiresolution segmentation dengan parameter skala yang berbeda untuk setiap level, baik level 1, level 2 dan level 3. Shape dan compactness juga disesuaikan untuk setiap level. Penentuan kelas pada level 1 menghasilkan tiga kelas yaitu daratan, perairan dangkal dan perairan dalam. Penentuan kelas pada level 2 untuk zona geomorfologi menghasilkan tiga kelas yaitu reef flat, reef crest dan reef slope. Klasifikasi habitat bentik pada level 3 menghasilkan 7 kelas dengan akurasi keseluruhan yaitu 66.40 %. Jurnal Teknologi Perikanan dan Kelautan, IPB_ _ E-mail: jurnalfpik.ipb@gmail.com Jurnal Teknologi Perikanan dan Kelautan Vol. 8 No. 2 November 2017: 209-219 PENDAHULUAN Teknik penginderaan jauh memiliki keunggulan untuk memetakan dan untuk melakukan pemantauan serta inventarisasi habitat perairan dangkal pada area yang luas, dengan biaya operasional relatif murah serta metode penginderaan jauh yang efektif untuk melengkapi pengamatan lapangan habitat bentik yang umumnya pada daerah yang sulit dijangkau. Oleh karena itu, teknik penggunaan teknik penginderaan jauh sangat memungkinkan untuk pemetaan dasar perairan dangkal (Siregar 2010), pemantauan kondisi terumbu karang (Green et al. 2000), deteksi perubahan dan dinamika sebaran terumbu karang (Klemas 2001), pemetaan geomorfologi dan ekologi terumbu karang (Phinn et al. 2011). Namun, pemanfaatan teknologi mempunyai kendala-kendala, yaitu kesulitan dalam mendeteksi habitat bawah air karena pengaruh kedalaman (Mumby & Edwards 2002). Permasalahan yang dihadapi dalam aplikasi penginderaan jauh yang lain adalah menentukan tingkat akurasi dan ketidakpastian (uncertainity) (Congalton & Green 2009). Beberapa penelitian menunjukkan bahwa dalam pemetaan geomorfologi laut dan habitat dasar perairan dangkal dengan akurasi keseluruhan >60 % sudah dapat menggambarkan bahwa klasifikasi yang dibangun sudah baik, maka pemilihan metode klasifikasi citra merupakan hal yang perlu dipertimbangkan untuk mendapatkan akurasi yang baik (Phinn et al. 2011).i Perkembangan metode klasifikasi data penginderaan jauh untuk berbagai kebutuhan semakin meningkat seiring 210 Jurnal Teknologi Perikanan dan Kelautan Vol. 8 No. 2 November 2017: 209-219 ISSN 2087-4871 Maret sampai April 2015. Peta Pulau Kotok Besar dapat dilihat pada Gambar 1. Maret sampai April 2015. Peta Pulau Kotok Besar dapat dilihat pada Gambar 1. Kombinasi beberapa teknik untuk mengekstraksi informasi dari data citra resolusi tinggi perlu mempertimbangkan beberapa faktor. Penerapan beberapa faktor koreksi untuk mempertajam interpretasi, metode klasifikasi citra dan survei lapangan yang tepat sangat mempengaruhi peningkatan akurasi dalam klasifikasi. Berdasarkan latar belakang di atas maka peneliti melakukan penelitian pemetaan geomorfologi dan habitat dasar perairan dangkal dari citra resolusi tinggi menggunakan klasifikasi berbasis objek. Alat yang digunakan dalam penelitian ini adalah perangkat keras, perangkat lunak dan peralatan lapangan. Perangkat keras yaitu personal laptop dan perangkat lunaknya adalah microsof office, microsoft excel add ins XLSTAT, Coral point with Excel Extention 4.0, Garmin GPSMAP 76csx, ENVI 5.1, ArcGIS Dekstop 10.1, dan Ecognition 9.0. Adapun peralatan lapangan tercantum pada Tabel 1. Bahan yang digunakan adalah citra satelit worldView-2 produk standar 2A dan dilengkapi dengan metadata (header file). Citra satelit worldview-2 ini akuisisi tanggal 5 Oktober 2013 dan sistem proyeksi koordinat UTM zona 48S – WGS1984 (DigitalGlobe 2010). Analisis data secara visual dengan acuan kode lifeform dari LIPI. Data hasil analisis dari CPCe ini disimpan dalam file excel untuk selanjutnya dilakukan analisis statistik. Analisis statistisk dengan menggunakan algoritma Agglomerative Hierarchical Clustering (AHC). Data lapangan berasal dari pengamatan langsung di lokasi penelitian. Zona geomorfologi ditentukan berdasarkan pengamatan visual dan interpretasi citra. Habitat bentik ditentukan berdasarkan identifikasi teknik foto transek quadrat (English et al. 1994; Roefselma & Phinn, 2008). Adapun karakteristik dari citra worldview-2 secara lengkap dapat dilihat pada Tabel 2. Tahapan pra pengolahan citra worldview-2 terlebih dahulu dilakukan sebelum klasifikasi citra. Worldview-2 dengan tipe produk standar 2A sudah terkoreksi geometrik. Kemudian dilakukan koreksi radiometrik dengan metode Dark Pixel Substraction. Prinsip metode ini adalah memperbaiki nilai radiometrik (pixel value) pada citra akibat gangguan atmosferik. Jika tidak ada atmosfer,objek berwarna gelap atau biasanya berupa air dan bayangan awan seharusnya memiliki nilai piksel 0 (Ardiansyah 2015). Analisis persentase tutupan dengan teknik pengamatan grid kuadran menggunakan aplikasi Coral Point Count With Excel Extension (CPCe) (Kohler & Gill 2006). Metode yang digunakan adalah metode uniform grid dengan sebaran titik sebanyak 25 titik setiap foto. Setiap titik diidentifikasi Tabel 2. Karakteristik citra worldview-2 Tabel 2. Karakteristik citra worldview-2 No Band Kisaran λ (nm) Resolusi 1 Pankromatik 0.5 2 Coastal 400-450 1.8 3 Blue 450-510 1.8 4 Green 510-580 1.8 5 Yellow 585-625 1.8 6 Red 630-690 1.8 7 Red-Edge 705-745 1.8 8 NIR-1 770-895 1.8 9 NIR-2 860-1040 1.8 citra berbasis objek menggunakan software eCognition 9.0. Input image layer yang digunakan adalah citra multispektral worldview-2 terkoreksi dengan 8 band (coastal, blue, green, yellow, red, red edge, NIR 1 dan NIR 2) dan input thematic layer adalah polygon klasifikasi. Membangun ruleset pada pohon proses (process tree) untuk setiap segmen menjadi kelas pada setiap level dan penentuan kelas (assign class) dengan menggunakan penentuan ambang batas (treshold). Koreksi radiometrik tujuannya adalah untuk memperbaiki kualitas visual citra, dalam hal ini memperbaiki nilai piksel yang tidak sesuai dengan nilai pancaran objek sebenarnya. Tahapan metode Dark Pixel Substraction ialah dengan menggunakan band minimum. Sejumlah piksel pada masing-masing kanal di laut dalam diambil dan rata-rata dari nilai-nilai piksel tersebut (nilai digital atau radiansi) digunakan sebagai faktor pengurang nilai piksel pada masing-masing kanal (Green et al. 2000), yang ditulis dengan persamaan : Algoritma yang digunakan untuk segmentasi adalah multiresolution segmentation (MRS). Algoritma MRS merupakan metode segmentasi berbasis region growing yang dikembangkan oleh Baatz & Schape ( 2000). METODE PENELITIAN Penelitian ini dilaksanakan di wilayah perairan Pulau Kotok Besar Kabupaten Kepulauan Seribu Provinsi DKI Jakarta. Penelitian ini dilaksanakan pada bulan Gambar 1. Lokasi penelitian Tabel 1. Peralatan lapangan No Peralatan Parameter 1 GPS Garmin 76csx Pengumpulan data koordinat : Ground truth point, Ground truth habitat 2 SCUBA Diving Pengambilan data habitat terumbu karang 3 Transek Kuadran 4 Meteran rol 50 m 5 Camera + Housing Underwater 6 Underwater slater dan paper Pencatatan data 7 GPS Floating Kit Pelampung 8 Dry Bag Pelindung GPS dari air Gambar 1. Lokasi penelitian Gambar 1. Lokasi penelitian Gambar 1. Lokasi penelitian Tabel 1. Peralatan lapangan No Peralatan Parameter 1 GPS Garmin 76csx Pengumpulan data koordinat : Ground truth point, Ground truth habitat 2 SCUBA Diving Pengambilan data habitat terumbu karang 3 Transek Kuadran 4 Meteran rol 50 m 5 Camera + Housing Underwater 6 Underwater slater dan paper Pencatatan data 7 GPS Floating Kit Pelampung 8 Dry Bag Pelindung GPS dari air ..(CANDRA et al.) 211 Jurnal Teknologi Perikanan dan Kelautan Vol. 8 No. 2 November 2017: 209-219 Klasifikasi level 2 Klasifikasi level 2 merupakan klasifikasi zona geomorfologi dimana class filternya diambil dari perairan dangkal level 1. Perairan dangkal ini pada level 2 disegmentasi ulang dengan algoritma Multiresolution Segmentation (MRS) dengan skala 10, shape 0.1 dan compactness 0.9. Dari hasil segmentasi ini dihasilkan sebanyak 1080 segmen. Selanjutnya dari segmen dilakukan assign class dengan penentuan ambang batas (treshold) untuk setiap zona geomorfologi. Treshold relatif border to deep water menghasilkan zona geomorfologi reef slope. Treshold relatif border to slope menghasilkan zona geomorfologi reef crest. Treshold ratio red edge menghasilkan zona geomorfologi reef flat. Klasifikasi level 2 menghasilkan 3 zona geomorfologi yaitu reef slope, reef crest dan reef flate. Phinn et al. (2011) mendeskripsikan bahwa rataan terumbu (reef flat) merupakan perairan dangkal, sebagian muncul ke permukaan terletak antara puncak terumbu (reef crest). Lereng terumbu (reef slope) merupakan wilayah yang memiliki kemiringan tertentu menghadap ke arah perairan atau biasa disebut tubir. Puncak terumbu (reef crest) merupakan zona yang muncul pada saat Klasifikasi level 1 Klasifikasi level 1 merupakan langkah awal dalam klasifikasi berbasis objek dengan menggunakan software eCognition 9.0. Langkah pertama dimulai dengan melakukan segmentasi citra dan assign class. Segmentasi level 1 menghasilkan segmen sebanyak 419 segmen. Kemudian dilakukan assign class dengan menggunakan treshold Normalize Difference Vegetation Index (NDVI) dan ratio green. Assign class menghasilkan 3 kelas utama yaitu kelas daratan (land) dengan luas 22.83 Ha, kelas perairan dangkal (shallow water) dengan luas 46.46 Ha dan kelas perairan dalam (deep water) dengan luas 52.60 Ha. Phinn et al. 2011 melaporkan bahwa klasifikasi pada level 1 (reef level) pada sistem klasifikasi hirarki yaitu kelas perairan dangkal menjadi batasan area kajian dan diproses menjadi segmen baru untuk klasifikasi pada level 2 (zona geomorfologi). Hasil klasifikasi level 1 ini nanti digunakan atau diperbanyak untuk proses segmentasi level 2. Hasil klasifikasi level 1 dapat dilihat pada Gambar 2. Klasifikasi level 3 yang merupakan klasifikasi habitat bentik menggunakan algoritma Support Vector Machine (SVM) dan input thematic layer untuk penentuan kelas. Menurut Wahidin et al. 2015 algoritma SVM mampu menghasilkan akurasi yang lebih baik pada metode berbasis OBIA. Klasifikasi level 3 dilakukan dengan mensegmentasi ulang hasil level 2. Langkah Selanjutnya assign class by thematic layer berikut atributnya, classifier algoritma SVM dan terakhir apply classifier. Hasil klasifikasi diekspor dalam bentuk raster dan vector lengkap dengan atribut kelasnya. Hasil klasifikasi level 3 dilakukan pengujian akurasi. Analisis data Proses segmentasi dijalankan berdasarkan lima parameter yaitu skala (scale), bentuk (shape), warna (colour), kehalusan (smoothness) dan kekompakan (compactness). Parameter skala merupakan L′i = Li – Lsi dimana: L′i = nilai piksel terkoreksi pada kanal i, Li = nilai piksel awal pada kanal I, dan Lsi = nilai piksel rata-rata sampling di laut dalam pada kanal i. dimana: L′i = nilai piksel terkoreksi pada kanal i, Li = nilai piksel awal pada kanal I, dan Lsi = nilai piksel rata-rata sampling di laut dalam pada kanal i. p Citra yang sudah terkoreksi digunakan sebagai input untuk pengolahan Jurnal Teknologi Perikanan dan Kelautan Vol. 8 No. 2 November 2017: 209-219 212 ISSN 2087-4871 ISSN 2087-4871 HASIL DAN PEMBAHASAN nilai abstrak untuk menentukan besarnya heterogenitas objek yang diperoleh dalam satu objek (Trimble 2014). Segmentasi level 1 menggunakan skala 25, level 2 skala 10 dan level 3 skala 5. Assign class level 1 dengan menggunakan treshold NDVI dan ratio band green. Assign class level 2 menggunakan treshold relatif border to deep water, relatif border to slope dan ratio red edge. Penggunaan skala dan treshold bersifat try and error dan belum ada framework teoritisnya. Pengguna harus mencari sendiri parameter terbaiknya (Blaschke & Hay 2001; Burnett &Blaschke 2003). Jurnal Teknologi Perikanan dan Kelautan Vol. 8 No. 2 November 2017: 209-219 Uji Akurasi Uji akurasi dilakukan pada peta hasil klasifikasi level 3 yaitu kelas hirarki habitat bentik. Uji akurasi diterapkan pada peta hasil klasifikasi untuk mengetahui akurasi dari teknik klasifikasi yang diterapkan. Uji akurasi yang umum dilakukan adalah matrik kesalahan (error matrix). Teknik matrik kesalahan yaitu membandingkan citra hasil klasifikasi sebagai peta terhadap kelas yang sebenarnya. Kelas yang sebenarnya diperoleh dari hasil pengamatan lapangan pada kelas-kelas yang telah didefinisikan berdasarkan skema klasifikasi. Penerapan uji akurasi menghasilkan persentase ketelitian pada sampel kelas yang diuji yaitu producer’s accuracy, user’s accuracy dan overall accuracy (Congalton & Green 2009). Persentase ketelitian suatu kelas diperoleh dari perbandingan jumlah piksel yang benar yang masuk pada training area suatu kelas. Persentase ketelitian klasifikasi secara keseluruhan dihitung dari perbandingan antara jumlah piksel yang benar setiap kelas dengan total piksel training area keseluruhan. 213 ..(CANDRA et al.) 2 Pemetaan Zona Geomorfologi.. crest 9.44 Ha dan zona reef slope 8.22 Ha. Jadi zona reef flat merupakan zona dengan luasan yang paling besar. Hasil klasifikasi level 2 dapat dilihat pada Gambar 3. surut terendah. Zona ini terletak pada bagian yang menghadap perairan dan mendapat energi yang tinggi dari gempuran gelombang. Dari hasil klasifikasi, zona reef flat memiliki luasan 27.79 Ha, zona reef Gambar 2. Klasifikasi level 1 (reef level) Gambar 3. Klasifikasi level 2 Gambar 2. Klasifikasi level 1 (reef level) Gambar 2. Klasifikasi level 1 (reef level) Gambar 2. Klasifikasi level 1 (reef level) Gambar 3. Klasifikasi level 2 Gambar 3. Klasifikasi level 2 Gambar 3. Klasifikasi level 2 214 Jurnal Teknologi Perikanan dan Kelautan Vol. 8 No. 2 November 2017: 209-219 ISSN 2087-4871 24.07 Ha (51.81%) dan yang paling sedikit adalah kelas habitat bentik karang mati + pasir yaitu seluas 1.77 Ha (3.81%).i 24.07 Ha (51.81%) dan yang paling sedikit adalah kelas habitat bentik karang mati + pasir yaitu seluas 1.77 Ha (3.81%).i Klasifikasi level 3 24.07 Ha (51.81%) dan yang paling sedikit adalah kelas habitat bentik karang mati + pasir yaitu seluas 1.77 Ha (3.81%).i Analisis habitat bentik di perairan dangkal Pulau Kotok Besar menggunakan Coral Point Count With Excel Extension (CPCe) 4.0 dan hasilnya dianalisis kluster menggunakan algoritma Agglomeretive Hierarchical Clustering (AHC) dengan truncation 0.4. Kehadiran kurang dari 4% diabaikan dalam menentukan jumlah kelas habitat bentik (Green et al. 2000). Dendogram hasil analisis kluster disajikan pada Gambar 4. p y ( ) Peta klasifikasi habitat bentik menunjukkan bahwa habitat bentik terdistribusi di perairan dangkal Pulau Kotok Besar. Zona geomorfologi reef slope didominasi oleh habitat bentik karang hidup (KH) diikuti habitat bentik karang mati (KM). Zona geomorfologi reef crest didominasi oleh habitat bentik rubble. Hal ini sesuai dengan klasifikasi hirarki zona geomorfologi dan habitat bentik berdasarkan Phinn et al. (2011), dimana terdapat asosiasi yang erat antara zona geomorfologi dengan keberadaan habitat bentik tertentu. Zona geomorfologi reef flat didominasi oleh habitat bentik pasir + FS (PFS) diikuti kelas lamun + pasir (LP). Zona geomorfologi ini memang cocok untuk habitat bentik FS dan lamun dikarenakan kondisinya terlindung dari gempuran gelombang laut yang kuat. Analisis kluster AHC ini menghasilkan 7 komposisi habitat bentik. Pelabelan kelas habitat bentik berdasarkan kelas centroid setiap kelas dan komposisi yang paling dominan. Kelas habitat bentik yang diberi label yaitu karang hidup (KH), karang mati (KM), karang mati + pasir (KHP), lamun + pasir (LP), rubble (R), pasir + FS (PFS), rubble + karang mati (RKM). Pelabelan komposisi habitat bentik dapat dilihat pada Gambar 5. Andrefouet et al. (2003) menjelas bahwa habitat bentik ditentukan berdasarkan persentase tutupan masing- masing komponen penyusun habitat. Hasil penggelompokkan 7 kelas habitat bentik dijadikan pedoman dalam klasifikasi level 3 habitat bentik dalam bentuk thematic layer. Uji akurasi Uji akurasi dengan matrik kesalahan (matrix error) diperoleh akurasi keseluruhan (overall accuracy) sebesar 66.40 % dan nilai kappa sebesar 0.58. Menurut Green et al. (2000) bahwa akurasi pemetaan habitat bentik yang dapat digunakan adalah dengan akurasi keseluruhan sebesar >60%. Nilai kappa merupakan ukuran kebenaran antara kelas yang direpresentasikan dan menunjukkan nilai kesesuaian hasil klasifikasi pada citra dan keadaan di lapangan. Nilai kappa akan selalu lebih rendah dari nilai overall. Nilai kappa 0.58 berarti 58% konsistensi akurasi pada klasifikasi acak. Menurut Landish & Koch (1997 dalam Congalton & Green 2009) nilai koefisien kappa antara 0.4 – 0.8 termasuk kategori sedang. Klasifikasi level 3 merupakan klasifikasi habitat bentik dengan melakukan segmentasi ulang klasifikasi level 2. Segmentasi menggunakan algoritma Multiresolution Segmentation dengan skala 5, shape 0.1 dan compactness 0.7. Hasil eksekusi segmentasi didapatkan sebanyak 4041 segmen. Selanjutnya assign class berdasarkan thematic layer di mana thematic layer merupakan polygon klasifikasi habitat bentik perairan dangkal. Klasifikasi habitat bentik menggunakan algoritma Support Vector Machine (SVM) dan menghasilkan 7 kelas habitat bentik yaitu karang hidup (KH),karang mati (KM), karang mati + pasir (KHP), lamun + pasir (LP), rubble (R), pasir + FS (PFS), rubble + karang mati (RKM). Hasil klasifikasi level 3 dapat dilihat pada Gambar 6.i (PA) Nilai producer accuracy (PA) dihasilkan dengan rentang antara 35% - 92.31% dan user’s accuracy (UA) dihasilkan dengan rentang antara 33% - 100%. Secara umum kelas habitat bentik dapat dipetakan dengan baik dan yang belum dipetakan dengan baik yaitu kelas habitat bentik karang hidup (KH), karang mati (KM). Kelas habitat bentik KH, PA dan UA masing-masing 66.67% dan 34.78%. Kelas habitat bentik KM, PA dan UA masing-masing 35.71% dan 33.33%. Uji akurasi dapat dilihat pada Tabel 3. Hasil klasifikasi level 3 pada Gambar 6 diperoleh luas area masing-masing habitat bentik yaitu : karang hidup 3.39 Ha (7.31%), karang mati 2.91 Ha (6.28%), karang mati + pasir 1.77 Ha (3.81%), lamun + pasir 2.51 Ha (5.41%), rubble 9.19 Ha (19.8%), pasir + FS 24.07 Ha (51.81%) dan rubble + karang mati 2.59 Ha (5.59%). Berdasarkan luas area dapat dilihat bahwa kelas habitat bentik pasir + FS mendominasi yaitu seluas Rendahnya akurasi disebabkan oleh beberapa faktor yaitu GPS dengan citra satelit resolusi tinggi pada saat pengambilan 215 .(CANDRA et al.) .(CANDRA et al.) Pemetaan Zona Geomorfologi.. data. GPS yang digunakan memiliki presisi 3-5 m dari posisi sebenarnya sedangkan citra satelit memiliki resolusi 1.84 m. Jurnal Teknologi Perikanan dan Kelautan Vol. 8 No. 2 November 2017: 209-219 Uji akurasi Diduga titik lapang yang menjadi acuan klasifikasi masih kurang dan tidak menyebar di seluruh wilayah penelitian. Pemetaan habitat bentik di Pulau Kotok belum begitu banyak dilakukan baik menggunakan metode piksel maupun metode OBIA. Kedepannya metode klasifikasi piksel dan OBIA dapat dilakukan secara bersamaan pada satu wilayah agar didapatkan perbandingan yang signifikan dan dilihat masing-masing keunggulannya. Khusus metode OBIA perlu diterapkan juga metode algoritma yang lain seperti Random Trees, KNN, Bayes dan Decision Tree. Gambar 4. Dendogram hasil kluster AHC Gambar 5. Pelabelan komposisi habitat bentik Gambar 4. Dendogram hasil kluster AHC Gambar 4. Dendogram hasil kluster AHC Gambar 5. Pelabelan komposisi habitat bentik Gambar 5. Pelabelan komposisi habitat bentik 216 Jurnal Teknologi Perikanan dan Kelautan Vol. 8 No. 2 November 2017: 209-219 ISSN 2087-4871 Gambar 6. Hasil Klasifikasi level 3 algoritma classifier SVM Gambar 6. Hasil Klasifikasi level 3 algoritma classifier SVM Tabel 3. Matrik Kesalahan Hasil Klasifikasi Tabel 3. Matrik Kesalahan Hasil Klasifikasi Tabel 3. Matrik Kesalahan Hasil Klasifikasi Lapang citra KH KM KM+P L+P R P+FS R+KM TOTAL UA KH 8 5 4 6 23 34.78 % KM 3 5 2 5 15 33.33 % KMP 8 8 100 % LP 8 2 10 80 % R 1 3 1 9 1 15 60 % PFS 3 2 1 36 42 85.71 % RKM 1 2 9 12 75.00 % TOTAL 12 14 12 10 18 39 20 125 34.78 % PA 66.67 % 35.71 % 66.67 % 80 % 50 % 92.31% 45 % 66.40% KESIMPULAN DAN SARAN Kesimpulan bentik dapat dipetakan dengan baik dan peta dengan akurasi lebih dari 60% dapat digunakan Tabel 3. Matrik Kesalahan Hasil Klasifikasi KESIMPULAN DAN SARAN bentik dapat dipetakan dengan baik dan peta dengan akurasi lebih dari 60% dapat digunakan. Kesimpulan Klasifikasi berbasis objek (OBIA) menggunakan citra resolusi tinggi worldview-2 di Pulau Kotok Besar menghasilkan klasifikasi pada 3 level. Klasifikasi level 1 menghasilkan kelas land, shallow water dan deep water. Klasifikasi level 2 menghasilkan 3 zona geomorfologi yaitu reef flat, reef crest dan reef slope. Klasifikasi level 3 menghasilkan 7 kelas habitat bentik yaitu KH, KM, KM + pasir, lamun + pasir, rubble, pasir + FS dan rubble + KM. Uji akurasi dari matrix error klasifikasi didapatkan akurasi keseluruhan (OA) sebesar 66.40% dan nilai kappa 0.58. Secara umum zona geomorfologi dan habitat Pemetaan Zona Geomorfologi.. UCAPAN TERIMA KASIH Kim M, Madden M, Warner T. 2008. Estimation of Optimal image object size for the segmentation of forest stand with multispectral IKONOS imagery. Object Based image nalysis. ‘Di Dalam’ : Blacschke T, Lang S, Hay G, ‘editor’, Academic Press. Volume 40: 81-251. Penulis mengucapkan terimakasih kepada Bapak Prof. Dr. Ir. Vincentius P. Siregar, DEA dan Dr. Syamsul Bahri Agus, S.Pi, M.Si yang telah banyak membantu penulis dalam hal penyediaan citra, pengerjaan data, membimbing dan memberi semangat kepada penulis. Penulis juga mengucapkan terimakasih kepada Tim Redaksi Jurnal Teknologi Perikanan dan Kelautan, Fakultas Perikanan dan Ilmu Kelautan, Institut Pertanian Bogor. Klemas V. 2001. Remote Sensing of landscape-level coastal environment indicators. ENVIRO manag. 85:159- 173. Kohler KE, Gill SM. 2006. Coral point count with excel extensions (Cpce): a visual basic program for the determination of coral and substrate coverage Using random point count methodology. Comput Geosci. 32: 1259-1269. Saran Pemetaan habitat bentik hendaknya melakukan pencocokan antara waktu perekaman citra dan perekaman pengambilan data lapang agar data lapang yang diambil sesuai dengan data citra yang sebenarnya. Pengambilan data diperlukan GPS yang lebih presisi dan akurat agar sesuai dengan citra satelit yang memiliki resolusi tinggi agar didapatkan data lapang yang lebih akurat. Data lapang yang lebih banyak dan tersebar secara merata sangat diperlukan di lokasi penelitian agar proses klasifikasi dan dapat menghasilkan uji akurasi yang lebih 217 .(CANDRA et al.) Pemetaan Zona Geomorfologi. resources, Australian Institute of Marine Science. tinggi. Teknik klasifikasi yang lain perlu dilakukan seperti algoritma classifier KNN, bayes, random tree dan decision tree agar didapatkan perbandingan teknik kualifikasi yang terbaik. Green EP, Mumby PJ, Edwards AJ, Clark CD. 2000. Remote sensing handbook for tropical coastal management: UNESCO. Jurnal Teknologi Perikanan dan Kelautan Vol. 8 No. 2 November 2017: 209-219 based image analysis for coral reef benthic habitat mapping with several classification algorithms. Procedia Environmental Sciences. 24: 222-227. Pemetaan Zona Geomorfologi.. based image analysis for coral reef benthic habitat mapping with several classification algorithms. Procedia Environmental Sciences. 24: 222-227. DAFTAR PUSTAKA Ardiansyah. 2015. 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https://openalex.org/W2974536228	https://inria.hal.science/hal-03769138/file/490001_1_En_5_Chapter.pdf	English	null	Using PVS for Modeling and Verification of Probabilistic Connectors	Lecture notes in computer science	2,019	cc-by	7,254	To cite this version: M. Saqib Nawaz, Meng Sun. Using PVS for Modeling and Verification of Probabilistic Connectors. 8th International Conference on Fundamentals of Software Engineering (FSEN), May 2019, Tehran, Iran. pp.61-76, 10.1007/978-3-030-31517-7_5. hal-03769138 M. Saqib Nawaz, Meng Sun. Using PVS for Modeling and Verification of Probabilistic Connectors. 8th International Conference on Fundamentals of Software Engineering (FSEN), May 2019, Tehran, Iran. pp.61-76, 10.1007/978-3-030-31517-7_5. hal-03769138 Distributed under a Creative Commons Attribution 4.0 International License Using PVS for Modeling and Veriﬁcation of Probabilistic Connectors M. Saqib Nawaz and Meng Sun LMAM & Department of Informatics, School of Mathematical Sciences, Peking University, Beijing, China {msaqibnawaz, sunm}@pku.edu.cn Abstract. Reo is a channel-based coordination language that allows the construction of connectors to coordinate behavior among diﬀerent components in distributed systems. Probabilistic connectors in Reo cap- ture the random and probabilistic behavior to deal with the uncertainty of the real world. In this paper we use PVS to provide a mechanical formalization for probabilistic connectors. We ﬁrst present the formal- ization of random/probabilistic channels and the composition operators in PVS. Random and probabilistic channels are modeled as relations on timed data distribution sequences that are observed at the source and sink ends of these channels. Composition operators are used to com- bine random/probabilistic channels together with primitive channels to construct complex component connectors. The approach can be used to naturally specify complex connectors and prove important properties for probabilistic connectors as well as the reﬁnement/equivalence relations between them with the PVS proof assistant. Keywords: Reo, PVS, Random/probabilistic connectors, Speciﬁcation, Veriﬁcation. HAL Id: hal-03769138 https://inria.hal.science/hal-03769138v1 Submitted on 5 Sep 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License This document is the original author manuscript of a paper submitted to an IFIP conference proceedings or other IFIP publication by Springer Nature. As such, there may be some differences in the official published version of the paper. Such differences, if any, are usually due to reformatting during preparation for publication or minor corrections made by the author(s) during final proofreading of the publication manuscript. This document is the original author manuscript of a paper submitted to an IFIP conference proceedings or other IFIP publication by Springer Nature. As such, there may be some differences in the official published version of the paper. Such differences, if any, are usually due to reformatting during preparation for publication or minor corrections made by the author(s) during final proofreading of the publication manuscript. 1 Introduction Large-scale distributed systems, that are generally transparent and heteroge- neous in nature, are built from components that interact with each other to per- form some speciﬁc tasks. Coordination languages oﬀer possible binding for com- ponents in a distributed environment to make the interactions possible. Reo [2,8] is a popular exogenous coordination language where exogenous coordination [1] means that the primitives that support the coordination of an entity with others reside outside of that entity. Reo allows the orchestration of complex connectors from simple ones (called channels) through composition operators. Connectors in Reo provide the protocols for controlling and organizing the communication, synchronization and cooperation among the components that they interconnect. Formal analysis and veriﬁcation of connectors have gained much interest in the past decade for component-based software engineering due to the recent evolution of software systems and advancements in cloud and grid technologies. It is also important to certify the correctness of connectors, which M. S. Nawaz and M. Sun 2 makes large-scale distributed systems more reliable. Some works have been done in this regard in the past years. For example, a modeling approach based on ﬁrst-order relational logic in Alloy modeling language was provided in [14] for Reo connectors. The symbolic model checker “Vereofy” has been developed in [6] to verify CTL-like properties for connectors. Moreover, a formal transformation from Reo to the speciﬁcation language mCRL2 that is based on process alge- bra was presented in [15]. The models were then veriﬁed conveniently with the mCRL2 model checker. Complex distributed systems need to incorporate many aspects of the com- munication and coordination between components, such as nondeterminism, probabilistic and stochastic interactions, real-time information and resource con- sumption, etc. The works reported in [3–5,9,12,17,22] extend classical Reo from diﬀerent perspective to deal with such requirements. The Unifying Theories of Programming (UTP) semantic framework was used in [21,23] to formalize con- nectors by providing design models for untimed and timed Reo connectors re- spectively, and recently extended in [24] to cover connectors that are composed from channels with random and probabilistic behavior. The theorem proving technique has been used in [18] to encode and reason about the design mod- els for untimed/timed Reo connectors in PVS [19]. In this paper, we extend the approach to cover the formalization for Reo connectors with random and probabilistic behavior. 1 Introduction The basic idea is to model the observable behavior of a probabilistic connector as a relation on the timed data distribution sequences being observed at the source (input) and sink (output) ends of the connector. The extended approach covers the scenarios for unpredictable, uncertain be- havior. Furthermore, the reﬁnement/equivalence relations between probabilistic connectors can be formalized and veriﬁed in PVS easily. Our mechanized veriﬁcation for probabilistic analysis of connectors is cer- tainly not the ﬁrst one. A variant of constraint automata called probabilistic constraint automata (PCA) has been developed in [5] to provide the operational semantics for probabilistic Reo connectors. Stochastic Reo automata was pro- posed in [17] to compositionally derive a QoS-aware semantics for Reo. The automata model was translated to Continuous-Time Markov Chains (CTMCs) so that third-party veriﬁcation tools can be used for stochastic analysis. Simi- larly, priced probabilistic timed constraint automata (pPTCA) was used in [12] for the reasoning about nondeterministic, probabilistic and timed behavior with aspects of energy consumption. Reo was also used in [7] to coordinate modules in the PRISM model checker. Although such formalisms scale up quite well, they suﬀer from the state space explosion problem as Reo connectors generally describe the manifold interactions among components that they interconnect, rather than simple input-output behavior on one individual interface. Moreover, the modeling and veriﬁcation of unbounded primitives or even bounded prim- itives with unbounded data domains always lead to the state space explosion problem, which cannot be solved with such ﬁnite automata models. However, such behavior can be speciﬁed and veriﬁed eﬃciently in theorem provers as shown in our previous works [13,18,25,26]. 3 Using PVS for Modeling and Veriﬁcation of Probabilistic Connectors Using PVS for Modeling and Veriﬁcation of Probabilistic Connectors Using PVS for Modeling and Veriﬁcation of Probabilistic Connectors Modeling and Veriﬁcation of Probabilistic Connector The remainder of the paper is organized as follows: The coordination lan- guage Reo is brieﬂy introduced in Section 2. In Section 3, we present the speciﬁca- tions in PVS for some basic deﬁnitions that are used later to model random/prob- abilistic channels. Section 4 presents the formal modeling of random/probabilis- tic channels and composition operators in PVS. Section 5 shows how to reason about properties of probabilistic connectors in PVS and reﬁnement/equivalence relations between them. Finally, Section 6 concludes the paper with some future work. The PVS dump ﬁle for this work can be found at [20]. 2 Preliminaries Reo oﬀers a compositional framework where component connectors can be con- structed from primitive channels of arbitrary types through composition opera- tors. Connectors provide the protocol for controlling and organizing the commu- nication, synchronization and cooperation between components. Each channel has two channel ends, with one of two types: source and sink. A source end provides input values to the channel via write actions and a sink end dispenses data out of the channel with read actions. A channel’s ends can also be both sinks or both sources. Figure 1 shows few primitive channel types in Reo. Sync Channel LossySync Channel FIFO1 Channel SyncDrain Channel t-Timer Channel t Fig. 1. Some primitive channels in Reo Fig. 1. Some primitive channels in Reo A synchronous (Sync) channel has one source and one sink end. Input/Out- put (I/O) operations can succeed only if the writing and reading operation is syn- chronized at source and sink end respectively. A lossy synchronous (LossySync) channel is a variant of the Sync channel. Data items in LossySync are transferred successfully if the write operation on the source end and the read operation on the sink end occur simultaneously, otherwise the data items are lost. A FIFO1 channel has one buﬀer cell of capacity 1, one source end and one sink end. FIFO1 accepts a data item whenever the buﬀer is empty. After accepting a data item from the source end, it is ﬁrst stored in the buﬀer and dispensed out of the channel through the sink end later in the FIFO order. The synchronous Drain (SyncDrain) channel is used for synchronizing the writing operations at its two source ends. It has no sink end and all written data items are lost. A t-timer channel accepts any data item at its source end and produces a timeout signal on its sink end after a delay of t time units. Further details on Reo and primitive untimed/timed channels can be found in [2, 3, 21, 23]. Furthermore, users can specify new channel types with their own requirements and interaction policies in Reo. For example, several probabilistic and stochastic extensions of Reo have been proposed in [5,9,16]. A connector can be depicted visually as a graph with some additional infor- mation. The nodes represent sets of the channel ends and the edges represent the 4 M. S. Nawaz and M. Sun established channels between the nodes. 2 Preliminaries Nodes can be categorized into source, sink or mixed nodes, depending on whether the node contains only source chan- nel ends, sink channel ends, or both. Therefore, source nodes are analogous to input ports and sink nodes to output ports. Data that ﬂow through source and sink nodes depends on the pending write and read operations of the environ- ment. For channel composition, three types of operators are used, which are (1) ﬂow-through, (2) replicate and (3) merge, as shown in Figure 2. Fig. 2. Operators for channel composition Fig. 2. Operators for channel composition Time: Type = posreal 3 Basic Deﬁnitions in PVS Time: Type = posreal 5 Using PVS for Modeling and Veriﬁcation of Probabilistic Connectors Data: TYPE [T -> real] Data: TYPE [T -> real] dfs?(F:[real->probability]):bool = EXISTS X: FORALL x: F(x) = P(X <= x) df: TYPE+ = (dfs?) CONTAINING (LAMBDA x: IF x < 0 THEN 0 ELSE 1 ENDIF) dfs?(F:[real->probability]):bool = EXISTS X: FORALL x: F(x) = P(X <= x) df: TYPE+ = (dfs?) CONTAINING (LAMBDA x: IF x < 0 THEN 0 ELSE 1 ENDIF) DD: TYPE = [Data, df] TDD: TYPE = [# T: sequence[Time], D: sequence[DD] #] DD: TYPE = [Data, df] DD: TYPE = [Data, df] TDD: TYPE = [# T: sequence[Time], D: sequence[DD] #] TDD: TYPE = [# T: sequence[Time], D: sequence[DD] #] Input, Output: VAR TDD Input, Output: VAR TDD Input, Output: VAR TDD A TDD is a record structure type that has two components: T and D. The T component is a sequence of time points being used to represent the time when the data in the D component is observed. The D component is a sequence of data distributions. The Input and Output are declared as variables of type TDD. The components of a record type can be accessed through the corresponding ﬁeld name. For example in our case, the T component of Input is accessed by Input‘T. Since the type of component T in TDD is deﬁned as sequence[Time], we have to deﬁne the operators “<” and “>” for sequences of times. A strict order (that is both transitive and irreﬂexive) is assumed for “<” and “>”. The type system of PVS is not algorithmically decidable and may lead to proof obligations called type correctness conditions (TCCs). Deﬁning “<,>” for sequence of time generated two TCCs. Proof steps for these two TCCs can be found at [20]. <: (strict_order?[sequence[Time]]) >: (strict_order?[sequence[Time]]) = >: (strict_order?[sequence[Time]]) = LAMBDA (s1, s2: sequence[Time]): s2 < s1 >: (strict_order?[sequence[Time]]) = LAMBDA (s1, s2: sequence[Time]): s2 < s1 LAMBDA (s1, s2: sequence[Time]): s2 < s1 Some more functions and predicates are used in PVS for concise modeling of probabilistic channels and composition operators. For example, Teq returns true if the time of two sequences are exactly equal to each other. Tle (Tgt) represents that time of the ﬁrst sequence is strictly less (greater) than the second sequence. next(T1): TDD = T1 WITH [T:=(suffix(T1‘T, 1)), D:=(suffix(T1‘D, 1))] 3 Basic Deﬁnitions in PVS The behavior of untimed and timed connectors are formalized by modeling their observable behavior as relations on the timed data sequences at their sink and source nodes. For random and probabilistic behavior, sequences of data dis- tributions where the data passes through connector nodes together with the time moments for data items observation emerges as the key building block to properly describe the connectors. Therefore, the observation on nodes can be speciﬁed naturally as timed data distribution (TDD) sequences for connectors that behaves probabilistically or randomly. The PVS library for probability [10] and some pre-deﬁned functions from PVS prelude are used in the modeling of random and probabilistic channels. The probability library is built on the ﬁrm foundations for probability theory [11]. Based on a σ-algebra, probability measure and probability space, the distribution function (df) for a real-valued random variable X is deﬁned in PVS. We are interested in the cumulative distribution function (CDF) of a random variable. To deal with continuous random variable, we would partially instantiate the sample space T with real, σ-algebra with borel_set (borel sets) and specify the probability measure to describe the distributions for random variable. A record structure in PVS is used to represent the TDD sequences on the sink and source nodes, where time is deﬁned as a positive real number (R+), which is natural and expressive enough for the modeling of connector behavior. For untimed/timed channels, data is deﬁned as a positive type. To capture the probabilistic behavior, data is deﬁned as a function of type [ T →real ] (where T is a positive (non-empty) type). With such a kind of functions, other abstract sets of data can be processed ﬁrst by mapping them to a set of real numbers in an appropriate way. Moreover, such mapping for data can be expanded easily in accordance with diﬀerent application domains. The data distribution DD is deﬁned as a Cartesian product with square brackets [_, _] in PVS. 4 Probabilistic Channels and Operators The modeling of some basic probabilistic/random channels and composition op- erators that are used to construct probabilistic connectors is presented in this section. 3 Basic Deﬁnitions in PVS Deq (Dneq) shows the equality of data: data sequence at one end is equal (not equal) to data sequence at the other end. For primitive (untimed) channels, the time of input sequence is a simulation of real time which means that time sequence is increasing as time passes by. For probabilistic connectors including timed channels, some more predicate formulas are deﬁned in a similar way. For example, Tltt (Tgtt) represents that the time of the ﬁrst sequence with a delay t is less (greater) than the second sequence. And the next function takes a TDD and returns the derivative of the sequence. Suﬃx function that is used in the next function is used to return a suﬃx sequence and its deﬁnition can be found in the prelude library of PVS. M. S. Nawaz and M. Sun 6 A rand(0,1) B RSync: Randomized synchronous channel (A rand(0,1) −−−−→B) is the randomized vari- ant of synchronous channel. When the channel is activated through an arbitrary write operation at source node A, it generates a random number b ∈{0, 1} at sink node B. Sink node synchronously takes the random number with equal probabil- ity for 0 (zero) and 1 (one). In RSync, zero and one are declared as random vari- ables. Their speciﬁcations generated two TCCs for expected type random vari- able, which is proved interactively with the PVS theorem prover. To prove both TCCs, we used already deﬁned judgment constant_is_measurable in the mea- sure space deﬁnition theory that can be found in the library measure_integration. The proofs for both TCCs are omitted here because of the page limitation and can be found at [20]. RSync is speciﬁed as follows: zero: random_variable = (LAMBDA t: 0) one: random_variable = (LAMBDA t: 1) RSync(Input, Output): bool = FORALL(n:nat): Output‘D(n) = (zero, oah) OR Output‘D(n) = (one, oah) & Teq(Input, Output) zero: random_variable = (LAMBDA t: 0) one: random_variable = (LAMBDA t: 1) RSync(Input, Output): bool = FORALL(n:nat): Output‘D(n) = (zero, oah) OR Output‘D(n) = (one, oah) & Teq(Input, Output) zero: random_variable = (LAMBDA t: 0) one: random_variable = (LAMBDA t: 1) RSync(Input, Output): bool = FORALL(n:nat): Output‘D(n) = (zero, oah) OR Output‘D(n) = (one, oah) & Teq(Input, Output) The universal quantiﬁcation and the ﬁrst disjunction capture the random be- havior being observed at the sink node. Each data element in the TDD sequence at the sink node can be either zero or one with probability oah, which is deﬁned as: 4.1 Random and Probabilistic Channels The behavior of probabilistic/random channels are speciﬁed in PVS with the disjunction or conjunction of diﬀerent predicates and constraints on the TDD sequences at source and sink nodes. We consider one random channel, randomized synchronous channel, and four probabilistic channels: message-corrupting syn- chronous channel, probabilistic lossy synchronous channel, faulty FIFO1 channel and lossy FIFO1 channel. oah(x): probability = 1/2 The synchronous behavior for this channel is satisﬁed with the predicate Teq, which shows that the time for the occurrence of data elements being observed at both channel ends are equal. 7 Using PVS for Modeling and Veriﬁcation of Probabilistic Connectors Using PVS for Modeling and Veriﬁcation of Probabilistic Connectors Modeling and Veriﬁcation of Probabilistic Connector CSync: Message corrupting synchronous channel (A −− p→B) is the probabilis- tic variant of synchronous channel, where with probability p, the delivered mes- sage can be corrupted. In such a channel, if a data element is written to the source end, then the probability that the exact correct data value will be ob- tained at the sink end is 1−p. A corrupted data value, represented with c in the speciﬁcation, will be obtained with probability p. CSync(Input, Output)(p:probability): INDUCTIVE bool = (Output‘T(0) = Input‘T(0) & Output‘D(0) = (Input‘D(0)‘1,(1-p)Input‘D(0)‘2)) OR (EXISTS(c:Data): Output‘D(0) = (c,(p)Input‘D(0)‘2) & CSync(next(Input), next(Output))(p)) The CSync channel is deﬁned inductively. Inductive deﬁnitions in PVS, which are predicates with eventual range type boolean, are similar to recursive deﬁni- tions as both involve induction and must satisfy some constraints to guarantee that they are total. The ﬁrst formula is for the time equality constraint for the synchronous behavior. We can also model the ﬁrst constraint with Teq predi- cate. The second and third formula with the disjunction reﬂects the probabilistic behavior. The sink node receives the same data that was written at the source node with the updated probability, where 1−p is multiplied with the probability for data at source node. On the other hand, sink node receives the corrupted value (c) with probability p multiplied with the probability for the written data. The last formula is for the recursive step that channel takes. PLSync: In the probabilistic lossy synchronous channel (A q −−→B), the trans- mission of the message from the source to sink fails with probability q. And with probability 1 −q, PLSync acts like a standard Sync channel where the message is successfully transmitted from the source end to the sink end. oah(x): probability = 1/2 In PVS, the PLSync channel is modeled as follows: PLSync(Input, Output)(q:probability): INDUCTIVE bool = (Output‘T(0) = Input‘T(0) & Output‘D(0) = (Input‘D(0)‘1,(1-q)Input‘D(0)‘2) & PLSync(next(Input), next(Output))(q)) & (Output‘D(0)‘2 = (q)Input‘D(0)‘2 => PLSync(next(Input),Output)(q)) PLSync(Input, Output)(q:probability): INDUCTIVE bool = (Output‘T(0) = Input‘T(0) & Output‘D(0) = (Input‘D(0)‘1,(1-q)Input‘D(0)‘2) & PLSync(next(Input), next(Output))(q)) & (Output‘D(0)‘2 = (q)Input‘D(0)‘2 => PLSync(next(Input),Output)(q)) The PLSync channel is deﬁned inductively but unlike CSync, it may take two diﬀerent routes in each step. Three conjuncted formulas are for the case when the data is successfully received by the sink end. First formula satisﬁes the time constraint that ensures the synchronous behavior. Second formula reﬂects that the data item is received by the sink end with probability 1 −q multiplied to the probability for that data at the source end. The third formula is the re- cursive step that channel takes when a data is transmitted successfully. For the case when the transmitted data is lost, the recursive behavior of the channel is reﬂected by the last two formulas with implication between them. In such case, M. S. Nawaz and M. Sun 8 no data is obtained at the sink end. FFIFO1: Faulty FIFO1 channel (A r· · ·<=→B) is a probabilistic variant of FIFO1 channel, that might loose (with probability r) the message when it is inserted into the buﬀer and the buﬀer remains empty. It can also behave as a normal FIFO1 channel where the insertion of the data into the buﬀer is successful with probability 1 −r. FFifo(Input, Output)(r:probability): INDUCTIVE bool = (Output‘T(0) > Input‘T(0) & Output‘T(0) < Input‘T(1) & Output‘D(0) = (Input‘D(0)‘1, (1-r)Input‘D(0)‘2) & FFifo(next(Input), next(Output))(r)) & (Output‘D(0)‘2 = (r)Input‘D(0)‘2 => FFifo(next(Input), Output)(r)) The FFIFO1 channel is also deﬁned inductively and like PLSync, it may take two diﬀerent routes in each step. For the case when data written at source end is inserted successfully into the buﬀer, the channel should satisfy four constraints which are speciﬁed with the conjunction of four predicates. The ﬁrst two formulas are for the time constraints, where the ﬁrst formula is for the time delay between data at source and sink ends. As FIFO1 has a buﬀering capacity of 1, next data item waits till the current data item in the buﬀer is taken out at the sink end. oah(x): probability = 1/2 This is speciﬁed with the second formula that the time of the next data item is greater than the time for the current data item in the buﬀer. The last two formulas are for the recursive behavior that channel takes when the written data is lost before its insertion in the buﬀer. Like PLSync, no data is received at the sink end in such case. LFIFO1: Another probabilistic variant called lossy FIFO channel (A−−<= r 99KB) might loose each stored data item with some ﬁxed probability (r) in any step. Compared to FFIFO1, this channel may loose the data in the process of taking the data from the buﬀer. As channels are modeled by the relations between observations on source and sink ends, therefore, the speciﬁcations for LFIFO1 and FFIFO1 are same. With this modeling approach, we can easily adjust the speciﬁcations for un- timed and timed channels in a proper way, where the observations on source and sink ends of all channels are speciﬁed by TDD sequences. Then the prob- abilistic/random channels as well as untimed/timed channels can be combined together to build connectors. A connector is probabilistic if it constitutes at least one probabilistic or random channel. Using PVS for Modeling and Veriﬁcation of Probabilistic Connectors Using PVS for Modeling and Veriﬁcation of Probabilistic Connectors Modeling and Veriﬁcation of Probabilistic Connector The ﬂow-through operator simply allows the data items to pass through mixed node(s) without any change. A component connected to a connector can write data items at source node and can obtain data items from the sink node. The replicate operator puts the source ends of diﬀerent channels together into one source node. A write operation by a component on source node succeeds only if all coinciding channels ends accept the data item. The behavior of ﬂow- through and replicate operators does not depend on the context of the data-ﬂow. The approach for the modeling of these two operators in [18] can be adopted here without any change. The structure of connectors allow us to specify both operators implicitly by means of nodes renaming and conjunction instead of writing a new function. This is explained with simple examples. For two channels PLSync(A, B) and FIFO1(B, C), the ﬂow-through operator acting on node B is implemented al- ready. For two channels PLSync(A, B) and FIFO1(C, D), the replicate operator can be implemented explicitly by renaming the C with A for the FIFO1 channel. Using conjunction and node renaming for these two operators make it possible to specify connectors directly as lemmas and theorems. Unlike ﬂow-through and replicate, the merge operator depends on the con- tent of the data-ﬂow. The time and data dimension is same for TD and TDD sequences, which means that these two dimensions do not need any change. As we are dealing with data distribution, so the equality relation for data is changed to the equality relation on data distribution. Thus, both data items and their associated probabilities should be equal. Using PVS for Modeling and Veriﬁcation of Probabilistic Connectors Merge is modeled as follows in PVS: Merge(s1,s2,s3:TDD): INDUCTIVE bool = (NOT s1‘T(0) = s2‘T(0)) & (s1‘T(0) < s2‘T(0) => s3‘T(0) = s1‘T(0) & s3‘D(0) = s1‘D(0) & Merge(next(s1),s2,next(s3))) & (s1‘T(0) > s2‘T(0) => s3‘T(0) = s2‘T(0) & s3‘D(0)= s2‘D(0) & Merge(s1,next(s2),next(s3))) Merge(s1,s2,s3:TDD): INDUCTIVE bool = (NOT s1‘T(0) = s2‘T(0)) & (s1‘T(0) < s2‘T(0) => s3‘T(0) = s1‘T(0) & s3‘D(0) = s1‘D(0) & Merge(next(s1),s2,next(s3))) & (s1‘T(0) > s2‘T(0) => s3‘T(0) = s2‘T(0) & s3‘D(0)= s2‘D(0) & Merge(s1,next(s2),next(s3))) Merge(s1,s2,s3:TDD): INDUCTIVE bool = (NOT s1‘T(0) = s2‘T(0)) & (s1‘T(0) < s2‘T(0) => s3‘T(0) = s1‘T(0) & s3‘D(0) = s1‘D(0) & Merge(next(s1),s2,next(s3))) & (s1‘T(0) > s2‘T(0) => s3‘T(0) = s2‘T(0) & s3‘D(0)= s2‘D(0) & Merge(s1,next(s2),next(s3))) The modeling approach provided in this section for probabilistic/random channels and composition operators can be used to construct diﬀerent proba- bilistic connectors according to their topological orders. 4.2 Operators Compositional operators can be applied on channels in various topological order for the construction of complex connectors. As already discussed in Section 2, there are three kinds of composition operators: (1) ﬂow-through, (2) replicate and (3) merge. 9 5 Reasoning After connectors modeling, we can analyze and prove their properties. In this sec- tion, some examples are provided for the reasoning about probabilistic/random connectors as well as the reﬁnement and equivalence relations between them. Example 1. Figure 3 shows a probabilistic Reo connector that distributed com- ponents can use for message communication. Component 1 can deliver its mes- sages to the connector via connecting to node in, while component 2 is connected to the node out to obtain the message from the connector. Messages are trans- mitted from component 1 to component 2 with FFIFO1 channel (AB). Other primitive channels (Sync, SyncD, FIFO1, LossySync) are organized in the con- nector to repeat component 1 message as often as necessary. The property that component 2 almost surely obtain the message via out from in is established with the following theorem in PVS. 10 M. S. Nawaz and M. Sun Fig. 3. A probabilistic Reo connector Theorem 1. Sync(in,D) & Sync(in,C) & Sync(C,A) & Fifo1(C,E) & Fifo1(E,F) & LSync(C,D)(n) & LSync(F,C)(n) & FFifo(A,B)(r) & Fifo1(D,H) & Sync(H,G) & SyncD(G,B) & Sync(G,E) & Sync(B,out) => out‘D(0)‘1 = in‘D(0)‘1 & Tle(in,out) 10 M. S. Nawaz and M. Sun Fig. 3. A probabilistic Reo connector M. S. Nawaz and M. Sun Fig. 3. A probabilistic Reo connector M. S. Nawaz and M. Sun 10 Fig. 3. A probabilistic Reo connector Theorem 1. Sync(in,D) & Sync(in,C) & Sync(C,A) & Fifo1(C,E) & Fifo1(E,F) & LSync(C,D)(n) & LSync(F,C)(n) & FFifo(A,B)(r) & Fifo1(D,H) & Sync(H,G) & SyncD(G,B) & Sync(G,E) & Sync(B,out) => out‘D(0)‘1 = in‘D(0)‘1 & Tle(in,out) Proof. Mathematical induction is used to prove theorem 1. After applying in- duction on n, main goal is split into two sub-goals. The ﬁrst sub-goal is for the base case and the other one is for the inductive case. Proof. Mathematical induction is used to prove theorem 1. After applying in- duction on n, main goal is split into two sub-goals. The ﬁrst sub-goal is for the base case and the other one is for the inductive case. For the base case, the antecedent formula is simpliﬁed by creating a free skolem variable and removing implies. The deﬁnitions of channels and predi- cates are expanded. Some irrelevant formulas in the antecedent are suppressed with the hide command. The sub-goal is divided into two more sub-goals: one for the data dimension and other for the time dimension. 5 Reasoning Both sub-sub-goals are proved with PVS proof commands and decision procedures. For the inductive case, the sequent formula is ﬁrst simpliﬁed with repeated skolemization and ﬂattening. In the ﬁrst antecedent formula, the universal quan- tiﬁers are instantiated automatically with inst? commands. Sometimes a single inst? can only ﬁnd a partial instantiation where successive invocations of inst? can succeed in fully instantiating all of the quantiﬁed variables. The rest of the proof is similar to the base case, where the sub-goal is split to two more sub- goals for data and time dimension. The detailed PVS proof for theorem 1 can be found at [20]. The notion of reﬁnement has been adopted widely in development of complex systems. Reﬁnement relation provides guarantee for the correctness of implemen- tation with respect to the abstract speciﬁcation of the same system, and thus helps in bridging the gap between requirements and the ﬁnal implementations. Here, we use the reﬁnement relation for connectors deﬁned in [23], where the reﬁnement order over connectors is established on the basis of the implication relation of predicates. As discussed already, connectors are represented by con- junction of a set of predicates, where the variables are bound by the universal and existential quantiﬁcation. Let C1 and C2 represent two connectors that are modeled by set of predicates. C2 is a reﬁnement of C1 only if C2 →C1, meaning the behavioral properties of C1 can be derived from the properties of C2. C2 properties are regarded as hypothesis and the properties of connector C1 as con- clusion. The reﬁnement relation between C1 and C2 is denoted as C1 ⊑C2. Next, we provide an example for reﬁnement relation between probabilistic connectors. Using PVS for Modeling and Veriﬁcation of Probabilistic Connectors 11 Example 2 (Reﬁnement). For the two connectors shown in Figure 4, connector P is a reﬁnement of connector Q (Q ⊑P). rand(0,1 ) rand(0,1 ) rand(0,1 ) rand(0,1 ) A D B C A B C E F Connector P Connector Q Fig. 4. Connectors reﬁnement example Example 2 (Reﬁnement). For the two connectors shown in Figure 4, connector P is a reﬁnement of connector Q (Q ⊑P). rand(0,1 ) rand(0,1 ) rand(0,1 ) rand(0,1 ) A D B C A B C E F Connector P Connector Q Fig. 4. Connectors reﬁnement example Fig. 4. 5 Reasoning Connectors reﬁnement example Given arbitrary input TDD sequence at node A and output TDD sequences at nodes B and C, connector P is a reﬁnement of another connector Q only if the behavior property of Q can be derived from the connector P property. In connector Q, the outputs are not synchronized and data is received asyn- chronously by the sink ends B and C respectively. There is no constraints on the relationship between the time sequence of the two output events. On the other hand, P reﬁnes the behavior of Q by synchronizing the two sink nodes, which ensures that the two output events must happen simultaneously. We use a, b, c to denote the time sequence at nodes A, B and C respectively. Let d denotes the random number d ∈{0, 1}, that ranges over all data items. Let β, γ represent the data sequence being observed at sink nodes B and C respectively. Probabilistic connector P satisﬁes the condition a < b ∧a < c ∧b = c ∧β = d∗ ∧γ = d∗. Whereas, Q satisﬁes a < b ∧a < c ∧β = d∗∧γ = d∗. The reﬁnement relation between Q and P is veriﬁed with following theorem. Theorem 2. ∀(A,B,C:TDD): (∃(D:TDD): Fifo1(A,D) & RSync(D, B) & RSync(D,C)) ⇒(∃ (E,F:TDD): (Fifo1(A,E) & RSync(E,B)) & (Fifo1(A,F) & RSync(F,C))) Proof. The ﬁrst suitable formula in the sequent (∀/∃A, B, C : TDD) is replaced to TDD[A!1/A1, B!1/B1, C!1/C1] by creating three skolem constants. Implies is removed from consequent with flattening. Now we have one existentially quantiﬁed formula in both antecedent and consequent. Quantiﬁed formula in an- tecedent is reduced by automatic introduction of skolem constant with skolem! command. In consequent, we need to ﬁnd the TDD sequences that specify the data ﬂow through mixed nodes E and F for connector Q. In other words, we need to ﬁnd an appropriate E and F that satisﬁes (Fifo1(A, E) ∧RSync(E, B)) ∧(Fifo1(A, F) ∧RSync(F, C)). With (inst 1 “D!1” “D!1”), the ﬁrst formula in consequent is instantiated where both E and F are substituted with D!1. Antecedent is divided into three formulas by removing logical &’s. Finally, the formula in the consequent is split into four sub-goals. All four sub-goals are trivially true and PVS proved those sub-goals automatically with propax propo- sitional axioms. PVS proof tree for theorem 2 is shown in Figure 5. 12 M. S. 5 Reasoning Nawaz and M. Sun (skolem!) (skolem * ("A!1""B!1""C!1")) (ﬂatten) (split) (propax) (propax) (ﬂatten) (propax) (propax) (inst 1 "D!1" "D!1") Fig. 5. Proof tree for connectors reﬁnement proof Fig. 5. Proof tree for connectors reﬁnement proof Generally, an equivalence relation is deﬁned as a binary relation that holds the reﬂexivity, symmetric as well as transitivity properties. The equivalence re- lation between two connectors C1 and C2 is deﬁned with mutual reﬁnement: C1 ≡C2 iﬀ C1 ⊑C2 ∧C2 ⊑C1 Here, the equivalence relation is represented with implications that goes both ways, such as C2 ↔C1. Example 3 (Equivalence). Figure 6 shows two probabilistic connectors that are constructed by composing ﬁve channels RSync, FIFO1, t-Timer, SyncD and Sync in diﬀerent topological orders. Both probabilistic connectors are equivalent (R1 ↔R2), which is proved in PVS. A R1 E rand(0,1 ) t D C t C rand(0,1 ) B A D E B R2 Fig. 6. Connectors equivalence example R1 Fig. 6. Connectors equivalence example The three untimed (Sync, SyncD and FIFO1) channels and one timed chan- nel (t-Timer) can be combined together to make a timed connector known as tFIFO1, that was also studied in [13]. In a primitive FIFO1 channel, the data distributions for the sequences at sink and source nodes are same with arbitrary 13 Using PVS for Modeling and Veriﬁcation of Probabilistic Connectors time delay. On the other hand, the time delay is ﬁxed by the parameter t in tFIFO1. Here, we call the tFIFO1 a sub-connector and is modeled as: time delay. On the other hand, the time delay is ﬁxed by the parameter t in tFIFO1. Here, we call the tFIFO1 a sub-connector and is modeled as: Tfifo(A, B)(t:Time)(d:Data): bool = EXISTS (R,S:TDD): Fifo1(A, R) & SyncD(R, S) & Timert(A, S)(t)(d) & Sync(R, B) Tfifo(A, B)(t:Time)(d:Data): bool = EXISTS (R,S:TDD): Fifo1(A, R) & SyncD(R, S) & Timert(A, S)(t)(d) & Sync(R, B) In general, the connectors build from same set of sub-connectors in commu- tative orders are not equivalent as the conﬁguration of connectors do not satisfy the commutative law. However, connectors R1 and R2 are equal for the above example. Unlike the general approach that we adopted previously to construct a con- nector from basic channels, connectors R1 and R2 are composed by connecting the tFIFO1 sub-connector with the RSync channel in diﬀerent topological order. 6 Conclusion The formalization approach for untimed/timed Reo connectors in PVS is ex- tended in this paper to model and reason about probabilistic/random connec- tors that are constructed from channels with random and probabilistic behavior. Probabilistic/random channels are modeled as relations on TDD sequences being observed at the source and sink nodes. Untimed/timed channels speciﬁcations are adjusted accordingly from TD sequences to TDD sequences. The speciﬁ- cations for probabilistic/random channels generated seven TCCs in total. Two TCCs are proved automatically by the prover and ﬁve are proved interactively. With formalised compositional operators and channels, complex connectors are modeled and their properties as well as the reﬁnement and equivalence relation between them are proved with the help of PVS proof-commands, inference rules and decision procedures. For future work, we would like to add more complex probabilistic and stochas- tic constraints in the connectors and reason about them. We also plan to extend the formalization approach further to deal with hybrid connectors, QoS (Quality of Service) and resource consumption aspects on connectors. Acknowledgement. The work has been supported by the National Natural Sci- ence Foundation of China under grant no. 61772038, 61532019 and 61272160, and the Guandong Science and Technology Department (Grant no. 2018B010107004). 5 Reasoning The main reason to use the reduced method (where a sub-connector is combined with a channel) for connectors construction is to make the proof process simpler and easier to understand. The equivalence relations between a channel linked with a sub-connector in diﬀerent positions are ﬁrst proved as lemmas. Lemma 1. ∀(A,B:TDD)(t:Time)(d:Data): ∃(E:TDD): RSync(A,E) & Tfifo(E,B)(t)(d) <=> RSync(A,E) & ∃ (C,D:TDD): Fifo1(E,D) & SyncD(D,C) & Timert(E,C)(t)(d) & Sync(D,B) Lemma 1. ∀(A,B:TDD)(t:Time)(d:Data): ∃(E:TDD): RSync(A,E) & Tfifo(E,B)(t)(d) <=> RSync(A,E) & ∃ (C,D:TDD): Fifo1(E,D) & SyncD(D,C) & Timert(E,C)(t)(d) & Sync(D,B) Lemma 1. ∀(A,B:TDD)(t:Time)(d:Data): ∃(E:TDD): RSync(A,E) & Tfifo(E,B)(t)(d) <=> RSync(A,E) & ∃ (C,D:TDD): Fifo1(E,D) & SyncD(D,C) & Timert(E,C)(t)(d) & Sync(D,B) Lemma 1 shows the equivalence relation between the reduced construction of a connector and a connector constructed from basic channels for R1. Similarly for R2, another lemma is provided. Lemma 2. ∀(A,B:TDD)(t:Time)(d:Data): ∃(E:TDD): Tfifo(A,E)(t)(d) & RSync(E,B) <=> ∃(C,D:TDD): Fifo1(A,D) & SyncD(D,C) & Timert(A,C)(t)(d) & Sync(D,E) & RSync(E,B) Lemma 2. ∀(A,B:TDD)(t:Time)(d:Data): ∃(E:TDD): Tfifo(A,E)(t)(d) & RSync(E,B) <=> ∃(C,D:TDD): Fifo1(A,D) & SyncD(D,C) & Timert(A,C)(t)(d) & Sync(D,E) & RSync(E,B) The main goal of equivalence relation between R1 and R2 is proved with following theorem: Theorem 3. ∀(A,B:TDD)(t:Time)(d:Data): ∃(E:TDD): RSync(A, E) & Tfifo(E,B)(t)(d) <=> ∃(R:TDD): Tfifo(A,R)(t)(d) & RSync(R,B) Theorem 3. ∀(A,B:TDD)(t:Time)(d:Data): ∃(E:TDD): RSync(A, E) & Tfifo(E,B)(t)(d) <=> ∃(R:TDD): Tfifo(A,R)(t)(d) & RSync(R,B) Theorem 3. ∀(A,B:TDD)(t:Time)(d:Data): Both lemmas are used to prove theorem 3 and the complete proof can be found at [20]. It is important to point out that one of the main limitations of using proof assistants such as PVS is that heavy user intervention is required in the proof development. 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https://openalex.org/W2913954080	https://open.library.ubc.ca/media/download/pdf/52383/1.0376259/3	English	null	Addressing the affordability of cancer drugs: using deliberative public engagement to inform health policy	Health research policy and systems	2,019	cc-by	6,955	RESEARCH Open Access © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. (2019) 17:17 (2019) 17:17 Bentley et al. Health Research Policy and Systems (2019) 17:17 https://doi.org/10.1186/s12961-019-0411-8 Addressing the affordability of cancer drugs: using deliberative public engagement to inform health policy Colene Bentley1* , Stuart Peacock1, Julia Abelson2, Michael M. Burgess3, Olivier Demers-Payette4, Holly Longstaff5, Laura Tripp2, John N. Lavis6 and Michael G. Wilson6 Abstract Background: Health system expenditure on cancer drugs is rising rapidly in many OECD countries given the costly new treatments and increased rates of use due to a growing and ageing population. These factors put considerable strain on the sustainability of health systems worldwide, sparking public debate among clinicians, pharmaceutical companies, policy-makers and citizens on issues of affordability and equity. We engaged Canadians through a series of deliberative public engagement events to determine their priorities for making cancer drug funding decisions fair and sustainable in Canada’s publicly financed health system. Methods: An approach to deliberation was developed based on the McMaster Health Forum’s citizen panels and the established Burgess and O'Doherty model of deliberative public engagement. Six deliberations were held across Canada in 2016. Transcripts were coded in NVivo and analysed to determine where participants’ views converged and diverged. Recommendations were grouped thematically. Results: A total of 115 Canadians participated in the deliberative events and developed 86 recommendations. Recommendations included the review and regular re-review of approved drugs using ‘real-world’ evidence on effectiveness and cost-effectiveness; prioritisation of treatments that restore patients’ independence, mental health and general well-being; ensuring that decision processes, results and their rationales are transparent; and commitment to people with similar needs receiving the same care regardless of where in Canada they live. Conclusions: The next steps for policy-makers should be to develop mechanisms for (1) re-reviewing effectiveness and cost-effectiveness data for all cancer drugs; (2) making disinvestments in cancer drugs that satisfy requirements relating to grandfathering and compassionate access; (3) ensuring fair and equitable access to cancer drugs for all Canadians; and (4) fostering a pan-Canadian approach to cancer drug funding decisions. Keywords: Public engagement, priority-setting, cancer, Canada Background Switzerland in terms of dollars spent on pharmaceuticals per capita [2]. Health system expenditure on cancer drugs is rising rap- idly in many OECD countries [1], sparking widespread debate in the public arena, as well as among clinicians, pharmaceutical companies and policy-makers world- wide. In Canada, the situation is no different. The af- fordability of cancer drugs is particularly concerning for Canada, which is third only to the United States and Several factors are driving up expenditures on cancer drugs, including prices for new cancer drugs increasing dramatically [3, 4], the use of systemic therapies in more patients [5, 6], the sharp rise in incident cases (due to, among other reasons, patients living longer) [7, 8] and the acceleration in the number of new cancer drugs (often with higher daily drug costs and longer duration of treat- ment) [9]. Together, these factors put considerable strain on the affordability and sustainability of Canada’s publicly financed provincial and territorial health systems. * Correspondence: cbentley@bccrc.ca 1Canadian Centre for Applied Research in Cancer Control (ARCC), 675 West 10th Avenue, Vancouver, British Columbia V5Z 1L3, Canada Full list of author information is available at the end of the article * Correspondence: cbentley@bccrc.ca 1Canadian Centre for Applied Research in Cancer Control (ARCC), 675 West 10th Avenue, Vancouver, British Columbia V5Z 1L3, Canada Full list of author information is available at the end of the article * Correspondence: cbentley@bccrc.ca 1Canadian Centre for Applied Research in Cancer Control (ARCC), 675 West 10th Avenue, Vancouver, British Columbia V5Z 1L3, Canada Full list of author information is available at the end of the article Participant recruitment The goal of recruitment was to identify a group of 20–25 citizens to participate in each panel who reflected a diversity of life experiences and social per- spectives based on the demographics of the province in which each panel was held. An online market research company, AskingCanadians™, was engaged to conduct recruitment. A letter of invitation to partici- pate in the deliberation was e-mailed to a randomly selected group of AskingCanadians™ online panel members. Interested individuals completed an eligibil- ity survey on the following criteria: age, income, education, ethno-cultural background, lived experience with chronic disease (as patient or caregiver; type of chronic disease was unspecified) and geographic loca- tion within their home province. Individuals were not eligible to participate if they were employees of healthcare organisations or health professionals, em- ployees or those with a direct financial relationship with a tobacco or pharmaceutical company, individ- uals who had lobbied for health advocacy groups, health policy-makers, including both elected officials and public servants, people who had worked for market research, advertising, public media or public relations firms, or individuals who had previously par- ticipated in a citizen panel hosted by one of the re- search collaborators. While relatively little is known about what Canadian clinicians and policy-makers value concerning different cancer programmes and interventions and their out- comes, even less is known about Canadians’ values on these matters [19]. To address this knowledge gap, we explored the concerns, perspectives and values of Cana- dians on the topic of cancer drug funding, and brought their recommendations to the attention of policy-makers who must confront issues of affordability, sustainability and fairness in their healthcare jurisdictions. Page 2 of 10 Page 2 of 10 Bentley et al. Health Research Policy and Systems (2019) 17:17 Bentley et al. Health Research Policy and Systems (2019) 17:17 Canada’s pan-Canadian Oncology Drug Review, which is part of the Canadian Agency for Drugs and Technolo- gies in Health (CADTH), utilises a national review process for new cancer drugs. It reviews the clinical and economic evidence (among other inputs) about each new cancer drug and makes funding recommendations to Canada’s provincial and territorial governments, which are responsible for healthcare funding decisions [10]. However, its recommendations do not address the economic and ethical challenges that provincial and territorial policy-makers face about how to cover in- creasingly expensive cancer drugs given limited budgets and competing health priorities. O’Doherty [20]. These approaches support citizens from a variety of backgrounds and perspectives to participate in meaningful dialogic exchange, where their deliberations are directed towards collective problem-solving while highlighting the acceptable trade-offs of a given policy initiative. Deliberative approaches draw on citizens’ perspectives to help define policy issues and advise on the social and normative aspects of decision-making [17, 21]. Empirical evidence has shown that the public can make coherent and sophisticated recommendations concerning values and health policy [22] and provide substantive knowledge to policy-makers. Deliberative approaches can enhance accountability in government decision-making [23], improve the legitimacy of deci- sions taken [24] and promote public understanding of complex healthcare issues [17]. There is relatively little guidance on if, when and how to consult the public on priority-setting and resource allocation decisions [11, 12]. In recent years, deliberative forms of public engagement have emerged as robust approaches to public consultation on many health policy initiatives [13, 14]. Deliberative public engagement brings members of the public together in a process of learning and dialogic exchange focused on collective problem-solving that is not consensus driven [13, 15]. In Canada, deliberative engagements have been used to advise provincial Ministries of Health on technology assessment [16], health services [17] and biobanks [18], among many other topics. Structure of deliberations All panels followed the same format. On Day 1, partici- pants heard from expert speakers and asked them ques- tions, viewed the video, and deliberated and made recommendations on Topic 1. On Day 2, participants deliberated on Topics 2 and 3 and made recommenda- tions on them. Panel discussions were led by trained facilitators in large and small group sessions. Each deliberation topic was discussed first in small group sessions, followed by collective deliberation and making recommendations in the large group. g The overarching deliberation topics and questions were: The overarching deliberation topics and questions were: Participants negotiated all recommendation state- ments. Once a recommendation was formulated, voting was used to gauge the degree of collective support for it, and as a technique to identify points of disagreement or tension that could be recorded and explored more fully. Participants who abstained or voted against a recom- mendation were prompted to explain their views. Provincial panels: Provincial panels: What should guide policy decisions about whether to fund new cancer drugs or change the funding provided for existing cancer drugs? What would make cancer drug funding decisions trustworthy? At least one, and often three, principal investigators attended each panel to answer questions of clarification from participants, observe the deliberations first hand and support the consistency of methods across the events. How can we improve existing approaches to decision-making about cancer drug funding? Pan-Canadian panel: What are important features of a pan-Canadian ap- proach to making funding decisions about cancer drugs? Methods Five 2-day citizen panels – or deliberative public engage- ment events – were convened in four Canadian prov- inces (Saskatchewan, Ontario, Quebec and Nova Scotia) between April and June 2016. Provinces were selected to reflect different geographic regions, cancer delivery pro- grammes and drug budgets across Canada. Two of the five panels were held in Quebec (one in English and one in French). A sixth ‘pan-Canadian’ panel was convened in October 2016 (and, as described below, drew on par- ticipants from the five panels as well as from an earlier, similar provincial event in British Columbia in 2014). Recruitment for the pan-Canadian event involved randomly selecting 3–5 participants from each of the provincial panels who had expressed their interest in be- ing invited to the pan-Canadian event. AskingCana- dians™oversaw the initial contact with prospective participants from each provincial panel and used the same stratified approach to recruitment. The panel design combined the strengths of two well- established deliberative public engagement approaches, namely that of the McMaster Health Forum (www. mcmasterhealthforum.org/citizens/citizen-briefs-and-panels) and the ‘mini-public’ approach of Burgess, Longstaff and Participants received a $125 honorarium per 8-hour day for the deliberation and their expenses were covered. Page 3 of 10 Page 3 of 10 Bentley et al. Health Research Policy and Systems (2019) 17:17 knowledge of cancer drug funding processes and budgets locally and/or at the pan-Canadian level. This provided experiential perspectives and local decision-making con- text for panel participants. Deliberation topics, questions and information supports Deliberation topics, questions and information supports Deliberation topics and questions were developed in con- sultation with provincial cancer policy-makers in each of the provinces where the panels were held, and with mem- bers of the project’s steering and advisory committees. In keeping with an ‘integrated knowledge translation’ or ‘co-- production’ model, the steering committee provided guid- ance and oversight on the project’s direction. It was comprised of three senior decision-makers in cancer con- trol, two of whom were from pan-Canadian organisations and one was in charge of a provincial drugs budget. The advisory committee included members of the research team with expertise in deliberative public engagement and recruitment methods, research ethics, and institutional oversight. They provided advice to ensure that the project was in compliance with relevant regulations (e.g. informed consent processes, financial reporting, etc.). Data collection and analysis What are the trade-offs associated with a pan- Canadian approach to making funding decisions about cancer drugs? All events were audio recorded and transcribed. The in- tegration of recommendations across panels was approached through constant comparison and consult- ation with transcripts to ensure appropriate interpret- ation. Differences across regional events and from the broader focus of the pan-Canadian event were consid- ered and reflected in the description of the themes. How might these trade-offs be addressed to produce trustworthy decisions? Several information sources were developed to support participants’ deliberations. Prior to each event, partici- pants received a link to the video ‘Cancer Dialogues’, which featured two oncologists, a health economist and a patient advocate, each speaking about cancer drug funding from their own perspective (see https://cc-arcc.ca/socie- tal-values-public-engagement-2/). Participants also re- ceived a plain-language citizen brief containing relevant research evidence on the topic of cancer drug funding de- cisions in Canada, how these decisions are made, the drug coverage plans for the province in which the panel was held, and several additional questions to encourage partic- ipants to reflect upon the information provided (see Additional file 1 for the citizen brief for the pan-Canadian panel). At each event, a cancer patient representative and an oncologist from the area gave brief remarks and answered questions. The oncologists also had expert p Transcript analysis began with a detailed review of the recommendations within their provincial or pan-Canadian context and through a comparative lens. This initial re- view by the study team led to the identification of a more thematically organised set of categories that aligned with the deliberative topics and questions, and which would provide helpful guidance to policy-makers. Within each thematic category, high-level agreement and divergent perspectives within and across the panel recommenda- tions were identified. A more thorough analysis of the event transcripts was then undertaken to assess how con- cepts and terms were understood across events and to fur- ther characterise the themes. Two qualitative researchers (CB, ODP) who also attended multiple panels analysed the transcripts, which were entered into NVivo 11 soft- ware. Weekly sessions were held to review the concepts Page 4 of 10 Bentley et al. Health Research Policy and Systems (2019) 17:17 and terms within each deliberative context. The study team also met weekly to provide feedback on the inter- pretation of the findings across panels. drug funding decisions. Results Participants articulated the principles they felt should guide funding decisions. At the provincial panels, partic- ipants were presented with decision scenarios in which they played the role of policy-makers. The scenarios were intentionally limited to force specific trade-offs be- tween cost and quality or quantity of life, and disinvest- ment (Fig. 1). Participants’ recommendations reflected what specific health benefit (i.e. improved duration or quality of life) they felt justified the significant additional cost (i.e. a doubling of the cost). A total of 115 citizens participated in the deliberative events, with 20–25 citizens per panel (Table 1). Across all panels, participants deliberated thoughtfully and respectfully on a range of complex issues related to the affordability, sustainability and fairness of cancer drug funding in Canada. They grasped the core issues under consideration, were able to identify acceptable cost-benefit and equity trade-offs, and provided relevant guidance on making cancer drug funding decisions in Canada. Participants developed 86 recommendations over the six panels on a range of themes. See Additional file 2 for recommendations from the provincial panels and Additional file 3 for recommendations from the pan- Canadian panel. The themes, described below, draw from the recommendations and transcripts to qualita- tively illustrate the areas of strength, convergence and disagreement within and across panels, and to capture important nuances from participants’ discussions. While the themes were supported by all panels, quotations or specific details are drawn from the recommendations of the panel, identified in brackets. Participants generally thought that modest life ex- tension alone, unless it was sustaining a pre-existing good quality of life, was insufficient justification for approving new or more expensive drugs over alterna- tives. Most considered quality and length of life inter- dependently. With respect to funding drugs that extend life, the majority of participants considered doubling the cost, i.e. going from $15,000 to $30,000 per patient, to be a significant cost increase and a worthwhile expenditure only if life was extended by at least 12 months. There was a small degree of support for significant cost being worthwhile for an extension of life of less than 12 months. Theme 1. Evidence and other inputs to support decision- making Participants in Saskatchewan and at both Quebec events (English and French) specified that, if a cancer drug restored independence for patients and/or im- proved their mental health, then a higher cost for that drug would be justified. There was strong support for clear criteria in drug fund- ing decisions. Participants recommended having “base- line criteria” (Nova Scotia) or a “decision-making tool” (Saskatchewan) containing specific data elements to guide decision processes so that decisions are based on adequate, identifiable and unbiased information. Ethics approval The project was approved by the Hamilton Integrated Research Ethics Board (#13-369) and the University of British Columbia-British Columbia Cancer Research Ethics Board (#H16-00623). All participants signed a written informed consent form prior to the event. Data collection and analysis However, the criterion of ‘age’ was explicitly rejected or considered discriminatory at all other provincial panels. Participants in Ontario supported considering drug costs in light of “other parts of the health system” and “opportunity costs”. They also considered “what the pub- lic is willing to pay for minimal survival improvements”, which indicates there may be a threshold beyond which some drugs are not funded. Theme 3. Disinvestment and re-review of data and past decisions The Saskatchewan panel specified the components of a “decision-making tool”: There was strong support across all panels for reviewing and reassessing currently approved and used cancer drugs. Participants recommended that reviews be based on clear and consistent principles and on real-world data on the drug’s effectiveness and cost-effectiveness, and that these data be compared with data about other cancer drugs. Participants strongly supported the principle that the health system should fund drugs that are more cost-effective and more clinically effective rela- tive to other cancer drugs. They accepted the need to make tough funding decisions, including stopping or scaling back funding for some currently funded drugs. “The criteria used in the decision-making tool should in- clude but are not limited to cost, quality and quantity of life, side effects, effectiveness, accessibility, incidence and type of cancer, mortality, age, duration of treatment, sustainability of the drug (uninterrupted supply), and ability to compare to other provinces’ decisions.” This recommendation reflects the types of information participants across provincial panels felt should guide Bentley et al. Theme 3. Disinvestment and re-review of data and past decisions Health Research Policy and Systems (2019) 17:17 Page 5 of 10 Table 1 Participant demographics Provincial panels Pan-Cana Provincial total Percentage of total Hamilton Halifax Montreal (English) Saskatoon Montreal (French) Participants 115 25 24 20 21 25 24 Sex Male 53 46% 12 13 8 9 11 13 Female 62 54% 13 11 12 12 14 11 Age, years 18–24 4 3% 2 0 0 1 1 0 25–34 21 18% 4 6 3 5 3 5 35–49 29 25% 10 6 2 5 6 3 50–64 34 30% 6 5 11 5 7 11 65+ 27 23% 3 7 4 5 8 5 Geography Urban area 56 49% 13 11 9 10 13 14 Suburban area 44 38% 10 10 8 6 10 7 Rural area 15 13% 2 3 3 5 2 3 Income Less than $20,000 11 10% 2 1 3 4 1 3 Between $20,000 and $34,999 25 22% 4 9 1 4 7 5 Between $35,000 and $49,999 15 13% 3 3 4 1 4 4 Between $50,000 and $79,999 28 24% 4 6 6 5 7 4 More than $80,000 14 12% 5 1 1 4 3 4 Prefer not to answer 22 19% 7 4 5 3 3 4 Education No certificate 0 0% 0 0 0 0 0 0 High school 14 12% 1 3 2 3 5 0 College or apprenticeship, non-university 23 20% 6 4 5 3 5 9 Some university 23 20% 4 4 4 2 9 3 University or above 55 48% 14 13 9 13 6 12 Children With children 62 54% 14 13 11 11 13 17 Without children 53 46% 11 11 9 10 12 7 Ethnicity Aboriginal 6 5% 2 1 0 3 0 2 Arab/Middle Eastern 8 7% 2 0 3 2 1 2 Black 10 9% 3 3 0 1 3 3 Chinese 4 3% 1 3 0 0 0 2 Filipino 5 4% 2 1 0 2 0 2 Japanese 3 3% 3 0 0 0 0 2 Korean 0 0% 0 0 0 0 0 0 Latin American 7 6% 3 1 2 0 1 0 South Asian 7 6% 3 2 2 0 0 5 White 58 50% 5 12 12 13 16 5 Other 4 3% 0 1 1 0 2 0 Prefer not to answer 3 3% 1 0 0 0 2 1 Are you currently living with a chronic disease? Theme 4. Ensuring fairness and access Fairness and equity of access to cancer treatment were im- portant principles across all panels. Participants’ concerns relating to fairness and equity included ensuring access for patients (1) undergoing treatment, (2) living in remote loca- tions and (3) who are members of vulnerable populations. Participants strongly supported involving citizens in decision processes, but the support was qualified by a concern that publics might not be adequately informed. Theme 3. Disinvestment and re-review of data and past decisions Do you now or have you in the past cared for someone with one or more chronic diseases? Yes/Yes (sufferer/caregiver) 16 14% 3 4 3 3 3 4 No/No (sufferer/caregiver) 48 42% 12 11 6 9 10 5 Yes/No (sufferer/caregiver) 27 23% 6 4 6 5 6 5 No/Yes (sufferer/caregiver) 24 21% 4 5 5 4 6 5 No/no answer (sufferer/caregiver) 2 Yes/no answer (sufferer/caregiver) 3 Bentley et al. Health Research Policy and Systems (2019) 17:17 Page 6 of 10 . 1 Decision scenarios Fig. 1 Decision scenarios i Page 7 of 10 Page 7 of 10 Bentley et al. Health Research Policy and Systems (2019) 17:17 industry in these processes so they could be made ac- countable for their data. Participants supported weighing the costs and benefits of comparable drugs to identify drugs for delisting. They supported replacing cancer drugs based on cost savings for drugs of similar safety and effectiveness. At both Quebec panels (English and French), some participants stipulated that any cost savings from disinvesting in cancer drugs be kept in the cancer drug funding budget envelope and either be re-invested in cancer drugs or in cancer research. There was some hesitation about the direct involve- ment of patients in drug funding decisions. For instance, some participants felt patients may be too emotionally tied to decisions under consideration (Nova Scotia, Quebec-French, Quebec-English). This led to some sup- port for recommending that decisions have input from “patient advocates” (Quebec-English) and “patient asso- ciations” (Quebec-French) to avoid placing individual patients under any emotional stress. Theme 6. Pan-Canadian approach to cancer drug funding and coverage decisions At the pan-Canadian event, panelists’ discussions tended to focus on removing provincial barriers to treat- ment and equitable access to cancer drugs for all Canadians. They also recommended implementing “equity audits” so that vulnerable populations are not overlooked. Theme 6. Pan-Canadian approach to cancer drug funding and coverage decisions Recommendations that focused on principles such as ‘compassionate access’ and ‘grandfathering’ reflected the notion that patients receiving treatment should not be disadvantaged by funding decisions and should have the opportunity to continue their treatment. Participants in Saskatchewan, Ontario and Nova Scotia stipulated that, if a drug is delisted, patients should be given the option to stay on the drug, i.e. grandfathering, while alternatives drugs are considered. Participants strongly supported the principle that people with similar needs should receive the same care regard- less of where in Canada they live, and that fairness should guide a pan-Canadian approach to cancer drug funding. While some participants were sceptical about the ability of Canadian provinces and territories to col- laborate on this goal, many were not, and even the scep- tics were supportive of the ideal. Several recommendations focused on improving access in remote regions and on funding for oral drugs because they can be more widely distributed. Personal responsi- bility for travel-related costs and for oral medications taken in ambulatory care was considered an inequity. Participants also recommended that marginalised popu- lations not be disadvantaged by funding decisions. At the pan-Canadian event, panelists’ discussions tended to focus on removing provincial barriers to treat- ment and equitable access to cancer drugs for all Canadians. They also recommended implementing “equity audits” so that vulnerable populations are not overlooked. Several recommendations focused on improving access in remote regions and on funding for oral drugs because they can be more widely distributed. Personal responsi- bility for travel-related costs and for oral medications taken in ambulatory care was considered an inequity. Participants also recommended that marginalised popu- lations not be disadvantaged by funding decisions. Participants in Nova Scotia and at both Quebec panels (English and French) recommended that the same cancer drugs be available in all the provinces and territories, and pan-Canadian panellists recommended a “mandatory pan-Canadian approach to cancer drug funding decisions”. At both Quebec panels, participants viewed a Canada-wide drug formulary as a way to mitigate regional differences in treatment access within the province, and all panels viewed it as a way to decrease costs through bulk purchasing. Participants in Saskatchewan considered a common drug formulary primarily as an opportunity to share information and avoid duplication of decision effort across provinces. Discussion Theme 5. Transparency of the decision-making process Transparency was regarded as foundational to trust- worthy governance. Nova Scotia participants stressed that the public needs to understand “how decisions are made and who is making them”, while Saskatchewan par- ticipants supported “decision-making that is sustainable, defensible, transparent, objective, accountable, and fair”. Our findings affirm many aspects of current decision- making practices in Canada related to the funding of cancer drugs. Recommendations that drug funding deci- sion processes consider a range of inputs and evidence, that increases in cancer drug spending be justified using clear and consistent principles, and that decision-making processes and their rationales should be transparent and publicly available are consistent with current approaches taken by pan-Canadian health technology assessment (HTA) bodies like CADTH. Participants emphasised that decision-making bodies should represent a range of different perspectives and appropriate expertise, with membership being a mix of citizens, cancer patients, health professionals and policy-makers. There was strong support in the Saskatch- ewan, Quebec-French, Quebec-English and pan-Canadian panels for excluding the pharmaceutical industry from decision processes due to conflict of interest concerns. However, some Ontario participants supported including Participants wanted drug funding decision processes supported by a range of inputs and evidence, including drug costs, clinical benefit of quality and quantity of life, potential side effects, and incidence rates. Again, CADTH currently considers clinical and economic Bentley et al. Health Research Policy and Systems (2019) 17:17 Page 8 of 10 Page 8 of 10 evidence in its drug assessments, as do Canadian prov- inces and territories, in their decisions about which drugs to place on their formularies. While CADTH con- siders input from patient groups in its drug assessment processes, participants expressed some concern about including patients and members of the public in such processes. collaborate on the goal of a common formulary, many felt that a pan-Canadian approach was still an important goal to pursue. Participants made several recommenda- tions related to improving access in rural and remote lo- cales, and strongly supported public funding for oral treatments over intravenous drugs as a means of redu- cing barriers to treatment access. g There are some limitations to the findings from this project. One limitation relates to the representativeness of the participants. Discussion While the participant sample reflected a balance of key demographic criteria across all six panels, with the exception of under-representation in the 18–24 age range, our recruitment goal was not to duplicate population distributions of life experiences but to over-represent the range of diversity in comparison to the general population. Although we did not recruit par- ticipants from every province and none were recruited from the territories, the provinces included across all six panels represented approximately 80% of the Canadian population in 2016 [26]. Participants wanted further assurances of trustworthy governance of decision-making processes, like avoiding conflicts of interest, committee membership renewal and disclosing reasons for granting compassionate access to drugs not listed on a provincial formulary. These recommendations applied not only to CADTH, but also to provincial and territorial HTA processes to help policy-makers improve how drug funding decisions are made and how their rationales are communicated to the public. Our findings suggest that new policy options be con- sidered. Participants supported developing processes for re-reviewing data and making disinvestments, ensuring fairness and equity are central principles, and that there should be a pan-Canadian approach to cancer drug funding decisions. While there are currently no formal mechanisms at the pan-Canadian, provincial or territor- ial levels to either re-review data on effectiveness and cost-effectiveness of currently approved and used cancer drugs or to disinvest in any area of cancer treatment, CADTH’s Strategic Plan for 2018–2021 has focused on HTA management, which includes reassessment and disinvestment of existing drugs and technologies [25]. Participants also recommended that real-world effective- ness and cost-effectiveness be considered in the ongoing re-review of approved drugs in order to achieve better value for money. These recommendations reflect that participants accepted the need for trade-offs and tough funding decisions. Participants also supported replacing an existing drug with a new drug on the basis of cost savings if the new drug has the same safety and effect- iveness. Finally, all jurisdictions should give priority to cancer drugs that offer improvements in survival and re- store patients’ independence, mental health and general well-being. A second limitation relates to the use of decision sce- narios, which showed that participants could make spe- cific cost-benefit trade-offs. The scenarios’ structure was intentionally limited in order to force a specific trade- off; however, this also meant that some trade-offs (e.g. Discussion involving rare cancers) were not explored in relation to fairness and sustainability, and some decision assump- tions (e.g. stage of disease, overall budget) constrained the trade-offs considered. Conclusion The findings from this project provide a set of perspec- tives on what participants collectively thought made for good, trustworthy decisions about funding for cancer drugs in Canada that are affordable, sustainable and fair. The results demonstrate that informed, deliberative pub- lics accept the need to make trade-offs when budgets are limited. Participants supported making disinvestments based on real-world effectiveness and cost-effectiveness if patients can remain on their current treatment while alternatives are considered. Our approach to public en- gagement has the strength of producing advice that is informed, considers different social perspectives and reflects collective priorities rather than aggregating indi- vidual preferences or highlighting diverse stakeholder perspectives. Policy-makers can use this advice along with other important inputs, including the expertise of clinicians, ethicists, health economists, policy analysts, policy-makers, patients and families. Participants recommended that all jurisdictions ensure that people with similar needs receive the same care re- gardless of where in Canada they live. Because health- care delivery in Canada, including reimbursement for cancer drugs, is a provincial and territorial responsibility, different provinces and territories have different drug formularies, resulting in disparities of drug coverage across the country. Participants across all panels consid- ered these disparities to be unfair, and called for a pan-Canadian approach to cancer drug funding as a matter of fairness. While some participants were scep- tical about the ability of provinces and territories to Canadian policy-makers might want to consider a more sustained public deliberation model, like a public panel or incorporating multiple members of the public into existing committees. While we, as researchers in the fields of public engagement and health policy, have a Bentley et al. Health Research Policy and Systems (2019) 17:17 Page 9 of 10 Competing interests clear interest in promoting the adoption of robust methods for engaging the public, we believe our ap- proach has demonstrated that civic-minded citizens can effectively contribute to health policy considerations. Competing interests Competing interests The authors declare that they have no competing interests. References l Additional file 3: List of recommendations from the pan-Canadian panel. (PDF 696 kb) 1. Vogler S, Vitry A, Babar ZU. Cancer drugs in 16 European countries, Australia, and New Zealand: a cross-country price comparison study. Lancet Oncol. 2016;17(1):39–47. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. The next steps for policy-makers should be to develop mechanisms for (1) re-reviewing effectiveness and cost-effectiveness data for all cancer drugs; (2) making disinvestments in cancer drugs that satisfy requirements relating to grandfathering and compassionate access; (3) ensuring fair and equitable access to cancer drugs for all Canadians; and (4) a pan-Canadian approach to cancer drug funding decisions. Author details 1 1Canadian Centre for Applied Research in Cancer Control (ARCC), 675 West 10th Avenue, Vancouver, British Columbia V5Z 1L3, Canada. 2Department of Health Research Methods, Evidence and Impact, McMaster University, 1280 Main Street West (CRL 203), Hamilton, Ontario L8S 4K1, Canada. 3School of Population and Public Health, University of British Columbia, 239 RHS, 1088 Discovery Avenue, Kelowna, British Columbia V1V 1V7, Canada. 4Institute National d’Excellence en Santé et en Services Sociaux (INESSS), 2021 avenue Union, 12th Floor, bureau 1200, Montreal, Quebec H3A 2S9, Canada. 5Engage Associates Consulting Firm, Vancouver, British Columbia, Canada. 6McMaster Health Forum, 1280 Main Street West, MML-417, Ontario, Hamilton L8S 4L6, Canada. Abbreviations 2. OECD. Pharmaceutical Spending (indicator); 2018. https://data.oecd.org/ healthres/pharmaceutical-spending.htm. Accessed 14 Jan 2019. 2. OECD. 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The project was approved by the Hamilton Integrated Research Ethics Board (#13-369) and the University of British Columbia-British Columbia Cancer Re- search Ethics Board (#H16-00623). All participants signed a written informed consent form in advance of the event and, before the event commenced, were provided with an in-person explanation of the project and its goals, a description of any risks involved for participants, and an opportunity to ask questions. 17. Abelson J, et al. Does deliberation make a difference? Results from a citizens panel study of health goals priority setting. Health Policy. 2003;66(1):95–106. 18. Burgess MM. From ‘trust us’ to participatory governance: deliberative publics and science policy. Public Underst Sci. 2014;23(1):48–52. publics and science policy. Public Underst Sci. 2014;23(1):48–52. 19. Regier DA, et al. Public engagement in priority-setting: results from a pan- Canadian survey of decision-makers in cancer control. Soc Sci Med. 2014; 122:130–9. Additional files Additional file 1: Citizen brief for pan-Canadian panel. (PDF 3689 kb) Additional file 2: List of recommendations by province and deliberative topic. (PDF 824 kb) Additional file 1: Citizen brief for pan-Canadian panel. (PDF 3689 kb) Additional file 2: List of recommendations by province and deliberative topic. (PDF 824 kb) Bentley et al. Health Research Policy and Systems (2019) 17:17 Big Picture Bioethics: Developing Democratic Policy in Contested Domains. The International Library of Ethics, Law and Technology, vol. 16. Cham: Springer International; 2016. p. 243–76. 21. Gutmann A, Thompson D. Deliberating about bioethics. Hastings Cent Rep. 1997;27(3):38–41. 22. Abelson J, et al. Effective Strategies for Interactive Public Engagement in the Development of Health Care Policies and Programs. Ottawa: Canadian Health Services Research Foundation; 2010. 23. Daniels N, Sabin J. Limits to health care: fair procedures, democratic deliberation, and the legitimacy problem for insurers. Philos Public Aff. 1997; 26(4):303–50. 24. Bohman J. Public Deliberation: Pluralism, Complexity and Democracy. Cambridge: MIT Press; 1996. 25. Canadian Agency for Drugs and Technologies in Health. Transforming How we Manage Health Technologies in Support of Better Health, Better Patient Experience, and Better Value. Ottawa: CADTH; 2018. 26. Statistics Canada. Census Profile – 2016 Census. Ottawa: Statistics Canada; 2017. 25. Canadian Agency for Drugs and Technologies in Health. Transforming How we Manage Health Technologies in Support of Better Health, Better Patient Experience, and Better Value. Ottawa: CADTH; 2018. Consent for publication Not applicable. Consent for publication Not applicable. 20. Burgess MM, Longstaff H, O’Doherty K. Assessing deliberative design of public input on British Columbia biobanks. In: Dodds S, Ankeny R, editors. Page 10 of 10 Bentley et al. Health Research Policy and Systems (2019) 17:17 Big Picture Bioethics: Developing Democratic Policy in Contested Domains. The International Library of Ethics, Law and Technology, vol. 16. Cham: Springer International; 2016. p. 243–76. 21. Gutmann A, Thompson D. Deliberating about bioethics. Hastings Cent Rep. 1997;27(3):38–41. 22. Abelson J, et al. Effective Strategies for Interactive Public Engagement in the Development of Health Care Policies and Programs. Ottawa: Canadian Health Services Research Foundation; 2010. 23. Daniels N, Sabin J. Limits to health care: fair procedures, democratic deliberation, and the legitimacy problem for insurers. Philos Public Aff. 1997; 26(4):303–50. 24. Bohman J. Public Deliberation: Pluralism, Complexity and Democracy. Cambridge: MIT Press; 1996. 25. Canadian Agency for Drugs and Technologies in Health. Transforming How we Manage Health Technologies in Support of Better Health, Better Patient Experience, and Better Value. Ottawa: CADTH; 2018. 26. Statistics Canada. Census Profile – 2016 Census. Ottawa: Statistics Canada; 2017. Big Picture Bioethics: Developing Democratic Policy in Contested Domains. The International Library of Ethics, Law and Technology, vol. 16. Cham: Springer International; 2016. p. 243–76. 21. Gutmann A, Thompson D. Deliberating about bioethics. Hastings Cent Rep. 1997;27(3):38–41. 22. Abelson J, et al. Effective Strategies for Interactive Public Engagement in the Development of Health Care Policies and Programs. Ottawa: Canadian Health Services Research Foundation; 2010. 23. Daniels N, Sabin J. Limits to health care: fair procedures, democratic deliberation, and the legitimacy problem for insurers. Philos Public Aff. 1997; 26(4):303–50. 25. Canadian Agency for Drugs and Technologies in Health. Transforming How we Manage Health Technologies in Support of Better Health, Better Patient Experience, and Better Value. Ottawa: CADTH; 2018. 25. Canadian Agency for Drugs and Technologies in Health. Transforming How we Manage Health Technologies in Support of Better Health, Better Patient Experience, and Better Value. Ottawa: CADTH; 2018. atistics Canada. Census Profile – 2016 Census. Ottawa: Statistics Canada; 20
https://openalex.org/W4282928595	https://www.frontiersin.org/articles/10.3389/fendo.2022.922640/pdf	English	null	From Isles of Königsberg to Islets of Langerhans: Examining the Function of the Endocrine Pancreas Through Network Science	Frontiers in endocrinology	2,022	cc-by	27,076	From Isles of Königsberg to Islets of Langerhans: Examining the Function of the Endocrine Pancreas Through Network Science Andrazˇ Stozˇer 1, Marko Sˇ terk 1,2, Eva Paradizˇ Leitgeb 1, Rene Markovicˇ 2,3, Masˇa Skelin Klemen 1, Cara E. Ellis 4, Lidija Krizˇancˇic´ Bombek 1, Jurij Dolensˇek 1,2, Patrick E. MacDonald 4 and Marko Gosak 1,2* 1 Institute of Physiology, Faculty of Medicine, University of Maribor, Maribor, Slovenia, 2 Department of Physics, Faculty of Natural Sciences and Mathematics, University of Maribor, Maribor, Slovenia, 3 Institute of Mathematics and Physics, Faculty of Electrical Engineering and Computer Science, University of Maribor, Maribor, Slovenia, 4 Department of Pharmacology and Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada Reviewed by: Reviewed by: Kazuki Harada, The University of Tokyo, Japan Xuelin Lou, Medical College of Wisconsin, United States *Correspondence: Marko Gosak marko.gosak@um.si Specialty section: This article was submitted to Diabetes: Molecular Mechanisms, a section of the journal Frontiers in Endocrinology Specialty section: This article was submitted to Diabetes: Molecular Mechanisms, a section of the journal Frontiers in Endocrinology Received: 18 April 2022 Accepted: 16 May 2022 Published: 15 June 2022 Islets of Langerhans are multicellular microorgans located in the pancreas that play a central role in whole-body energy homeostasis. Through secretion of insulin and other hormones they regulate postprandial storage and interprandial usage of energy-rich nutrients. In these clusters of hormone-secreting endocrine cells, intricate cell-cell communication is essential for proper function. Electrical coupling between the insulin- secreting beta cells through gap junctions composed of connexin36 is particularly important, as it provides the required, most important, basis for coordinated responses of the beta cell population. The increasing evidence that gap-junctional communication and its modulation are vital to well-regulated secretion of insulin has stimulated immense interest in how subpopulations of heterogeneous beta cells are functionally arranged throughout the islets and how they mediate intercellular signals. In the last decade, several novel techniques have been proposed to assess cooperation between cells in islets, including the prosperous combination of multicellular imaging and network science. In the present contribution, we review recent advances related to the application of complex network approaches to uncover the functional connectivity patterns among cells within the islets. We ﬁrst provide an accessible introduction to the basic principles of network theory, enumerating the measures characterizing the intercellular interactions and quantifying the functional integration and segregation of a multicellular system. Then we describe methodological approaches to construct functional beta cell networks, point out possible pitfalls, and specify the functional implications of beta cell network examinations. We continue by highlighting the recent ﬁndings obtained through advanced multicellular imaging techniques supported by network-based analyses, giving special emphasis to the current developments in both mouse and human islets, as well as outlining challenges offered by the multilayer network formalism in exploring the collective activity of islet cell populations. Finally, we emphasize that the combination of these imaging techniques and Islets of Langerhans are multicellular microorgans located in the pancreas that play a central role in whole-body energy homeostasis. Through secretion of insulin and other hormones they regulate postprandial storage and interprandial usage of energy-rich nutrients. In these clusters of hormone-secreting endocrine cells, intricate cell-cell communication is essential for proper function. Electrical coupling between the insulin- secreting beta cells through gap junctions composed of connexin36 is particularly important, as it provides the required, most important, basis for coordinated responses of the beta cell population. June 2022 \| Volume 13 \| Article 922640 REVIEW published: 15 June 2022 doi: 10.3389/fendo.2022.922640 REVIEW published: 15 June 2022 doi: 10.3389/fendo.2022.922640 The increasing evidence that gap-junctional communication and its modulation are vital to well-regulated secretion of insulin has stimulated immense interest in how subpopulations of heterogeneous beta cells are functionally arranged throughout the islets and how they mediate intercellular signals. In the last decade, several novel techniques have been proposed to assess cooperation between cells in islets, including the prosperous combination of multicellular imaging and network science. In the present contribution, we review recent advances related to the application of complex network approaches to uncover the functional connectivity patterns among cells within the islets. We ﬁrst provide an accessible introduction to the basic principles of network theory, enumerating the measures characterizing the intercellular interactions and quantifying the functional integration and segregation of a multicellular system. Then we describe methodological approaches to construct functional beta cell networks, point out possible pitfalls, and specify the functional implications of beta cell network examinations. We continue by highlighting the recent ﬁndings obtained through advanced multicellular imaging techniques supported by network-based analyses, giving special emphasis to the current developments in both mouse and human islets, as well as outlining challenges offered by the multilayer network formalism in exploring the collective activity of islet cell populations. Finally, we emphasize that the combination of these imaging techniques and Edited by: Quan Zhang, University of Oxford, United Kingdom Edited by: Quan Zhang, University of Oxford, United Kingdom 1In the case of points with only even numbers of lines attached to them, it does not matter where the walk starts. In the case with two points with odd numbers of lines attached to them, the walk must start in one of them. INTRODUCTION Strogatz pointed out the omnipresence of small world connectivity patterns that are highly clustered and exhibit small characteristic path lengths. Such topological structures were identiﬁed in electric power grids, neuronal and brain networks, protein-protein interactions, metabolic pathways, transportation, social networks, and food webs, to name only a few (7–10). The next decisive breakthrough was the growth and preferential attachment model that describes the universal scaling in degree distributions by Barabási and Albert (11), along with the notion that highly heterogeneous and scale-free- like networks likewise pervade biological, social, and technological systems (12–14). It is clear that the potential of graph theory to solve real-world problems is only beginning to be realized. From Isles of Königsberg to Network Science Graph theory has its roots in the 18th century. In 1735, Leonhard Euler became interested in a then popular brainteaser of Königsberg, today’s Kaliningrad. Kaliningrad´s center was built on four land masses, two isles on the river Pregel and two riverbanks. At that time, they were connected by seven bridges (Figure 1A). The problem was to cross all seven bridges in a continuous stroll, crossing each bridge exactly once. To solve the problem, Euler redrew the geographical situation of the city by constructing a graph with the land masses being represented as four nodes and the bridges being represented as seven links between them (Figure 1B). Euler analyzed the possibility of walking through a graph (city) using each edge (bridge) only once by considering the degree (number of edges connecting to each isle) of the vertices (isles). A path in a graph, which contains each edge only once, is called an Euler’s path. He realized that for such a walk to be feasible, there must be none or exactly two nodes with an odd number of links attached to them since any walker must both enter and leave all nodes, except the ones where the walk begins and ends1. All four nodes possessed an odd number of links; thus, the proposed walk was impossible (1). Interestingly, due to bombing, reconstruction, and construction of new bridges, today, there are altogether 9 bridges, one node has 3 links, two have 4 links, and the fourth has 7 links (Figure 1C). Therefore, a modern walk is possible in theory, but it must start on one and end on the other isle, making it hardly feasible in practice (Figure 1C). By solving the brainteaser in this way, Euler laid the foundation of graph theory and showed that functional implications of a particular structure can be understood and even predicted without working out all particularities. Frontiers in Endocrinology \| www.frontiersin.org Keywords: pancreatic islets, beta cells, calcium imaging, intercellular communication, functional networks, multilayer networks Citation: Stozˇer A, Sˇ terk M, Paradizˇ Leitgeb E, Markovicˇ R, Skelin Klemen M, Ellis CE, Krizˇancˇ ic´ Bombek L, Dolensˇek J, MacDonald PE and Gosak M (2022) From Isles of Königsberg to Islets of Langerhans: Examining the Function of the Endocrine Pancreas Through Network Science. Front. Endocrinol. 13:922640. doi: 10.3389/fendo.2022.922640 June 2022 \| Volume 13 \| Article 922640 1 Frontiers in Endocrinology \| www.frontiersin.org Multicellular Networks Within Pancreatic Islets Stozˇer et al. network-based analyses does not only represent an innovative concept that can be used to describe and interpret the physiology of islets, but also provides fertile ground for delineating normal from pathological function and for quantifying the changes in islet communication networks associated with the development of diabetes mellitus. network-based analyses does not only represent an innovative concept that can be used to describe and interpret the physiology of islets, but also provides fertile ground for delineating normal from pathological function and for quantifying the changes in islet communication networks associated with the development of diabetes mellitus. Keywords: pancreatic islets, beta cells, calcium imaging, intercellular communication, functional networks, multilayer networks From Network Science to Islets of Langerhans (A) In Euler’s time, one part of Königsberg (a) was connected with two bridges to the smaller isle (b) in the Pregel River and with one bridge to the larger isle (d). The same was true for the other part of the city (c). The problem was to create a path that would cross each bridge only once. (B) Euler solved the problem by representing the system as a graph in which the isles were represented as nodes and the bridges as links between them. (C) Euler´s path is shown on the current map of Kaliningrad. B A A B C FIGURE 1 \| The “Königsberg bridge problem” as a graph. (A) In Euler’s time, one part of Königsberg (a) was connected with two bridges to the smaller isle (b) in the Pregel River and with one bridge to the larger isle (d). The same was true for the other part of the city (c). The problem was to create a path that would cross each bridge only once. (B) Euler solved the problem by representing the system as a graph in which the isles were represented as nodes and the bridges as links between them. (C) Euler´s path is shown on the current map of Kaliningrad. introduced to the islet community a decade ago and are increasingly seen as a valuable tool to examine and quantify collective activity patterns in the pancreatic islets of Langerhans (18, 54–69). In these microorgans, communication among a variety of cells with unique functions and characteristics must occur to ensure proper control of metabolic homeostasis (70– 73). The most prevalent cell type are the insulin-secreting beta cells, which are electrically coupled through gap junctions composed of connexin36 (Cx36) to ensure mediation of intercellular signals (74, 75). Because beta cells are intrinsically highly heterogeneous and operate in a continuously changing environment, they exhibit complex yet coherent activity patterns, that are essential to tightly controlled insulin secretion. Inspired by and contributing to the increasing awareness that both synchronized islet activity and cell-to-cell communication are altered during the pathogenesis of diabetes (76–80), tools from the armamentarium of the complex network theory are now recognized as a powerful computational framework to assess the multicellular activity in islets and to study the progression of islet dysfunction in diabetes. Therefore, we review the recent advances related to the application of network theory to study islet physiology. From Network Science to Islets of Langerhans g Complex networks are widely applicable because they can represent and study the relationships between individual components in virtually any discrete system. In the last two decades, along with the advances in high-throughput technologies, including increasing computational power, biometric data acquisition, imaging techniques, and bioinformatics, network concepts have become an indispensable tool in biomedical research (15–23). In biomedicine, network science allows us to analyze, integrate and interpret several types of data, as well as to link them together in novel ways. Therefore, it complements the reductionist approach of systems biology, which focuses on identifying the key elements and their role in isolation. In addition to studies of gene-gene interactions (24), network science is also being used to analyze interactions between individual molecules, proteins (25, 26), signaling pathways, and more recently, to study the underlying pathophysiology of various disease processes (27–29), both at the level of the individual organs and at the systemic level. Network analyses nowadays allow the study of complex signaling pathways in health and disease, with the integrative concept being especially valuable in complex diseases with different comorbidities and multiple phenotypes (30). By understanding the bigger picture, it allows, on the one hand, the detection of potential drug targets, while at the same time offering the possibility of a systems approach to diagnosis and therapy (21, 27, 31, 32). Since its beginning, graph theory has occupied a place at the ‘pure’ end of pure mathematics. However, during the last century, analyses of social networks started gaining prominence apart from the developments in the ﬁeld of mathematical graph theory (2, 3). These advances, along with a data deluge at the turn of the 21st century, have led to the birth of a new interdisciplinary ﬁeld that has come to be known as network science (4, 5). This inherently multidisciplinary new discipline branch of study is now established as a backbone for describing various natural, social, and technological systems. It has catalyzed one of the most striking discoveries in complex systems research, i.e., that interactions of real-world networks follow some universal features. In a seminal paper (6), Watts and June 2022 \| Volume 13 \| Article 922640 2 Multicellular Networks Within Pancreatic Islets Stozˇer et al. A B C FIGURE 1 \| The “Königsberg bridge problem” as a graph. June 2022 \| Volume 13 \| Article 922640 Frontiers in Endocrinology \| www.frontiersin.org TYPES OF NETWORKS AND NETWORK METRICS Small-world networks can be divided into three structural classes: (i) single-scale networks, (ii) broad-scale networks, and (iii) scale-free networks (8). This classiﬁcation relies on the computation of the degree distribution, meaning the proportion of nodes that have a certain degree or number of edges (8, 92). Figure 2 shows three different types of networks along with their degree distributions that were obtained from the SNAP database (93). Figure 2A represents the road network in California, which is a homogeneous single-scale network that is characterized by a degree distribution with a fast decay, i.e., there are no nodes with a very high degree, meaning there are no cities or towns with extremely high numbers of roads connecting to them. Figures 2B, C represent a disease network and the air transport network in the USA, respectively. Both of these networks are heterogeneous, as evidenced by the heavy-tailed degree distributions, indicating thereby that there are a few nodes with a very high number of connections, i.e., hub nodes. However, only the air transportation network exhibits a scale- free structure with a clear power-law degree distribution. In contrast, despite being heterogeneous, the disease network structure deviates from the pure power-law behavior. Therefore, it can be categorized as a broad-scale or a weakly scale-free network. Notably, such structures are much more The birth of modern network science began with the objective of characterizing the topology of real-world systems consisting of many interacting units. In its purest form, a graph or a network is an abstract mathematical object consisting of nodes (or vertices) which are connected by links (or edges or connections). Because of this rather simple deﬁnition, complex networks represent a general and very useful framework to describe a large variety of social (3, 8, 81), biological (17, 18, 82–85), and technological systems (86–88). Nodes and edges can be regarded as a manifestation of some properties of a system and the corresponding network is a simpliﬁed mathematical representation of the relationships (edges) between variables (nodes), that does not necessarily encompass all the details of the underlying system. For example, in online social platforms, people (nodes) are connected through friendships (links); studying these social networks can allow the study of how information spreads without knowing details about each speciﬁc person in the network. From Network Science to Islets of Langerhans We ﬁrst provide a comprehensible introduction to the basic principles of network theory to introduce physiologists to this rather new theoretical paradigm. Then, we demonstrate how the complex network approaches can be used Furthermore, under the framework of the recently emerging ﬁeld of network physiology, network approaches have proven valuable to describe the structural organization and functional interrelations between different physiological and organ systems on the whole-organism level (18, 22, 33–35). Noteworthy, in the last decade perhaps the most remarkable breakthroughs of complex network analysis were achieved in neuronal physiology, where these approaches are used to quantify the brain’s functional and anatomical organization and have reached the maturity to be translated into clinical practice (36–39). Of most importance for our present review is the fact that in recent years, network theory has been used to assess intercellular interactions in multicellular systems where nodes represent individual cells, and their locations correspond to physical positions of cells in the tissue. Functional connections between cells are created based on the temporal similarity of the measured cellular dynamics, as evaluated through the calculation of the correlation coefﬁcient or other metrics for statistical similarity of time series. Multicellular recordings of membrane potential or intracellular calcium concentration are typically used as input signals. Network principles have thus been used to elucidate the connectivity patterns in neurons (40–44), astrocytes (45), pituitary endocrine cells (46, 47), lens epithelium cells (48, 49), hepatocytes (50), mammary epithelium cells (51), endothelial cells (52), and pancreatic acinar cells (53). Network analyses were June 2022 \| Volume 13 \| Article 922640 Frontiers in Endocrinology \| www.frontiersin.org 3 Multicellular Networks Within Pancreatic Islets Stozˇer et al. lead to a systems level understanding of the cell. Furthermore, data abstraction in networks facilitates the identiﬁcation of general topological characteristics in interaction patterns. The observation of small-world characteristics, universal scaling, and heterogeneity in the degree distributions, as well as the existence of community structure, are the most important features observed in a plethora of empirical networks (6, 11, 89–91). to uncover the functional connectivity patterns among cells within the islets and specify their functional implications. We conclude by pointing out some challenges and possible directions for future investigation of islets and the multicellular dynamics of endocrine cells in general. DESIGNING FUNCTIONAL BETA CELL NETWORKS To estimate whether nodes in the network are connected in a more segregated or a more integrated way, we can calculate the so-called local clustering coefﬁcient Ci, which is a measure of how well adjacent nodes are interconnected, while the average clustering coefﬁcient is deﬁned as the average of all local clustering coefﬁcients and indicates the overall interconnectedness. From a functional point of view, high clustering imparts resilience to removal of nodes since a signal will reach all neighbors of a removed cell if they are connected with each other. A metric for evaluating the quality of integration is the global efﬁciency of the networks, which is deﬁned as the inverse sum of all shortest path lengths between all accessible pairs of nodes. Higher values of global efﬁciency (i.e., shorter average path lengths) indicate better communication capability between nodes. Importantly, the trade-off between clustering coefﬁcient and the global efﬁciency is also used to measure the degree of small-worldness in the network, small-world networks are expected to simultaneously display both high integration and segregation, i.e., high global efﬁciency and clustering (97). Moreover, it is sometimes important to ﬁnd communities or subsets of nodes that are densely connected, and the extent to which the network is divided into communities can also be evaluated using the so-called modularity metric. A well- pronounced community structure in a network indicates a functional specialization of speciﬁc regions or subnetworks. In islets of Langerhans, this could indicate presence of islets within islets or islets that merged during ontogenesis. Lastly, network analysis can be used to identify connected components, deﬁned as a subset of nodes that can reach any other node by traversing links with the goal of describing the reachability of the network structure and ﬁnding isolated subnetworks. Of particular importance is the network’s largest component Smax, i.e., the maximal set of mutually connected nodes, which reﬂects the degree of integration and resilience of the network. All network measures described here and in Figure 3 can easily be applied to intercellular interaction patterns within the pancreatic islets once multicellular networks are established from experimental data. Here, we focus exclusively on functional multicellular networks, which derive from the system’s dynamics. In functional connectivity maps, nodes represent individual beta cells and connections between them are established based on the temporal similarity of the measured cellular dynamics, as typically assessed by statistical similarity of calcium signals (18). DESIGNING FUNCTIONAL BETA CELL NETWORKS common in real-world networks than rigorous scale-free networks. More speciﬁcally, due to different constraints and ﬁnite sizes, scale freeness of networks is not a ubiquitous phenomenon and therefore the term scale free network is in the literature sometimes loosely applied to different types of heterogeneous networks with heavy-tailed degree distributions (14). Nevertheless, from the functional point of view, when dealing with real-life networks, it is much more important to know whether a network´s degree distribution is heavy-tailed, so that we are aware of the existence of hub nodes, than whether it exactly follows a power-law (94, 95). Networks are a convenient abstraction tool for exploring how relationships and interactions between individual components give rise to emergent dynamics. When mapping cellular associations, an important distinction must be drawn between how cells are physically connected and how the actual information transfer occurs, affecting the collective activity of cellular populations. Formally, we distinguish between structural and functional networks, even though it is known that the structure and function in networks are closely intertwined (98). Structural intercellular networks describe the patterns of cellular morphology arrangement and provide the mechanistic substrate for intercellular signal transfer. Structural network analysis has already been utilized to study the cytoarchitecture of the islets. Speciﬁcally, network-based methods have been used to assess the spatial organization of cells and their homo- and hetero-typic contacts (99–104), to elucidate the principles of beta cell arrangement in normal and diabetic islets (105), to infer the structural basis for paracrine regulation of delta cells (106), and to study the arrangement of endocrine cells regarding the vascular network in the islets (107). While structural network analysis represents a vibrant topic in the islet community, it is beyond the scope of this review. The most commonly used network metrics applicable to studying multicellular networks are schematically presented in Figure 3 (for more details see (18, 96)). By simply counting the number of links of a node, we deﬁne its degree ki, and by averaging the degrees of all nodes, we can calculate the average degree of the network. The degree of a node is an indicator of its importance. By drawing a degree distribution, we can then distinguish between different types of networks, as already outlined above. TYPES OF NETWORKS AND NETWORK METRICS Similarly, protein-protein interaction networks, where nodes in networks represent proteins and links represent interactions between them, can C A B C D E F FIGURE 2 \| Examples of real-world networks along with their corresponding degree distributions. Road network in California (A), disease-disease network in which nodes represent inherited, developmental, or acquired diseases and links represent associations between them (B), and air transport network in the US (C). In panels (A-C) highly connected nodes, i.e., hubs, are colored purple, all other nodes are colored yellow. The lower panels feature the corresponding degree distributions of networks which are categorized as single-scale (D), broad-scale or weakly scale-free (E), and scale-free (F). C A B B C E F D F E FIGURE 2 \| Examples of real-world networks along with their corresponding degree distributions. Road network in California (A), disease-disease network in which nodes represent inherited, developmental, or acquired diseases and links represent associations between them (B), and air transport network in the US (C). In panels (A-C) highly connected nodes, i.e., hubs, are colored purple, all other nodes are colored yellow. The lower panels feature the corresponding degree distributions of networks which are categorized as single-scale (D), broad-scale or weakly scale-free (E), and scale-free (F). June 2022 \| Volume 13 \| Article 922640 4 Frontiers in Endocrinology \| www.frontiersin.org Multicellular Networks Within Pancreatic Islets Stozˇer et al. Frontiers in Endocrinology \| www.frontiersin.org June 2022 \| Volume 13 \| Article 922640 DESIGNING FUNCTIONAL BETA CELL NETWORKS (F) A group of nodes that are directly or indirectly connected to each other forms a connected component and stands for reachability within the network. The relative largest connected component representing the fraction of nodes that are connected either directly or indirectly is denoted by Smax. connected, weighted and undirected graph of the system and extracts only the sub-network which contains N-1 strongest connections (N – total number of nodes) with no disconnected nodes or circular connections. It has been, for example, successfully applied in brain research (113) and in assessing the collective dynamics of cryptocurrencies (114) or energy consumption proﬁles (115). The lack of disconnected nodes and the simple, barebone representation are the main advantages of this method, since disconnected nodes can represent a problem for previously described approaches. The main drawback is the lack of circular connections, consequently zero clustering coefﬁcient and an overall poorer description of the underlying system. Networks can also be constructed from binarized time series and in this case the so-called coactivity coefﬁcient is used to evaluate synchronicity between cells (64, 66, 116). While this method does objectively capture the level of synchronicity between cell pairs, it should not be used when there are notable differences in the activities of individual cells, as it might lead to spurious results. Another approach, popular in neuroscience for inferring the connectivity between brain regions, is the Granger causality (117). As the name suggests, threshold value should be chosen at R>0.7, such that more than half of variance is explained by the correlation (R2>0.49). Alternatively, one can ﬁx the average node degree, such that it reﬂects a physiologically relevant value (i.e., the expected number of connections in the observed tissue). By this means, the correlation threshold value for intercellular connectivity is iteratively adjusted until the resulting network has the desired edge density. In this context, the average node degree itself no longer has any descriptive meaning when comparing different networks, however, the underlying network parameters and degree distributions can differ greatly. If, for example, individual nodes of a network are poorly correlated, the resulting network will have more random-network-like attributes (high efﬁciency, very low clustering, high modularity), compared to highly correlated nodes which result in a more ordered network structure (high clustering, lower efﬁciency, and lower modularity). Therefore, this methodology should not be used when the signals in different islets differ substantially. DESIGNING FUNCTIONAL BETA CELL NETWORKS The creation of functional beta cell networks has become feasible with the development of functional multicellular calcium imaging techniques. Two earliest adopters of these techniques in conjunction with network-based analysis were Hodson et al. (54) in isolated islets and our group in tissue slices (62), where we represented cells as network nodes and functional connections between cells as network edges. In both studies as well as in most studies that followed, a thresholding of the correlation matrix was used to construct functional networks, where two cells were deemed functionally connected if their correlation coefﬁcient exceeded a preset threshold level. The procedure is schematically presented in Figure 4 and encompasses the processing of the recorded Ca2+ time series. In most of our previous works, we focused exclusively on the fast Ca2+ oscillations that propagate across the islets in the form of intercellular waves. To this aim, the signals were band-pass ﬁltered and smoothed to remove noise and variations in basal Ca2+ levels. We will discuss how preprocessing of recorded traces affects the network characteristics in the next chapter. The methodology to create such functional connectivity maps originates from neuroscience, where similar approaches have been used to study functional associations between brain regions or neurons (36, 108). However, a drawback of the method is that it relies on a somewhat arbitrary connectivity threshold selection (109–112). As one of the criteria it was suggested that the selected June 2022 \| Volume 13 \| Article 922640 Frontiers in Endocrinology \| www.frontiersin.org 5 Multicellular Networks Within Pancreatic Islets Stozˇer et al. A B C D E F FIGURE 3 \| Quantifying intercellular connectivity patterns with conventional network metrics. (A) Schematic representation of a multicellular system as a network composed of nodes (cells) and edges (connections or functional associations between cells). (B) Nodes are scaled based on the number of the neighbors to which they are directly connected, i.e., their degree. This feature is shown schematically for the yellow node, with the direct neighbors colored purple. (C) The clustering coefﬁcient describes the tendency of nodes to cluster together and is deﬁned as the number of existing connections between the neighbors of that node divided by the number of all possible connections between them. This property is illustrated by the example of the yellow-colored node, to which non-existing yellow edges between the purple neighboring nodes have been added. DESIGNING FUNCTIONAL BETA CELL NETWORKS (D) The shortest path length between any two nodes is the number of edges that form the shortest path between them. Lower average shortest paths of a network indicate more efﬁcient communication abilities. This feature is highlighted by two yellow nodes representing the source and destination nodes and the purple-colored nodes forming the shortest path. (E) A community consists of a group of nodes that have a higher connection density compared to the whole network. A well-pronounced community structure implies an internal network organization into functional modules. In panel (E), nodes are color-coded according to their membership in a community. (F) A group of nodes that are directly or indirectly connected to each other forms a connected component and stands for reachability within the network. The relative largest connected component representing the fraction of nodes that are connected either directly or indirectly is denoted by Smax. A B D E C F B C B D E F E D F FIGURE 3 \| Quantifying intercellular connectivity patterns with conventional network metrics. (A) Schematic representation of a multicellular system as a network composed of nodes (cells) and edges (connections or functional associations between cells). (B) Nodes are scaled based on the number of the neighbors to which they are directly connected, i.e., their degree. This feature is shown schematically for the yellow node, with the direct neighbors colored purple. (C) The clustering coefﬁcient describes the tendency of nodes to cluster together and is deﬁned as the number of existing connections between the neighbors of that node divided by the number of all possible connections between them. This property is illustrated by the example of the yellow-colored node, to which non-existing yellow edges between the purple neighboring nodes have been added. (D) The shortest path length between any two nodes is the number of edges that form the shortest path between them. Lower average shortest paths of a network indicate more efﬁcient communication abilities. This feature is highlighted by two yellow nodes representing the source and destination nodes and the purple-colored nodes forming the shortest path. (E) A community consists of a group of nodes that have a higher connection density compared to the whole network. A well-pronounced community structure implies an internal network organization into functional modules. In panel (E), nodes are color-coded according to their membership in a community. Frontiers in Endocrinology \| www.frontiersin.org DESIGNING FUNCTIONAL BETA CELL NETWORKS As an alternative, the minimum spanning tree network can be used. This method represents an unbiased network construction method which relies on the fully June 2022 \| Volume 13 \| Article 922640 Frontiers in Endocrinology \| www.frontiersin.org 6 Multicellular Networks Within Pancreatic Islets Stozˇer et al. A B C D E F FIGURE 4 \| Workﬂow of functional beta cell network extraction. (A) Confocal image of islet of Langerhans in acute tissue slice with three indicated beta cells (gray, yellow and violet dots) and indicated islet outline (white curve), exported raw Ca2+ signals of the three beta cells (B) and processed Ca2+ signals (C) of the same three cells. (D) Pearson correlation coefﬁcient matrix based on ﬁltered and smoothed Ca2+ signals of all cell pairs in the islet. (E) Extracted binary connectivity matrix based on the thresholded correlation matrix; only cell pairs whose correlations exceeded Rth = 0.82 were considered connected. (F) The corresponding functional beta cell network. Blue dots denote individual beta cells and gray lines represent functional connections between highly synchronized cells. C B A B F D E F D F E FIGURE 4 \| Workﬂow of functional beta cell network extraction. (A) Confocal image of islet of Langerhans in acute tissue slice with three indicated beta cells (gray, yellow and violet dots) and indicated islet outline (white curve), exported raw Ca2+ signals of the three beta cells (B) and processed Ca2+ signals (C) of the same three cells. (D) Pearson correlation coefﬁcient matrix based on ﬁltered and smoothed Ca2+ signals of all cell pairs in the islet. (E) Extracted binary connectivity matrix based on the thresholded correlation matrix; only cell pairs whose correlations exceeded Rth = 0.82 were considered connected. (F) The corresponding functional beta cell network. Blue dots denote individual beta cells and gray lines represent functional connections between highly synchronized cells. between slow Ca2+ oscillations can be driven by metabolic coupling of neighboring cells, via feedback onto the slow dynamics by the fast dynamics due to same electrical coupling (77, 135–139), as well as by intrinsic metabolic characteristics of beta cells (61). Since both fast and slow oscillations are synchronized among the cells in an islet, they both contribute to functional connectivity networks derived from Ca2+ signals (56, 133, 140, 141). DESIGNING FUNCTIONAL BETA CELL NETWORKS this method is not only used for determining functional connectivity between brain regions, but also for the direction (causality) of these connections and was also successfully utilized in beta cell research for identifying ﬁrst responder cells in mouse islets following glucose stimulation (67). Finally, it should be noted that particularly in neuroscience more advanced methods to quantify statistical interdependencies between active nodes are gaining attention, such as the partial cross-correlation (114, 118, 119), dynamic time warping (120, 121), and minimum jump cost (122, 123). In our ﬁrst study in which we employed network analysis to investigate functional connectivity we showed that beta cells are most synchronized and densely connected during the activation phase following the administration of stimulatory levels of glucose and during the deactivation phase after glucose stimulation has ceased. During these periods, the functional connections were mostly independent of the Euclidean distance between cells within the islet. This behavior may be because beta cells individually yet synchronously move from basal [Ca2+]IC to the much higher “plateau” level required to trigger insulin secretion upon stimulation, and back to baseline upon termination of stimulation, yielding highly correlated signals. Notably, similar observations were later also made in measurements on zebraﬁsh islets in vivo (67). Furthermore, in the phase of sustained activity (plateau phase), an intermediate level of synchronization was observed, and the length of functional connections was considerably lower, suggesting that the cells are predominantly synchronized via calcium waves which spread across the islet (62). Moreover, initial studies of Frontiers in Endocrinology \| www.frontiersin.org TOPOLOGICAL ANALYSIS OF FUNCTIONAL BETA CELL NETWORKS Beta cells respond to stimulation with tightly coupled oscillations in membrane potential and intracellular calcium concentration [Ca2+]IC leading to insulin secretion (124–128). At present, Ca2+ oscillations can be observed in two distinct time domains: i) fast Ca2+ oscillations under 20 s long, appearing with a frequency of about 5 min-1 and corresponding to beta cell bursting electrical activity (55, 124, 129, 130), and ii) the underlying slow oscillations with a frequency of around 0.1-0.2 min-1 (56, 131, 132). Gap junctional coupling of beta cells via Cx36 allows for the electrical linkage as well as the exchange of small molecules between neighboring cells and mediates principally the coherence of fast Ca2+ dynamics (133, 134). Correlations June 2022 \| Volume 13 \| Article 922640 Frontiers in Endocrinology \| www.frontiersin.org 7 Multicellular Networks Within Pancreatic Islets Stozˇer et al. beta cell networks have revealed that functional beta cell connectivity patterns are small-world networks dominated by a small subset of cells with a high degree offunctional connectivity, i.e., hub cells (62, 64). The high level of heterogeneity and the presence of long-range correlations were perhaps the most striking observations of the earliest studies. The existence of hub cells was also linked with metabolic proﬁles, which will be addressed in more detail in section 5. The presence of long-range connections that give rise to small-world characteristics was attributed to various reasons associated with a complex multicellular dynamics, whereby heterogeneity and a heterogeneous coupling were highlighted also in theoretical models (142, 143). dynamics comprises different temporal domains (Figure 5A), whose activity is coordinated across the islets in a different manner. In other words, beta cells have ﬂuctuations in intracellular calcium concentrations at both faster and slower oscillatory rates, and the networks constructed from these separate temporal domains have differing properties. Figure 5A shows a representative beta cell response, where the raw, unﬁltered [Ca2+]IC dynamics is shown in gray and yellow and violet traces represent the ﬁltered signal with only the slow and only the fast oscillatory component, respectively. In Figure 5B, the corresponding raster plots of the binarized fast and slow activity are presented and it can be noticed that both types of oscillatory dynamics display coordination across the islet. With these different input signals, three different functional networks can be constructed and characterized (Figure 5C). The structure of functional networks depends heavily on the type of the input signal. From Classical Physiological Parameters to Functional Connectivity Collective activity of beta cells achieved through tightly regulated electrical coupling of neighboring beta cells (and perhaps also other cell types) via Cx36 is vital to well-regulated insulin secretion (152). Functional multicellular imaging has allowed deeper insight into the importance of multicellular cooperation and functional connectivity of beta cells within an islet (73, 153– 155). The response of islets to glucose depends on the level of stimulation. With increasing glucose concentration from substimulatory (< 6 mM) to supraphysiological glucose concentrations (> 10mM), the delays to activation of cells shorten (advancement), and the fraction of beta cell population involved in the response increases (recruitment) (55, 145, 156– 159). Also, the delay between the activation of cells that respond ﬁrst (called ﬁrst responders) and beta cells in the same islet that activate later is shortened (55, 156, 160). Compared with dissociated or uncoupled cells, the probability density of cell activation in coupled beta cells in islets is narrower and thus the dose response curve is much steeper. Importantly, all of these features of the activation phase agree with model predictions, as excellently reviewed recently by Peercy and Sherman (60). In the plateau phase, the glucose dependency or the dose response is evident as an increase in active time or so-called duty cycle, which is in good agreement with insulin secretion (55, 160–164). During deactivation, the delay before cells turn off is longer for higher preceding glucose stimulation, however, the heterogeneity between cells is less than during activation (55, 156, 160). Importantly, a coordinated cessation of activity is important, as it prevents hypoglycemia. Data suggest that this last phase of response may be disrupted in uncoupled beta cells and under diabetogenic conditions (66, 152). Looking at the functional connectivity of beta cells, a clear transition from more segregated to more integrated networks can be observed with increasing glucose concentrations (55, 63). These changes in activity and functional connectivity are accompanied by a switch from more local to more global [Ca2+]IC waves (58, 147). However, despite the highly integrated functional connectivity in high glucose, an islet cannot be regarded as a uniform supercell sharing identical activity in all its parts. Rather, based In what follows we focus on the fast component of Ca2+ response. Beta cells respond to glucose stimulation in a biphasic manner (144–146). TOPOLOGICAL ANALYSIS OF FUNCTIONAL BETA CELL NETWORKS As explained above, the measured Ca2+ A B C FIGURE 5 \| Constructing functional beta cell networks from different types of Ca2+ signals. (A) Raw average Ca2+ signal of a representative islet (gray line) with extracted slow (yellow line) and fast (blue line) signal components. (B) Raster plots showing the activity of the slow (left, yellow dots) and fast (right, blue dots) Ca2+ signals of all cells in the islet. (C) Correlation-based functional networks constructed from unprocessed (left), slow- (middle) and fast (right) Ca2+ signals. Networks were constructed with a ﬁxed average network node degree kavg = 8.3. The following parameters for each functional network are provided: average correlation coefﬁcient (Ravg), average clustering coefﬁcient (Cavg), global efﬁciency (Eglob), largest connected component (Smax), small-world coefﬁcient (SW), average physical length of functional connections (Davg) and the number of communities (Ncom). A B C B C C FIGURE 5 \| Constructing functional beta cell networks from different types of Ca2+ signals. (A) Raw average Ca2+ signal of a representative islet (gray line) with extracted slow (yellow line) and fast (blue line) signal components. (B) Raster plots showing the activity of the slow (left, yellow dots) and fast (right, blue dots) Ca2+ signals of all cells in the islet. (C) Correlation-based functional networks constructed from unprocessed (left), slow- (middle) and fast (right) Ca2+ signals. Networks were constructed with a ﬁxed average network node degree kavg = 8.3. The following parameters for each functional network are provided: average correlation coefﬁcient (Ravg), average clustering coefﬁcient (Cavg), global efﬁciency (Eglob), largest connected component (Smax), small-world coefﬁcient (SW), average physical length of functional connections (Davg) and the number of communities (Ncom). June 2022 \| Volume 13 \| Article 922640 8 Frontiers in Endocrinology \| www.frontiersin.org Multicellular Networks Within Pancreatic Islets Stozˇer et al. Metabolically driven slow dynamic component gives rise to substantially longer functional connections, due to global slow Ca2+ waves occurring on a broader temporal scale (56). The slow component gives rise to a higher average correlation (Ravg), while the fast component results in higher clustering (Cavg), pointing out a denser interconnectivity between neighboring cells. Moreover, the network built upon the fast Ca2+ component results in a lower global efﬁciency (Eglob) compared to both the slow and the unﬁltered signal, denoting a longer characteristic path length between beta cells. TOPOLOGICAL ANALYSIS OF FUNCTIONAL BETA CELL NETWORKS Despite the higher clustering in the fast component, the lack of long-range connections leads to a decreased measure of small-worldness (SW). This indicates that the slow component importantly contributes to the level of small-worldness of the beta cell functional connectivity networks, which is an important aspect to consider when unprocessed signals are used for the analysis. Therefore, in addition to the insights gained by separating Ca2+ signals into various components (fast vs. slow vs. a combination of both), further methodological steps, i.e., signal ﬁltration, affect the characteristics of functional networks and their implications in islet function. Finally, it should be emphasized that functional connectivity describes a statistical relationship between the measured signals and therefore long enough intervals should be used to obtain relevant results, particularly when slow oscillations are analyzed, which display a relatively low temporal density of events (typically on the order of magnitude of 0.1 min-1). heterogeneity in the degree distribution implies the existence of highly connected beta cells, i.e., hub cells that are in this islet connected to more than 20% of other cells. While some studies have demonstrated a particular importance of this highly connected subpopulation of beta cells in orchestrating collective beta cell activity in healthy islets (148), as well as their crucial role in pathogenesis of diabetes (54, 58, 64), the evidence is not unanimous and many details about their characteristics and the part they play in islet coordination remain to be elucidated (55, 149–151). Frontiers in Endocrinology \| www.frontiersin.org From Classical Physiological Parameters to Functional Connectivity Following exposure to, there is an activation phase with a transient rise in [Ca2+]IC and fast Ca2+ oscillations, during which beta cells are progressively recruited and begin to operate more synchronously. Heterogeneous activation of beta cell clusters during activation is reﬂected in local Ca2+ waves that are heterogeneous in size (55, 145, 147). Activation is then followed by a stable plateau phase with synchronized, regular oscillations and islet-wide activation of beta cells, as indicated with the replacement of local Ca2+ waves with global Ca2+ waves (55, 147). This behavior is demonstrated in Figures 6A, B. The corresponding temporal evolution of functional networks throughout stimulation is reﬂected in its characteristics (Figure 6C), with a higher average node degree, clustering coefﬁcient, largest component, and a smaller number of communities during the plateau phase. In other words, with the increase in activity and coordination cells become more connected, the network becomes denser and more integrated, with fewer and bigger communities. The node degree distribution during the plateau phase decreases roughly potentially with a cut-off in the tail, i.e., following an exponentially truncated power law signifying the broad-scale nature of functional networks (Figure 6D) (55, 62). We wish to point out that physical limitations prevent the emergence of truly scale-free networks, i.e., a cell cannot be functionally connected to more cells than there are in a tissue, and consequently, they are not as ubiquitous in real world as is often presumed (14). It is worth emphasizing that the noted June 2022 \| Volume 13 \| Article 922640 9 Multicellular Networks Within Pancreatic Islets Stozˇer et al. A B C D FIGURE 6 \| Characteristics of the functional beta cell network in response to stimulation. (A) Average Ca2+ signal of an islet subjected to a 6-9-6 mM glucose stimulation protocol. Black line represents the unprocessed Ca2+ signal and the gray line represents the extracted fast component of cellular activity. Colored horizontal arrows ndicate the selected activation and plateau phases for further analysis. (B) Raster plots showing the binarized fast component of beta cell activity for the activation (left) and plateau (right) phase as indicated in panel (A). (C) Functional beta cell networks constructed for both phases (left, activation phase; right, plateau phase) based on fast signal components of all cells with a correlation coefﬁcient threshold for connectivity Rth = 0.8. From Classical Physiological Parameters to Functional Connectivity The following parameters for each functional network are provided: average correlation coefﬁcient (Ravg), average clustering coefﬁcient (Cavg), average network node degree (kavg), global efﬁciency (Eglob), largest connected component (Smax) and the number of communities (Ncom). (D) Node degree distributions for the networks extracted in the activation (yellow dots) and plateau phase (blue dots). A B B D C C D D FIGURE 6 \| Characteristics of the functional beta cell network in response to stimulation. (A) Average Ca2+ signal of an islet subjected to a 6-9-6 mM glucose stimulation protocol. Black line represents the unprocessed Ca2+ signal and the gray line represents the extracted fast component of cellular activity. Colored horizontal arrows indicate the selected activation and plateau phases for further analysis. (B) Raster plots showing the binarized fast component of beta cell activity for the activation (left) and plateau (right) phase as indicated in panel (A). (C) Functional beta cell networks constructed for both phases (left, activation phase; right, plateau phase) based on fast signal components of all cells with a correlation coefﬁcient threshold for connectivity Rth = 0.8. The following parameters for each functional network are provided: average correlation coefﬁcient (Ravg), average clustering coefﬁcient (Cavg), average network node degree (kavg), global efﬁciency (Eglob), largest connected component (Smax) and the number of communities (Ncom). (D) Node degree distributions for the networks extracted in the activation (yellow dots) and plateau phase (blue dots). insulin content, thus resembling a transcriptionally immature phenotype due to the low expression levels of signature beta cell transcription factors. Notably, energetic demands of the functionally most connected cells can also be estimated by analyzing details of [Ca2+]IC dynamics. More speciﬁcally, we employed methods of nonlinear time series analysis to reconstruct the phase space and to compute the dissipative characteristics of individual cells within the network (167). Hub cells exhibit the highest energy dissipation rates, which was also predicted theoretically. This concept is demonstrated in Figure 7, where we show an exemplary islet with color-coded energy dissipation rates of individual beta cells. In addition, the coordinated collective response of beta cells to elevated glucose was blunted after silencing the hubs, and restored after speciﬁc stimulation, thus suggesting that they may be necessary for coordinated islet [Ca2+]IC dynamics (64, 67). From Classical Physiological Parameters to Functional Connectivity At present, these ﬁndings cannot be completely reconciled with electrophysiological on differences in activation times, activity during the plateau phase, the origin of [Ca2+]IC waves, and the number of connections in functional networks, distinct beta cell subpopulations presumed to have distinct impacts on islet activity have been deﬁned (54, 55, 59, 62, 64, 67, 165). Beta Cell Subpopulations and Functional Heterogeneity Similarly, in human islets, during the ﬁrst phase of glucose response, small clusters of beta cells with high activity govern the response, whereas during the second phase, the electrical coupling becomes more important to synchronize large multicellular functional clusters (146). Moreover, from the network point of view, cells with the most functional connections tend to activate sooner (55), but not necessarily be the ﬁrst responders (68). Interestingly, the ability of cells to activate ﬁrst in response to glucose seems to depend less on glucokinase activity than on the resting KATP and junctional conductance, which may in addition to intra-islet differences between cells (60, 68) also explain some inter-strain differences in activation (152, 160). At present, there is not enough consistent evidence for a signiﬁcant overlap between hubs, ﬁrst responders, and wave initiators. However, one feature which is consistently present in both experimental and modeling studies in hub cells is their higher-than-average active time or duty cycle. This may be due to the fact that the hub cells participate in the great majority of [Ca2+]IC waves and that their [Ca2+]IC oscillations may be a bit longer and perhaps more stable than the ones in other cells, which is also able to explain why their signals are similar to signals from many other cells and thus their functional connectivity is high (55, 58, 60, 68, 170). and modelling ﬁndings, for reasons that arguably originate in different methodological approaches [for a recent review see (60)]. Our current understanding suggests that in a system of coupled cells of which all are intrinsically able to oscillate, silencing a small proportion of cells with the highest number of functional connections cannot prevent or importantly change the activity of the remaining cells, except close to the threshold for stimulation where the majority of cells would otherwise not be active by themselves (60, 143, 148, 149). Further experiments in a near- threshold glucose milieu with ablation of hub cells rather than their silencing may further aid to our understanding of the role of hub cells in the islet syncytium. Importantly, similar strategies could be used in future studies to assess the importance of other subpopulations of cells discussed below. Second, a large degree of beta cell heterogeneity is also evident from coordinated [Ca2+]IC waves that propagate across islets and seem to consistently emerge from speciﬁc subpopulations of beta cells named wave initiators or pacemakers. Beta Cell Subpopulations and Functional Heterogeneity Beta cells with a high number of links in functional networks have been termed hub cells. They were suggested to govern the functioning of islet networks by initiating [Ca2+]IC waves (64, 67). According to this view, non-hub cells are believed to only follow metabolic, electrical, and other signaling cues from the hub cells (166). Advanced optogenetic and photopharmacological investigations indicated that hub cells are metabolically highly active, exhibit hyperpolarized mitochondria and have a lower June 2022 \| Volume 13 \| Article 922640 Frontiers in Endocrinology \| www.frontiersin.org 10 Multicellular Networks Within Pancreatic Islets Stozˇer et al. A B FIGURE 7 \| Functional network architecture of beta cells and the corresponding energy dissipation rates. (A) Each circle represents the physical position of a cell inside the islet and the connections signify functional connections. Colors of circles denote the average dissipation rates calculated as the sum of Lyapunov exponents (see Ref (167). for further details). (B) The average dissipation rate reﬂecting the rate of energy consumption of individual cells as a function of the node degree, i.e., number of functional connections. The grey line denotes the linearly decreasing trend showing that highly connected nodes exhibit higher dissipation and energy consumption rates. B A FIGURE 7 \| Functional network architecture of beta cells and the corresponding energy dissipation rates. (A) Each circle represents the physical position of a cell inside the islet and the connections signify functional connections. Colors of circles denote the average dissipation rates calculated as the sum of Lyapunov exponents (see Ref (167). for further details). (B) The average dissipation rate reﬂecting the rate of energy consumption of individual cells as a function of the node degree, i.e., number of functional connections. The grey line denotes the linearly decreasing trend showing that highly connected nodes exhibit higher dissipation and energy consumption rates. the one on the plateau. Recent data suggest that the so-called leaders or ﬁrst responders tend to cluster in groups that become larger in higher glucose and that tend to be more active, i.e., have longer active times, also during the plateau phase (55). Their removal seems to be able to delay the onset and diminish the amplitude of subsequent oscillations (67), but they are dispensable in the sense that if they are ablated another cell will become a ﬁrst responder (68). Frontiers in Endocrinology \| www.frontiersin.org Beta Cell Subpopulations and Functional Heterogeneity In the case of different or the same cells being ﬁrst responders for different secretagogues, a cell´s role as a ﬁrst responder may be determined by the relative importance of a given component in the stimulus-secretion coupling cascade in this cell. In addition to resolving the controversies regarding the importance of speciﬁc subpopulations for normal and pathological islet functioning, future studies shall therefore also shed light on the temporal persistence of these roles and their sensitivity to experimental conditions, such as different secretagogues. Moreover, they shall pinpoint additional subpopulations of cells and dissect into more detail their molecular, structural, and additional functional signatures. Additionally, a consensus among different research groups would be welcome on the nomenclature and cut-off values for determining subpopulation sizes. For additional details on the role of subpopulations in human islets, please see Human Islets and Coordinated Beta Cell Activity in Health and Disease, and for some suggestions on how mathematical modelling and multilayer networks may help address the issue of different subpopulations, please see Computational Models of Beta Cell Networks and Islets as Multilayer Networks below. Finally, for more details on the role of heterogeneity and different beta cell subpopulations in islets, as well as some suggestions for future studies, we wish to refer the reader to some other recent articles addressing this topic in detail (55, 58–60). Altered Connecti it in Beta Cell Net orks gap-junctional coupling for the coordinated collective response (60, 77, 78). Importantly, prolonged exposure to high concentrations of glucose and fatty acids, as expected in diabetes, was found to downregulate Cx36 in mice, rats and humans, and may disrupt the pattern of intercellular synchronization (172–174). Similarly, studies on Cx36 KO mouse models have also shown an impairment of normal oscillatory patterns of insulin secretion elicited by glucose and a diabetic phenotype (77, 78, 152, 175). Moreover, a decrease in Cx36 protein and a smaller size of gap junction plaques were also observed in prediabetic C57BL/6 mice fed a high-fat-diet for 60 days (176). Similarly, in diabetic ob/ob mice the Cx36 protein level is signiﬁcantly reduced despite a preserved Cx36 mRNA level, pointing to a decrease in protein synthesis and/or inappropriate gap junction organization, leading to deﬁcient electrical coupling between cells (172). Also the synchronicity of Ca2+ oscillations in ob/ob mice is disturbed, disrupting the normal insulin secretion pattern (172, 177). Beta Cell Subpopulations and Functional Heterogeneity These cells could correspond to highly glucose responsive beta cells that have increased glucokinase activity (165, 168, 169). Interestingly, depending on the model employed, they can also exhibit a lower NAD(P)H response and a faster natural oscillation frequency than other cells in the islet, including hub cells. Therefore, they are more likely to depolarize ﬁrst in response to stimulating glucose concentrations and send depolarizing currents to neighboring cells (165, 170). At present, it is undisputable that the great majority of [Ca2+]IC waves are initiated in a limited number of cells or regions, which do not necessarily overlap with the regions with most functional connections (58, 170). Also, it remains to be shown, whether their removal critically impacts the islet as a whole and which mechanistic substrates make them initiate the waves. Finally, functional differences between beta cell subpopulations may change with time and experimental conditions. For instance, heretofore, the described functional roles have not been tracked over long periods of time and are Third, analyzing with single cell resolution also the activation phase enables a comparison of behavior during this phase with June 2022 \| Volume 13 \| Article 922640 Frontiers in Endocrinology \| www.frontiersin.org 11 Multicellular Networks Within Pancreatic Islets Stozˇer et al. thus not necessarily stable properties. Additionally, the cells that are ﬁrst responders to glucose may not necessarily be ﬁrst responders to some other secretagogue, and the same holds true for the role of pacemakers and hubs. Moreover, it is reasonable to assume that to some extent some traits may be shared between different subpopulations, e.g., some of the ﬁrst responders to glucose may be among the ﬁrst responders to GLP- 1 or among the hub cells. This may again depend on the experimenter’s deﬁnition of the size of subpopulation, with larger subpopulations possibly generating more overlap than more narrowly deﬁned subpopulations. In other words, some of the ﬁrst responders will with large probability be among the hubs if one sixth or even one third of the fastest responding cells and cells with the highest degree are deﬁned as ﬁrst responders and hubs, respectively, compared with a lower probability for overlap if the cut-off is set at one tenth. On the other hand, the overlap may have to do with genuine biological properties of beta cells. Beta Cell Subpopulations and Functional Heterogeneity From a mechanistic point of view, in (pre-)diabetes, a decrease in Cx36 coupling could be a consequence of increased concentrations of pro-inﬂammatory cytokines. Interestingly, in both mouse and human islets a cytokine-mediated decrease in coupling can be prevented with pharmacological interventions which increase intracellular cAMP. In this case, in mouse islets the glucose- stimulated calcium signaling was preserved by increasing the levels of gap junction protein Cx36 on the plasma membrane using exendin-4, a glucagon-like peptide (GLP-1) receptor agonist (178). To protect islets from high-fat-diet induced impairment of beta cell gap junction coupling and to preserve proper Ca2+ signaling, including Ca2+ oscillation coordination and amplitude, a relatively simple intervention, namely a 40% caloric restriction, seems to be very efﬁcient, mirroring similar beneﬁcial effects of caloric restriction in human patients (179). Additionally, trying to decipher the effects of vertical sleeve gastrectomy (VSG) on beta cell function in obese mice, Akalestou et al. have shown that Ca2+ dynamics as well as the number and strength of connections between beta cells increase within 8-10 weeks post-surgery, which could be attributed to the strong inﬂuence of GLP-1 on islet functioning (57). GLP-1 in physiological picomolar concentrations augments postprandial insulin secretion. However, it signiﬁcantly enhances beta cell cluster activity, coupling, and coordination only in the second phase of insulin secretion as shown using a microﬂuidic system with multielectrode arrays (146). As evident from the above ﬁndings, an important impact on Ca2+ oscillations is mediated by cAMP acting downstream through at least two different pathways, namely the guanine nucleotide exchange factor Epac2A and the protein kinase A (PKA) pathway (178). We wish to point out that in addition to physiological or pharmacological stimulation of cAMP production by neurohormonal secretagogues, glucose itself induces cAMP signaling in beta cells. This effect is most probably mediated by a Ca2+- dependent arm through Ca2+-dependent adenylate cyclase isoforms and by a Ca2+-independent arm through direct ATP availability or since the concentration of ATP is possibly Frontiers in Endocrinology \| www.frontiersin.org Human Islets and Coordinated Beta Cell Activity in Health and Disease y During the response to glucose, human beta cells have been described to display a more regional coordination between cell clusters compared with responses in mice (189, 190), which could reﬂect differences in islet architecture and gap junction coupling among beta cells in different species (191–193), but at least partly also due to differences in donor age and health, mode of preparation, as well culture duration and conditions (191, 192, 194–197). In human islets, diabetes results in disrupted cytoarchitecture with altered homotypic and heterotypic communication between different cell types within an islet (105, 198). The coordinated responses of islets to stimulation with glucose, glucagon-like peptide 1 (GLP1), and glucose- dependent insulinotropic polypeptide are also altered possibly due to a reduction in Cx36 expression. Moreover, perturbed beta-cell coupling and dysregulation of Cx36-dependent [Ca2+]IC signaling were observed in islets from donors with a high BMI, suggesting that lipotoxicity may result in lowered insulin secretion and progression to diabetes (54). Furthermore, during ageing, which is associated with increased risk of diabetes, a signiﬁcant decline was found in gap junctional coupling. This was accompanied by reduced overall coordination of [Ca2+]IC activity, which became restricted to islet subregions, and diminished insulin secretion. However, activating gap junctional communication using modaﬁnil, a pharmacologic activator of Cx36 electrical coupling, successfully reversed the age-related decline in synchronization of [Ca2+]IC signals (190). Supporting these observations, advanced optogenetic methods in combination with complex computational tools, conﬁrmed differences in the coordinated responses to glucose between mouse and human beta cells. In human islets, [Ca2+]IC waves seem to be initiated from speciﬁc subregions called pacemaker or leader regions with speciﬁc metabolic proﬁle and local excitability (54, 64, 71, 165). The synchronization patterns are more clustered compared to mice and the collective behavior of beta cells becomes altered in diabetes (67, 145). Our own research indicates that in type 2 diabetic islets glucose-dependence is still present, but beta cell activity seems to be reduced, mostly due to a reduced oscillation frequency. Furthermore, human beta cell networks are more segregated than mouse networks and in diabetic islets more than in islets from control donors. This is accompanied by smaller and more locally restricted [Ca2+]IC waves. Importantly, the hub regions seem to lose a disproportionately large fraction of connections, especially the long-range ones (58). Altered Connectivity in Beta Cell Networks and Its Relevance in Diabetes Adequate intercellular electrical coupling through gap junctions is essential for synchronized beta cell activity. In basal glucose, insulin release is low since the less active beta cells keep the intrinsically more active beta cells quiescent through junctional hyperpolarization or clamping (152, 171). In contrast, during increasing stimulation, more and more cells become active and intercellular coupling may facilitate recruitment of the least active cells. In Cx36 knock-out (Cx36 KO) mice, the response of beta cells to glucose resembles that of dispersed cells in culture including increased basal and lower stimulated insulin secretion, thereby conﬁrming the theoretically predicted role of June 2022 \| Volume 13 \| Article 922640 Frontiers in Endocrinology \| www.frontiersin.org 12 Multicellular Networks Within Pancreatic Islets Stozˇer et al. saturating for adenylate cyclases even in basal glucose, through a decrease in inhibitory AMP. Moreover, cAMP oscillates in beta cells in response to stimulation by both glucose and extracellular primary messengers with the same period as electrical activity and [Ca2+]IC. The mechanism underlying these oscillations may differ depending on the experimental protocol. As [Ca2+]IC can inﬂuence cAMP production and degradation, oscillations in cAMP may at least partly be due to oscillations in [Ca2+]IC. This seems to be supported by the observation that oscillations in [Ca2+]IC and cAMP are typically in phase or occur with a slight shift. Pharmacological activation of GLP-1 receptors and/or direct activation of adenylate cyclase by forskolin are unable to stimulate beta cells in low glucose but can signiﬁcantly enhance glucose-stimulated Ca2+ oscillations (180–182). Indeed, by constructing functional connectivity networks we have recently shown that increasing cAMP levels using forskolin increases beta cell activity as well as enhances synchronicity and coordination of intercellular signaling, evident as denser and more integral networks (180, 183). While both slow and fast Ca2+ oscillations rely on periodic entry of Ca2+ from extracellular space into the beta cells through voltage-activated Ca2+ channels, the fast Ca2+ oscillations may also depend on mobilization of intracellular Ca2+ stores from the endoplasmic reticulum. Therefore, pharmacological agents that increase cAMP may promote the appearance of fast Ca2+ waves (184, 185). inhibition also stabilizes the course of [Ca2+]IC waves and the role of wave initiators (69). Altered Connectivity in Beta Cell Networks and Its Relevance in Diabetes Given the importance of connectivity for normal and pathological functioning of islets and the fact that the network analyses are able to detect effects of non- pharmacological and pharmacological interventions beyond the classical physiological measures of beta cell activity, they are a promising tool to detect early changes during progression to diabetes and non-classical effects of new therapeutic approaches (188). Frontiers in Endocrinology \| www.frontiersin.org Human Islets and Coordinated Beta Cell Activity in Health and Disease Loss of gap junction connectivity in Cx36 KO mouse islets desynchronizes beta cell nectivity in Cx36 KO mouse islets desynchronizes beta cell cells, and an overall decrease in local connectedness, as well as i A B C D URE 8 \| Multicellular activity and functional connectivity maps in a control (wild type) mouse islet (A), an uncoupled (Cx36 KO) mouse islet (B), human islet from ormal donor (C), and human islet from a donor with T2D (D). Left column visualizes Ca2+ activity with indicated stimulation intervals. Bars indicate 3- (light gray), gray) and 12- (dim gray) mM glucose stimulation. Note that 3 mM glucose was used as the substimulatory concentration in (B-D), as 6 mM can already evoke vity in these islets (unlike in control mouse islets). Inserts show short intervals of selected cellular signals. Middle column features the correlation matrices acted from Ca2+ signals. Correlation levels between cell pairs are color-coded as indicated with the color bar (the same scale refers to all four panels). In the right umn the corresponding functional networks are presented, which were obtained by thresholding the correlation matrices (Rth = 0.7). Along with networks, the key ameters are provided: average correlation (Ravg), average clustering coefﬁcient (Cavg), average network node degree (kavg), global efﬁciency (Eglob), largest nected component (Smax), and the number of communities (Ncom). A B B C C D D D FIGURE 8 \| Multicellular activity and functional connectivity maps in a control (wild type) mouse islet (A), an uncoupled (Cx36 KO) mouse islet (B), human islet from a normal donor (C), and human islet from a donor with T2D (D). Left column visualizes Ca2+ activity with indicated stimulation intervals. Bars indicate 3- (light gray), 6- (gray) and 12- (dim gray) mM glucose stimulation. Note that 3 mM glucose was used as the substimulatory concentration in (B-D), as 6 mM can already evoke activity in these islets (unlike in control mouse islets). Inserts show short intervals of selected cellular signals. Middle column features the correlation matrices extracted from Ca2+ signals. Correlation levels between cell pairs are color-coded as indicated with the color bar (the same scale refers to all four panels). In the right column the corresponding functional networks are presented, which were obtained by thresholding the correlation matrices (Rth = 0.7). Human Islets and Coordinated Beta Cell Activity in Health and Disease Figure 8 Many other pharmacological substances and drug candidates for diabetes treatment can affect beta cell connectivity patterns by targeting different signaling pathways, but to the best of our knowledge, apart from the cAMP-elevating agents only a few have been investigated using network measures. In this regard, glutamate signaling via N-Methyl-D-Aspartate receptors (NMDARs) has been demonstrated to play an important role in beta cell function by shortening the duration of bursts of electrical activity and therefore fast [Ca2+]IC oscillations, with NMDAR antagonists being able to prolong the duty cycle by prolonging these bursts (186, 187). More speciﬁcally, following glucose metabolism, increased ATP inhibits KATP channels and causes plasma membrane depolarization, resulting in activation of voltage-dependent Ca2+ channels, increase in [Ca2+]IC, and insulin secretion. A negative regulation of this pathway is mediated by NMDARs. Under physiological conditions, these receptors are probably fully saturated with glutamate originating from glutamate in blood, from glutamate secreted by alpha cells, or from glutamate exported by beta cells through excitatory amino acid transporters. Membrane depolarization is therefore able to activate NMDARs, which in turn activate KATP channels and Ca2+- dependent K+ channels (SK4 channels) through a not fully understood functional interaction, thus hyperpolarizing the membrane, terminating the bursts of membrane depolarization and shortening Ca2+ oscillations. NMDAR inhibition interferes with this negative feedback loop and prolongs bursts and Ca2+ oscillations (69, 186–188). Representing the spreading [Ca2+]IC waves as network layers, we have recently shown that the inhibition of NMDARs not only increases beta cell activity but also stabilizes and synchronizes intercellular connectivity patterns. Within consecutive [Ca2+]IC waves, NMDAR June 2022 \| Volume 13 \| Article 922640 Frontiers in Endocrinology \| www.frontiersin.org 13 Multicellular Networks Within Pancreatic Islets Stozˇer et al. [Ca2+]IC responses, resulting in poor cell-cell correlation and sparse functional networks (Figure 8B). The changes in intercellular coupling are reﬂected in key network parameters, e.g., a lower average correlation (Ravg), scarcely connected beta cells, and an overall decrease in local connectedness, as well as in summarizes how differences in structural connectivity between control and Cx36 KO islets, as well as between mouse and human islets from normal and diabetic donors, translate to differences in functional connectivity. Human Islets and Coordinated Beta Cell Activity in Health and Disease In human islets, [Ca2+]IC waves are heterogeneous in size and mostly encompass only smaller regions of the islet, in contrast to global, islet-wide [Ca2+]IC waves in mice (58, 147). Both can be at least partly contributed to structural differences, particularly a higher proportion of alpha cells and a more lobular structure in human islets, promoting a higher number of heterologous contacts between alpha and beta cells and possibly introducing bottlenecks for spreading of global [Ca2+]IC waves between the more segregated subregions (189, 192, 193). We wish to point out that despite this larger degree of segregation, human islets still seem to be able to produce [Ca2+] IC waves that involve the great majority of islet beta cells but do so much less often compared with mouse islets where globally synchronized oscillations are the rule rather than an exception (58, 197). Due to the abovementioned structural and functional differences, human islets may even be more prone to losing the ability to produce globally synchronized [Ca2+]IC with decreases in intercellular coupling under pathological conditions. Along this line, the functional connectivity patterns in islets from diabetic human donors (Figure 8D) are indeed much sparser and more segregated compared with islets from healthy controls, indicating thereby a higher fraction of inactive regions and a lack of larger and coherent [Ca2+]IC waves (58). Finally, while much of the evidence on the role played by decreased intercellular coupling in development of diabetes presented in the last two chapters may be circumstantial, we wish to point out that when taken together, some excellent recent studies nevertheless suggest that decreased Cx36 coupling is at least partly causative. More speciﬁcally, knockout of Cx36 leads to a diabetic phenotype (78), some degree of decrease in Cx36 is present in diabetic islets and improving this by pharmacological (178, 190) or dietary interventions (178) is sufﬁcient to improve islet function. However, more studies in this direction are needed to quantify the contribution of decreased coupling and determine whether improving coupling is a viable treatment option for diabetes. Human Islets and Coordinated Beta Cell Activity in Health and Disease Along with networks, the key parameters are provided: average correlation (Ravg), average clustering coefﬁcient (Cavg), average network node degree (kavg), global efﬁciency (Eglob), largest connected component (Smax), and the number of communities (Ncom). June 2022 \| Volume 13 \| Article 922640 14 Frontiers in Endocrinology \| www.frontiersin.org Multicellular Networks Within Pancreatic Islets Stozˇer et al. research and have also served as a paradigmatic case study for emergent networks [for excellent reviews see (60, 125, 199–201)], but the interest for the design of multicellular models has increased over the last few years. This is in part due to growing computational resources, which facilitate multiscale simulations of beta cell populations, but also due to advances in experimental techniques, which have provided valuable new data on microarchitecture and intercellular dynamics. Several studies have utilized computational models to investigate how intercellular coupling facilitates beta cell synchronization (138, 202–206) and the propagation of [Ca2+]IC waves (74, 207, 208). In recent years, speciﬁc attention has been given to cellular heterogeneity and to how the collective activity emerges from functionally heterogeneous beta cell subpopulations (71, 143, 145, 147, 148, 165, 169, 209). In the context of complex beta cell networks, it has been demonstrated that the coupling scheme between the beta cells represents the basis for the functional connectivity patterns, but the relation relies heavily on various physiological determinants, such as the level of stimulation, cellular noise, and the coupling parameters (210). Multicellular models have also been employed to study the occurrence of long- range connections in functional beta cell networks. Importantly, it has been demonstrated that long-range synchronicity can in principle be established solely by the propagation of excitation waves through nearest-neighbor-coupled networks, if the cells and the coupling strengths are heterogeneous (142, 143, 211). In recent years, particular emphasis has also been given to the role of functional subpopulations, such as hubs and pacemaker cells, although their exact roles remain somewhat debatable. This is in part also due to the inconsistent use of terminology in the literature, but with some more recent papers this aspect is improving (55, 59, 60). It has been shown by numerical simulations that the inclusion of specialized hub cells importantly affects the collective activity on different temporal scales (212). Human Islets and Coordinated Beta Cell Activity in Health and Disease Furthermore, by silencing a few hub cells within the islet it was possible in principle to abolish whole-islet [Ca2+]IC activity in simulations, similarly to what was noted experimentally, but as mentioned before, only close to the threshold glucose stimulation (148). Later studies incorporating in-depth numerical analyses alleviated these ﬁndings a bit by showing that removing the metabolically most active and heavily connected cells or cells with the highest intrinsic frequencies does not diminish whole-islet activity (60, 143, 170). These results suggest that the whole-islet [Ca2+]IC activity is probably not driven by a very small (<10%) subpopulation of extraordinary cells and that the islets are robust to loss of small groups of cells, even if they have special attributes, as would be expected for evolutionary robust assemblies of intrinsic oscillators (60, 213). To sum up, the recent theoretical works have partly conﬁrmed some of the recent experimental ﬁndings and thereby contributed to our understanding of the complex signaling mechanisms within the global efﬁciency compared to the control islet (Figures 8A, B). Conversely, despite having a comparable average correlation as Cx36 KO islets, human islets have better connected beta cells, are more locally integrated, and have a signiﬁcantly higher number of communities. Their global integration, reﬂected in Eglob, however, is comparable to Cx36 KO mice islets, indicating a lack of long-range functional connections between subregions of the islet due to the relative lack of global [Ca2+]IC waves compared with mice (Figure 8C). Clusters of beta cells in human islets form communities (while Cx36 KO islets do not), these different communities are also connected, but much less than in islets in control mice, where global [Ca2+]IC waves and thus globally synchronized [Ca2+]IC oscillations are the rule rather than the exception. In human islets, [Ca2+]IC waves are heterogeneous in size and mostly encompass only smaller regions of the islet, in contrast to global, islet-wide [Ca2+]IC waves in mice (58, 147). Both can be at least partly contributed to structural differences, particularly a higher proportion of alpha cells and a more lobular structure in human islets, promoting a higher number of heterologous contacts between alpha and beta cells and possibly introducing bottlenecks for spreading of global [Ca2+]IC waves between the more segregated subregions (189, 192, 193). Human Islets and Coordinated Beta Cell Activity in Health and Disease We wish to point out that despite this larger degree of segregation, human islets still seem to be able to produce [Ca2+] IC waves that involve the great majority of islet beta cells but do so much less often compared with mouse islets where globally synchronized oscillations are the rule rather than an exception (58, 197). Due to the abovementioned structural and functional differences, human islets may even be more prone to losing the ability to produce globally synchronized [Ca2+]IC with decreases in intercellular coupling under pathological conditions. Along this line, the functional connectivity patterns in islets from diabetic human donors (Figure 8D) are indeed much sparser and more segregated compared with islets from healthy controls, indicating thereby a higher fraction of inactive regions and a lack of larger and coherent [Ca2+]IC waves (58). Finally, while much of the evidence on the role played by decreased intercellular coupling in development of diabetes presented in the last two chapters may be circumstantial, we wish to point out that when taken together, some excellent recent studies nevertheless suggest that decreased Cx36 coupling is at least partly causative. More speciﬁcally, knockout of Cx36 leads to a diabetic phenotype (78), some degree of decrease in Cx36 is present in diabetic islets and improving this by pharmacological (178, 190) or dietary interventions (178) is sufﬁcient to improve islet function. However, more studies in this direction are needed to quantify the contribution of decreased coupling and determine whether improving coupling is a viable treatment option for diabetes. Computational Models of Beta global efﬁciency compared to the control islet (Figures 8A, B). Conversely, despite having a comparable average correlation as Cx36 KO islets, human islets have better connected beta cells, are more locally integrated, and have a signiﬁcantly higher number of communities. Their global integration, reﬂected in Eglob, however, is comparable to Cx36 KO mice islets, indicating a lack of long-range functional connections between subregions of the islet due to the relative lack of global [Ca2+]IC waves compared with mice (Figure 8C). Clusters of beta cells in human islets form communities (while Cx36 KO islets do not), these different communities are also connected, but much less than in islets in control mice, where global [Ca2+]IC waves and thus globally synchronized [Ca2+]IC oscillations are the rule rather than the exception. June 2022 \| Volume 13 \| Article 922640 FRONTIERS OF ISLET NETWORK SCIENCE: ASSESSING MULTICELLULAR ACTIVITY BY MULTILAYER NETWORKS AND GOING BEYOND CALCIUM To date, biomedical endeavors employing the MLN concepts have been mostly limited to molecular and brain networks. In the former, the MLN formalism is predominantly used to assess the interdependent biochemical networks extracted from linked genomic, proteomic, and metabolomic data (227–230), whereas in the latter, these methods are used to address the temporal evolution of brain networks and rewiring dynamics (231–234), associations among different frequency bands (235–238), and to explore the longstanding issue about the interplay between brain structure and dynamics (36, 239, 240). On the level of tissues and intercellular interactions, the MLN methodological directions are still rather unexplored, even though the number of potential applications is large, particularly in pancreatic islets. We therefore present here some speciﬁc examples on how the MLN theory has been and could be further integrated into islet research (Figure 10). They are explained in continuation and encompass the assessment of different temporal scales of oscillatory activity, tracking the network evolution during prolonged stimulations or pharmacological interventions, following the course of the intercellular signals, characterization of heterologous interactions between different cell types, and the evaluation of the multicellular beta cell function by simultaneously acquiring multiple measured variables. The Many Layers of Multicellular Networks Pancreatic islets are non-stationary complex systems governed by different oscillatory subsystems, they are characterized by different types of interactions, and their function can be captured with different measures and parameters. Therefore, the standard network approach focusing on single networks in isolation might be insufﬁcient to unveil the functional regulatory patterns originating from complex interactions across multiple layers of physiological relationships and processes. In the last few years, the multilayer network (MLN) formalism has emerged as a new research direction to engage with such multi-dimensional systems (19, 218–221), including in the area of biomedical research (18, 222–224). By means of the MLN formalism, it is possible to track the evolution of interactions among entities over prolonged periods of time, evaluate precisely the changes caused by altered experimental conditions, such as addition of pharmacological substances or the development of a disease, explore the associations between different temporal and spatial scales, and characterize different types of interactions. In this way, a much more precise insight into the architecture and dynamics of biological systems can be acquired, compared to single-layer analyses only. While standard networks can be represented by adjacency matrices (see Figures 4, 8), for MLNs higher-order matrices, i.e., tensors, are required. Computational Models of Beta Cell Networks The new ﬁndings by network science about the complex nature of intercellular activity patterns and the speciﬁc roles played by certain cells were recognized as theoretically very appealing and have inspired the development of multicellular beta cell models. Mathematical modelling approaches have a long tradition in islet June 2022 \| Volume 13 \| Article 922640 Frontiers in Endocrinology \| www.frontiersin.org Multicellular Networks Within Pancreatic Islets Stozˇer et al. supra-adjacency with a block structure matrix is used, in which diagonal blocks encode intra-layer connectivity and off-diagonal blocks encode inter-layer connectivity (Figures 9A, B). This framework allows for expansion of the traditional network analysis by examining interlayer similarity, overlapping (weighted) degrees and other measures, detection of modular super-units, identiﬁcation of most central units, etc. (225, 226). In the context of beta cell networks, these MLN metrics can be used for example to assess the spatio-temporal beta cell network persistency, to quantify the effects of pharmacological interventions or to identify signal-speciﬁc functional subpopulations, as well as to determine how they change with time, as speciﬁed in more detail below. effect of incretins (217), as well as long feedback loops, for instance between the liver and the pancreas (137). We ﬁrmly believe that the tools developed in the ﬁeld of computational physiology and network science will help us to address these issues and will provide us further insights into functional properties, as well as the underlying mechanisms that guide the multicellular dynamics of endocrine cells. FRONTIERS OF ISLET NETWORK SCIENCE: ASSESSING MULTICELLULAR ACTIVITY BY MULTILAYER NETWORKS AND GOING BEYOND CALCIUM Formally, to represent connectivity within and between network layers, a Islets as Multilayer Networks Within this new framework, each region of the brain is mapped into a network node and replicated across all layers encoding frequency bands. Such representation of functional connectivity is better able to distinguish between brain functional connectivity patterns in health and disease (246). induced pathogenesis by monitoring the activity in islets transplanted into the anterior chamber of the eye (67, 249). In Figure 10B, a hypothetical scenario is presented, illustrating how the average temporal activity and the corresponding functional connectivity maps evolve when islets are subjected to stimulation by candidate drugs. Third, [Ca2+]IC waves are the main synchronizing mechanism between beta cells and their course depends heavily on cellular heterogeneity and intercellular interaction patterns. Because of the rich information encoded in individual waves, they represent the perfect candidates for being studied via MLN analysis. By this means, each individual wave can be regarded as an individual layer in which the connections are weighted and directed. We wish to point out that this is in stark contrast with the traditional way of constructing networks based on longer time series that include many waves and are based on similarity measures. The directionality of connections indicates the path of the intercellular signal, and the weights reﬂect the temporal delay between cell pairs that are subsequently activated along the course (69). We schematically present this approach in Figure 10C. On the left panel the activation sequence of 9 cells in 5 waves is shown in the form of a raster plot, and on the right panel the corresponding directed and weighted networks of waves are shown in their temporal order. Notably, we leveraged this approach in a recent study where we quantiﬁed the differences in collective beta cell activity between prolonged glucose stimulation only and stimulation by glucose and the NMDA receptor inhibitor MK-801 (69), which was previously shown to increase beta cell activity and synchronicity (186, 187). In our study, we focused on the velocity of intercellular [Ca2+]IC waves and their temporal stability. Since each layer encodes the exact path of the wave (direction of connections) and the time delays between cells (weights of connections), one can compute the exact wave propagation velocity based on the geodesic path length between wave initiator cells and the last cells that activated as the sum of the weights of this path. Islets as Multilayer Networks As explained above, mouse beta cells in isolated islets and in pancreatic tissue slices oscillate at different temporal scales when A B FIGURE 9 \| Multilayer representation of a multicellular network. (A) A multilayer network consists of different network layers, each one represented by an adjacency matrix. (B) A rank-2 tensor, generally known as the supra-adjacency matrix, can be used to describe and analyze both intra- and inter-layer connectivity. B A B FIGURE 9 \| Multilayer representation of a multicellular network. (A) A multilayer network consists of different network layers, each one represented by an adjacency matrix. (B) A rank-2 tensor, generally known as the supra-adjacency matrix, can be used to describe and analyze both intra- and inter-layer connectivity. June 2022 \| Volume 13 \| Article 922640 Frontiers in Endocrinology \| www.frontiersin.org 16 Multicellular Networks Within Pancreatic Islets Stozˇer et al. exposed to stimulatory glucose concentrations (56, 130, 241, 242). The slow oscillatory component with a period of several minutes is believed to reﬂect metabolic activity and drives the oscillatory ATP production (243, 244). Superimposed on the slow and sometimes occurring without them are so-called fast [Ca2+]IC oscillations with a glucose-dependent frequency of around 5 min-1 and duration of around 2-15 s (55, 131, 159, 160), which reﬂect the bursting pattern of electrical activity. Both slow and fast oscillations are synchronized between different beta cells of the same islet and contribute to proper secretion patterns (133, 137, 140, 141, 245). To quantify how the multimodal oscillatory pattern manifests itself on the multicellular level, a multiplex network representation can be used (56). More speciﬁcally, using a band-pass ﬁlter, the slow and fast oscillatory component can be extracted separately from the [Ca2+]IC recordings, and these signals can be used to construct individual network layers, as demonstrated in Figure 10A. Previous analyses have shown that the slow oscillations are more global, resulting in several long-range connections and networks extracted from them have a more cohesive structure compared to the networks based on fast oscillations. Moreover, there was only a weak relation between the fast and slow network layer characteristics, which suggests that different synchronization mechanisms shape the collective cellular activity in islets (56). Of note, a conceptually similar approach is commonly applied in neuroscience to construct frequency band-speciﬁc multi-layered functional brain networks (236, 237). Frontiers in Endocrinology \| www.frontiersin.org Islets as Multilayer Networks Our analysis revealed that the propagation velocity did not change either under prolonged stimulation with glucose or under the action of NMDA receptor inhibitor. However, when quantifying the inter-layer similarity, we found that the wave initiator regions and the stability of wave paths increased dramatically when the NMDA receptors were inhibited. In this manner, we were able to identify the key factor underlying the more synchronous behavior. As such, the proposed methodology can help quantitatively evaluate the impact of pharmacological interventions on multicellular dynamics beyond classical physiological or network parameters and is applicable to other secretagogues and multicellular systems as well. The second conceivable frontier of network analyses is the investigation of network dynamics. The so-called temporal networks offer mathematically principled models of evolving networks. as well as a battery of statistical variables to characterize their evolution (19, 218). Layers are to that purpose typically generated on the basis of a series of (possibly overlapping) time windows and the nodes are linked only across sequential replicas to indicate identity. Mapping the temporal changes in connectivity patterns is meaningful in biomedical sciences as well (231, 247, 248) and could be very beneﬁcial for the description of information ﬂow and dynamic interaction patterns within the islets. More speciﬁcally, the activity patterns in these mini-organs are remarkably complex already under constant stimulation (147), and even more in a dynamic in vivo environment (67, 249, 250). In this vein, the multilayer model for time-varying networks could be used to explore ﬂuctuations in functional connectivity during prolonged or variable stimulation of cells, as well as to assess the functional adaptation and plasticity after repeated stimulations (48, 63). Moreover, from the viewpoint of the recent developments in the ﬁeld, MLN could represent a viable approach to track the network evolution after targeting speciﬁc cells via optogenetic and photopharmacological strategies (59, 64). In addition, with this approach one could track the changes in islets during diet- Fourth, the pancreatic islets are multicellular micro-organs that comprise predominantly alpha, beta, delta cells and communication among them is essential for proper function (73, 214, 251, 252). June 2022 \| Volume 13 \| Article 922640 Islets as Multilayer Networks While direct electrical coupling through gap- junctions is the key determinant ensuring synchronous beta cell activity (76, 77) and was also found to be present between beta and delta cells (253, 254), islet cells also communicate by paracrine, autocrine, and other juxtacrine signaling pathways June 2022 \| Volume 13 \| Article 922640 Frontiers in Endocrinology \| www.frontiersin.org 17 Multicellular Networks Within Pancreatic Islets Stozˇer et al. (73, 215, 252). It is well known that this variety of intercellular signal mechanisms is essential for a synergistic cooperation between islet cells and this represents one of the most vibrant issues in islet cell biology. With the recent advances in multicellular imaging of islet networks, our ability to dissect individual components of cell-cell communication has much improved (73). In this regard, the MLN formalism could be advantageous by providing the means to decipher the complexity of islet communication. In Figure 10D we illustrate a MLN representation of the endocrine cell crosstalk within the islets with some of their paracrine interactions. Within the framework of the MLN paradigm, networks of different types of cells could be constructed based on their [Ca2+]IC signals and compared with networks based on the physical positions of cells and their paracrine or other interactions. Additionally, the relative contributions of different local signals could be dissected using the MLN approach through application of different agonists and antagonists as described in the second paragraph above. Interestingly, in a similar context, the MLN approaches have already been used to study the neural network of C. elegans by using multiple layers to represent different types of connections between neurons, which has led to novel ﬁndings about the functional organization of this famous neuronal network (255, 256). Very recently, muscle cells and further modes of interactions between cells have been incorporated as additional layers to this scheme, providing thereby an even deeper knowledge of the C. elegans connectome (257). Along similar lines, Virkar et al. proposed a multilayer network to study diffusive transport of metabolic resources among the glial network and to the synapses in the neuronal network layer (258). example, glucose stimulation can stimulate insulin secretion even when [Ca2+]IC is clamped (270). Moreover, in (pre) diabetes increased efﬁcacy of Ca2+ on the secretory apparatus can directly enhance insulin secretion (174, 271). Thus, to what extent multi-modal proﬁling of islet network activity should be undertaken to better understand islet behavior and dysfunction in disease? Islets as Multilayer Networks The most obvious methodological approaches to provide additional layers of data would involve simultaneous imaging of physiological processes either up- or down-stream of [Ca2+]IC dynamics. Although the candidates described below are certainly not an exhaustive list, several measures may be considered when adding additional layers of information to these networks. More speciﬁcally, individual steps of the stimulus-secretion coupling cascade could form individual layers of MLNs and the interactions between the steps would constitute inter-layer connectivity. To date, several candidates for individual layers meet the criterion of being experimentally measurable and having an adequate spatial and temporal resolution, as well as ranging from the most proximal to the most distal steps. We wish to point out that genetically encoded sensors (including for Ca2+) may allow greater speciﬁcity in measurements when targeted to the cell cytosol, mitochondria, or other relevant intracellular organelles or sub-cellular locations, and greater cell speciﬁcity when expressed under the control of relevant promotors (for example the insulin promoter) in engineered model animals, such as mice or zebraﬁsh, or when delivered to human islet cells, typically by adenoviral vectors (181). If we follow the stimulus-secretion coupling cascade, although cellular glucose uptake can be measured using a ﬂuorescent probe (272), and this can be combined with [Ca2+] IC responses in islet cells (273), additional information on the metabolic activity of islet cells can also be gained using approaches that more directly assess relevant metabolic signals. In this regard, sensors reporting glycolysis (the most proximal signaling step) have provided insight into the regulation of oscillatory metabolic activity and can be characterized by oscillations in phosphofructokinase-1 (PFK1) activity and its product fructose-1,6-bisphosphate (FBP) (274), and pyruvate kinase activity (244, 275). Moreover, several approaches to monitor mitochondrial or cellular metabolic function have been adopted for use in pancreatic islets over the last decades. NAD(P)H autoﬂuorescence is a long-used indicator of islet cell metabolic function (276–278), the signal which is generally dominated by mitochondrial activity (279). Chemical probes reporting mitochondrial membrane potential have been used to monitor mitochondrial activity within islets coincident with [Ca2+]IC responses (244, 280, 281). Additionally, mitochondrial oscillations can be optically monitored by measuring the signals from mitochondrial ﬂavins (244). Furthermore, sensors for NADPH (282) or H2O2 (283) have provided insight into redox-dependent control of islet function. Genetically encoded probes for ATP have revealed signaling microdomains (284) and the relationship between mitochondrial Ca2+ and cytosolic ATP/ ADP ratios (285). Frontiers in Endocrinology \| www.frontiersin.org Islets as Multilayer Networks (D) ologous and homologous interactions within the islets visualized as a multilayer network. (E) Hypothetical simultaneously measured dynamics of [Ca2+]IC, intracellular and insulin release along with the corresponding functional network layers. Intralayer connections represent functional connections between time series of the same les, whereas the interlayer connections stand for associations between different measured variables. A B B B C D E FIGURE 10 \| The MLN formalism can be used to study various aspects of collective dynamics and cellular activity patterns within the pancreatic islets. (A) A hypothetical raw [Ca2+]IC signal and the extracted slow and fast components (left panel). The two different oscillatory components are then used to construct a two-layered multiplex functional beta cell network (right panel). (B) Typical [Ca2+]IC activity in a pancreatic islet subjected to a hypothetical protocol (upper panel) and the corresponding temporal functional network layers extracted from speciﬁc time intervals (lower panel). (C) A schematic presentation of how network layers are designed from individual intercellular [Ca2+]IC waves. The left panel features a raster plot indicating the onsets of oscillations of cells within speciﬁc waves and the right panel visualizes the corresponding network layers. The direction and weights of connections within each layer denote the course and the temporal lag between subsequent oscillation onsets, respectively. (D) Heterologous and homologous interactions within the islets visualized as a multilayer network. (E) Hypothetical simultaneously measured dynamics of [Ca2+]IC, intracellular ATP, and insulin release along with the corresponding functional network layers. Intralayer connections represent functional connections between time series of the same C C E D E E D D FIGURE 10 \| The MLN formalism can be used to study various aspects of collective dynamics and cellular activity patterns within the pancreatic islets. (A) A hypothetical raw [Ca2+]IC signal and the extracted slow and fast components (left panel). The two different oscillatory components are then used to construct a two-layered multiplex functional beta cell network (right panel). (B) Typical [Ca2+]IC activity in a pancreatic islet subjected to a hypothetical protocol (upper panel) and the corresponding temporal functional network layers extracted from speciﬁc time intervals (lower panel). (C) A schematic presentation of how network layers are designed from individual intercellular [Ca2+]IC waves. The left panel features a raster plot indicating the onsets of oscillations of cells within speciﬁc waves and the right panel visualizes the corresponding network layers. Islets as Multilayer Networks Combined imaging of these and other Finally, as illustrated at length above, intracellular [Ca2+]IC responses of islet beta cells represent an accessible and well- characterized approach to assess islet activity, with tremendous insight to be gained about islet behavior. However, stimulus- secretion coupling involves several steps from receptor binding or metabolism of a secretagogue to membrane depolarization and secretion of insulin granules (133, 259–261). These intracellular signaling steps interact with each other and are ﬁnely tuned by additional physiological, pathological, and pharmacological triggering and amplifying factors (133, 141, 262, 263). The [Ca2+]IC signal itself is determined by a number of upstream processes that include glucose entry, cellular and mitochondrial metabolism, and electrical responses leading to action potential-dependent Ca2+ entry and an interplay with intracellular buffering and storage mechanisms (216, 261, 264). It is important to note that Ca2+ is not just an indicator of cellular activity but is indeed a key mediator for downstream processes required for insulin secretion itself (265), where a glucose-stimulated uptake of Ca2+ (266) triggers the fusion of insulin granules with the cell plasma membrane to elicit insulin release by exocytosis (267–269). Thus, while Ca2+ is generally a good indicator of islet cell activity, it is not reporting all relevant cell physiological processes, and indeed may not always be directly concordant with insulin secretory function. As one June 2022 \| Volume 13 \| Article 922640 Frontiers in Endocrinology \| www.frontiersin.org 18 Multicellular Networks Within Pancreatic Islets Stozˇer et al. A B C D E RE 10 \| The MLN formalism can be used to study various aspects of collective dynamics and cellular activity patterns within the pancreatic islets. (A) A hypothetical Ca2+]IC signal and the extracted slow and fast components (left panel). The two different oscillatory components are then used to construct a two-layered multiplex onal beta cell network (right panel). (B) Typical [Ca2+]IC activity in a pancreatic islet subjected to a hypothetical protocol (upper panel) and the corresponding temporal onal network layers extracted from speciﬁc time intervals (lower panel). (C) A schematic presentation of how network layers are designed from individual intercellular IC waves. The left panel features a raster plot indicating the onsets of oscillations of cells within speciﬁc waves and the right panel visualizes the corresponding network The direction and weights of connections within each layer denote the course and the temporal lag between subsequent oscillation onsets, respectively. Frontiers in Endocrinology \| www.frontiersin.org CONCLUSION Since the end of the 20th century, complex networks have become a common and irreplaceable language in interdisciplinary studies and over the last two decades, the ﬁeld of network science has experienced an explosive growth (98, 307). The research interests have evolved in several directions, from social and economic systems (3, 308, 309), to a wide range of engineered and technological systems (86, 87, 310), and to the more recently developing ﬁeld of multilayered networks (19, 219). Guided by the advances in high-throughput data-collection and imaging techniques, network analyses are also becoming an indispensable tool in biomedical sciences across multiple disciplines and levels of organization (15, 36, 82, 85), including in studies of intercellular interactions in tissues (18, 59). Understanding how heterogeneous populations of interconnected cells in a dynamic and noisy environment operate to ensure proper function is very appealing and challenging to investigate. In the present contribution, we focused on how the network approaches have been and can be used to study the pancreatic islets of Langerhans. Work in this ﬁeld has stimulated research collaborations across different disciplines, from experimental to advanced modelling and computational approaches (58–60, 124, 141, 242, 263). For such collaborations to be even more fruitful in the future, experimentalists need to have a good basic understanding of network science and network scientists need to have a detailed understanding of islet biology. In our view, this is today even more important than it was at the birth of islet networks a decade ago, since the number of experimental and analytical options is increasing at an unprecedented pace. Our wish for the present review is to serve as an information hub for islet biologists and network scientists, helping them navigate through the complex network of existing knowledge and future options and ﬁnding the way to each other. Network-based analyses of islets of Langerhans are typically studied in islets isolated from the pancreas and therefore lacking in the extracellular matrix, vascularization, and innervation that play important roles in signal transduction (300). However, other models may allow the observation and construction of islet cell networks during development, after transplantation into a live animal, and in the context of disease. The tissue slice approach can be regarded as a ﬁrst in the series of possible upgrades to conditions that are closer to the in vivo situation (301–303). Islets as Multilayer Networks The direction and weights of connections within each layer denote the course and the temporal lag between subsequent oscillation onsets, respectively. (D) Heterologous and homologous interactions within the islets visualized as a multilayer network. (E) Hypothetical simultaneously measured dynamics of [Ca2+]IC, intracellular ATP, and insulin release along with the corresponding functional network layers. Intralayer connections represent functional connections between time series of the same variables, whereas the interlayer connections stand for associations between different measured variables. and improved genetically-encoded probes for key signaling molecules, for example recent new probes for citrate (287) and lactate (288), may also provide for improved imaging to allow collection of additional layers of data relating metabolic and [Ca2+]IC networks in islets. relevant metabolic signals and mitochondrial function (including lactate, glutamate, and mitochondrial pH) have recently provided important insight into the metabolic control of insulin secretion and prompted a reconsideration of the consensus model for stimulus-secretion coupling (286). New June 2022 \| Volume 13 \| Article 922640 Frontiers in Endocrinology \| www.frontiersin.org 19 Multicellular Networks Within Pancreatic Islets Stozˇer et al. readily visualized (304). By introducing stable ﬂuorescent probes, zebraﬁsh can therefore be a model for studying glucose-stimulated [Ca2+]IC dynamics in islet cells within the local microenvironment. A similarly transparent site is the anterior chamber of the eye of rodents, where islets can be transplanted, become highly vascularized, and [Ca2+]IC dynamics can be visualized (249). This site can be used to study not only human or mouse islet networks in an environment closer to the native pancreas, but also the characteristics of networks and cell-cell signaling in developing stem cell-derived islet-like clusters (305). Moreover, this approach can be employed in the context of a model for type 1 diabetes, wherein beta cells are attacked by the immune system (306), and in the context of type 2 diabetes using high fat diet or other rodent models (271), or donor islets from people who lived with the disease (249). Combining and comparing network properties from isolated islets and islets analyzed in situ will continue to offer insights into islet communication, both in non- diabetic and diabetic states. Although the abovementioned approaches can all be combined with Ca2+ signaling networks, it is worth noting that other relevant signals can be assessed. Islets as Multilayer Networks Imaging of islet cell membrane potential has been accomplished using small molecule voltage-sensitive probes (289), and may perhaps be more robustly measured using newer genetically encoded voltage indicators (290). Moreover, optogenetic control of islet function is also feasible in transgenic models (291). Further, as already mentioned, signaling by G-protein coupled receptors via second messengers, such as cAMP, is important in both physiology and diabetes treatment (292), and such second messengers are even important for the maintenance of beta-cell activity at a baseline level (293). Genetically encoded probes for cAMP demonstrate glucose- dependent cAMP responses (182) and intra-islet signaling (294). Finally, approaches to monitor the downstream process of insulin secretion from islet cells of intact islets typically involve the visualization of extracellular probes at a cellular resolution. This can include the visualization of zinc ions (Zn2 +) released into the extracellular space concomitantly with insulin (295). Indeed, many Zn2+-binding dyes suitable for this purpose are available (296). Markers of the extracellular volume, such as sulphorhodamine B, have also been used to monitor insulin secretion from individual cells within islets (297), including evaluation of the spatiotemporal control of individual insulin exocytosis event (298). Thus, in the context of intact islet cellular network activity as measured by [Ca2+]IC responses, abundant opportunity exists to add additional layers assessing upstream (metabolism, cAMP), coincident (voltage responses), and downstream (exocytosis) physiological processes. Figure 10E shows an example of an MLN that comprises oscillations in [Ca2+]IC (upper panel), out-of-phase oscillations in [ATP]IC (middle panel), and in-phase oscillations in secreted insulin (lower panel). Along the same line, the MLN approach has already been used to elucidate the relationship between functional connectivity patterns based on membrane potential and [Ca2+]IC signals (299). In perspective, such approach may be for instance instrumental in dissecting the properties of ﬁrst responders, wave initiators, hubs, and other cells in terms of their biochemical properties, their sensitivity to glucose, and their secretory potential. 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June 2022 \| Volume 13 \| Article 922640 REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 299. Gosak M, Dolenšek J, Markovič R, Slak Rupnik M, Marhl M, Stožer A. Multilayer Network Representation of Membrane Potential and Cytosolic Calcium Concentration Dynamics in Beta Cells. Chaos Solitons Fractals (2015) 80:76–82. doi: 10.1016/j.chaos.2015.06.009 June 2022 \| Volume 13 \| Article 922640 Frontiers in Endocrinology \| www.frontiersin.org 28
https://openalex.org/W2888321306	https://europepmc.org/articles/pmc6173346?pdf=render	English	null	Anger and Coronary Artery Disease in Women Submitted to Coronary Angiography: A 48-Month Follow-Up	Arquivos Brasileiros de Cardiologia	2,018	cc-by	5,430	Original Article Original Article Mailing Address: Márcia Moura Schmidt • Avenida Princesa Isabel, 370. Postal Code 90620-000, Santana, Porto Alegre, RS – Brazil E-mail: mmoura.pesquisa@gmail.com, mouramarcia050@gmail.com Manuscript received August 11, 2017, revised manuscript December 20, 2017, accepted April 11, 2018 Abstract Background: Anger control was significantly lower in patients with coronary artery disease (CAD), regardless of traditionally known risk factors, occurrence of prior events or other anger aspects in a previous study of our research group. Objective: To assess the association between anger and CAD, its clinical course and predictors of low anger control in women submitted to coronary angiography. Methods: This is a cohort prospective study. Anger was assessed by use of Spielberger’s State-Trait Anger Expression Inventory (STAXI). Women were consecutively scheduled to undergo coronary angiography, considering CAD definition as ≥ 50% stenosis of one epicardial coronary artery. Results: During the study, 255 women were included, being divided into two groups according to their anger control average (26.99). Those with anger control below average were younger and had a family history of CAD. Patients were followed up for 48 months to verify the occurrence of major cardiovascular events. Conclusion: Women with CAD undergoing coronary angiography had lower anger control, which was associated with age and CAD family history. On clinical follow-up, event-free survival did not significantly differ between patients with anger control above or below average. (Arq Bras Cardiol. 2018; 111(3):410-416) Keywords: Anger; STAXI; Personality Inventory; Coronary Artery Disease/mortality. Keywords: Anger; STAXI; Personality Inventory; Coronary Artery Disease/mortality. Karine Elisa Schwarzer Schmidt, Alexandre Schaan de Quadros, Mauro Regis Moura, Carlos Antonio Mascia Gottschall, Márcia Moura Schmidt Instituto de Cardiologia / Fundação Universitária de Cardiologia (IC/FUC), Porto Alegre, RS – Brazil Introduction in addition to high in-hospital, early and late mortality rates as compared to men.2,6-11 There is growing evidence that psychological factors and emotional stress, such as anger and hostility, can interfere with health behaviors and influence the onset and clinical course of ischemic heart disease.2 At the biological level, the expression of anger can lead to a chronic increase in the levels of catecholamines, evoking an inflammatory response, increasing interleukin-6 levels,12 leading to the progression of atherosclerosis, and, eventually, to the clinical manifestation of cardiovascular diseases.13-15 Cardiovascular diseases (CVD) remain the leading cause of morbidity and mortality of women in several countries, such as USA and Brazil.1 There are more deaths from CVD (41.3%) than from the next seven causes of death combined, and the risk of dying from CVD is six-fold greater than that from breast cancer, the major concern among women.1 There are sex-specific differences regarding CVD presentation, pathophysiology and clinical outcomes; however, as observed by Shivpuri S. et al.2, in a meta-analysis only 5 of 21 studies provided information specific to the female sex, and only a few reported sex-specific differences.2-4 In a previous study, we have reported a significantly lower control of anger in patients with coronary artery disease (CAD), independently of the traditional risk factors, the occurrence of previous events or other aspects of anger.16 Recent data have shown a significant increase in the incidence of cardiovascular disease and deaths among women aged 45 to 54 years, in contrast to the declining trend observed in Brazil and worldwide.2,5 According to the American Heart Association, women show a worse risk factor profile and higher mortality among the youngsters as compared to the elderly, This study aimed at assessing the association between anger and CAD, its clinical course and predictors of low anger control in women undergoing cine coronary angiography. Outcomes valvular heart disease; congenital heart disease; severe diseases with life expectancy <6 months; severe aortic stenosis; and ejection fraction <30%. The project was submitted to the Ethics Committee in Research of the institution and was in accordance with the Declaration of Helsinki and the Resolution 466/12 of the National Council of Health. The outcome primary cardiovascular event was a combination of cardiovascular death, acute myocardial infarction (AMI), myocardial revascularization or hospitalization due to angina. Cardiovascular death was defined as any death due to immediate cardiac causes (AMI, cardiogenic shock, arrhythmia), or death of unknown cause. Acute myocardial infarction was considered in the presence of: 1) increase and/or gradual decrease in cardiac biomarkers (preferably troponin) with at least one measure over the 99th percentile and at least one of the following criteria: 1) chest pain > 10 minutes or new ST-T changes or new left bundle-branch block; or 2) development of pathological Q waves (duration ≥ 0.03 seconds; depth ≥1 mm) in at least two contiguous precordial leads or at least two leads of adjacent limbs; or 3) evidence of viable myocardial loss or new regional wall motion abnormality on any imaging test. Myocardial revascularization comprised primary percutaneous coronary intervention (PCIp) or coronary artery bypass grafting (CABG) occurring after entrance into the study. Hospitalization due to angina was defined as hospital length of stay longer than 24 hours to assess or treat cardiac chest pain, with neither AMI nor need for myocardial revascularization. Coronary angiography Coronary angiography was performed according to the Judkins technique, all analyses were performed in at least two views, and the severity of the coronary obstructions was assessed by use of a digital calibration system previously validated (Siemens AxiomArtis - Munich, German). Prior to the measurements, intracoronary nitroglycerin was administered routinely at the dose of 100-200 μg. Coronary artery disease was defined as ≥ 50% stenosis of at least one major epicardial artery. Assessment of anger Anger is an emotional state that consists of feelings that vary in intensity from mild irritation or annoyance to intense fury and rage, and changes over time spams depending on what is perceived as injustice or frustration.17 Anger assessment was performed by use of Spielberger’s State-Trait Anger Expression Inventory (STAXI),17 a tool translated to several languages, validated in Brazil and recommended by the Federal Council of Psychology. It comprises 40 statements about the intensity of anger, how patients usually feel and how often they experience anger. Each item is rated on a four-point Likert-type scale, scored as follows: 1 for “rarely”; 2 for “sometimes”; 3 for “often”; and 4 for “almost always”. The test is subdivided into subscales: state anger, trait anger (temperament and reaction) and anger expression (anger expression-in, anger expression-out, and anger control). Trait anger is defined as a predisposition to experiencing anger, indicating lasting personality trends. It is assessed by use of questions such as: “I get angry easily”, “I get angry when my good work is not recognized”. Anger expression provides an assessment of how anger is experienced: suppression, expression or control. (Examples: “I keep things to myself”, “I do things such as slam the door”, “I boil inside, but I do not show”). As state anger assesses the amount of anger that is experienced at a particular time, that subscale was not used in the sample of in-hospital patients. Statistical analysis and justification of the sample size The sample size was calculated with power of 80, alpha of 0.05 and 95% confidence interval. At least 250 individuals were necessary to detect a relative risk of 1.60,18 considering the 30% incidence of MCVE in the total group of women. Continuous variables were expressed as mean ± standard deviation, while categorical variables, as absolute number and percentage. The characteristics of the patients with CAD were compared to those of patients without CAD, using Student t test for independent samples for continuous variables and chi‑square test for categorical variables. The women were divided into two groups according to their scores being above or below average range (26.99). Their demographic characteristics, risk factors, previous history and STAXI scores were compared by use of Student t test or chi-square test. Cronbach’s alpha was used to assess the internal consistency of the STAXI subscales. Kolmogorov-Smirnov test was used to assess the normality of the distribution of the scores. Multiple logistic regression analysis was used to assess the variables associated with CAD on baseline angiography and control of anger. The Kaplan‑Meier curves and the log-rank test were used to compare event-free survival between patients with anger control scores above and below the sample’s average range. For all tests, a p value < 0.05 was considered statistically significant. All data were recorded in an Excel database for analysis with the SPSS program, version 24.0 for Windows. Follow-up The participants were followed up for 48 months by use of visits and telephone contacts, to assess the occurrence of major cardiovascular events (MCVE), defined as cardiovascular death, AMI, myocardial revascularization (CABG or PCI) and hospitalization due to angina > 24 hours. Arq Bras Cardiol. 2018; 111(3):410-416 Patients This is a prospective cohort study. All women scheduled for elective coronary angiography because of suspected CAD during the study period were consecutively assessed. This study included women aged 18 years and older, who provided written informed consent to participate in the study. The exclusion criteria were: indication for catheterization for DOI: 10.5935/abc.20180165 DOI: 10.5935/abc.20180165 410 Schmidt et al Anger and coronary artery disease in women Schmidt et al Anger and coronary artery disease in women Original Article Results Table 3 shows the baseline characteristics of the patients regarding anger control, with 36.3% of the women with anger control below the average range and 63.7%, over the average range. Those with anger control below the average range were younger (58.1 ± 8.9 vs 62.2 ± 10.9, p < 0.001) and had a higher prevalence of family history of CAD (53.9% vs 29.5%, p < 0.001) than those whose control of anger was above the average range. Other characteristics, such as weight, diabetes, previous coronary events (AMI, PCI, CABG) and other risk factors did not differ between the two groups. However, the patients with anger control below the average range had a tendency towards lower prevalence of hypertension (p = 0.09) and previous CABG (p = 0.11). On logistic regression (Table 4), only age and the family history of CAD were predictors of poor anger control. Figure 1 shows no significant difference in event-free survival in patients with anger control below and above 27 points (p = 0.62). Table 3 shows the baseline characteristics of the patients regarding anger control, with 36.3% of the women with anger control below the average range and 63.7%, over the average range. Those with anger control below the average range were younger (58.1 ± 8.9 vs 62.2 ± 10.9, p < 0.001) and had a higher prevalence of family history of CAD (53.9% vs 29.5%, p < 0.001) than those whose control of anger was above the average range. Other characteristics, such as weight, diabetes, previous coronary events (AMI, PCI, CABG) and other risk factors did not differ between the two groups. However, the patients with anger control below the average range had a tendency towards lower prevalence of hypertension (p = 0.09) and previous CABG (p = 0.11). On logistic regression (Table 4), only age and the family history of CAD were predictors of poor anger control. Figure 1 shows no significant difference in event-free survival in patients with anger control below and above 27 points (p = 0.62). From November 29, 2009, to february 3, 2010, 255 participants were included. Table 1 shows the results according to the presence of CAD, clinical history and different STAXI subscales. The patients with CAD most frequently had previous cardiac procedures (CABG and PCIp) and a lower mean level of anger control than patients without CAD, who most often were married as compared to the former. Results Other risk factors, previous medical history and anger subscales showed no significant differences. The multiple logistic regression analysis (Table 2) identified a relationship between CAD and low anger control, previous CABG or PCI, and marital status. The patients were followed up for 48 [39-49] months to assess the occurrence of MCVE. From the initial sample of 255 patients, 10 women (3.9%) could not be reached, leaving 245 to participate in this study, 89 with anger control below the average range, and 156, over the average range. Table 1 – Clinical characteristics, medical history and STAXI scales according to the presence of coronary artery disease (CAD) Characteristics CAD n = 115 (45.1%) No CAD n = 140 (54.9%) Total: 255 p* Age (years), mean ± SD 61.0 ± 10.5 60.5 ± 9.7 0.65 White, n (%) 97 (85.1) 111 (79.9) 0.27 Married, n (%) 50 (43.5) 81 (57.9) 0.02 Schooling, years 6.2 ± 5.4 5.9 ±4.4 0.65 Current job, n (%) 26 (22.6) 28 (20.0) 0.61 Living alone, n (%) 28 (24.3) 32 (22.9) 0.78 Risk factors Hypertension, n (%) 102 (88.7) 120 (85.7) 0.48 DM, n (%) 41 (35.7) 20 (27.9) 0.18 Dyslipidemia, n (%) 71 (61.7) 73 (52.1) 0.12 Smoking, n (%) 28 (24.3) 23 (16.4) 0.11 Family history of CAD, n (%) 44 (38.3) 52 (37.1) 0.85 Depression, n (%) 38 (33.0) 55 (39.3) 0.30 BMI (kg/m2), mean ± SD 27.6 ± 5.3 28.4 ± 6.0 0.25 Previous medical history Previous AMI, n (%) 30 (26.1) 27 (19.3) 0.19 Previous PCI, n (%) 19 (16.5) 11 (7.9) 0.03 Previous CABG, n (%) 9 (7.8) 0 (0.0) < 0.001 STAXI subscales Trait of anger (points), mean ± SD 20.0 ± 7.9 20.7 ± 8.5 0.54 Angry temperament (points), mean ± SD 8.6 ± 4.1 8.4 ± 4.0 0.69 Angry reaction (points), mean ± SD 8.0 ± 3.5 8.6 ± 4.2 0.20 Anger expression-In (points), mean ± SD 16.03 ± 4.26 16.6 ± 5.2 0.34 Anger expression-Out (points), mean ± SD 13.2 ± 4.6 12.9 ± 4.0 0.58 Control of anger (points), mean ± SD 26.2 ± 5.00 27.7 ± 3.7 < 0.001 Anger expression (points), mean ± SD 19.0 ± 10.3 17.8 ± 9.0 0.29 SD: standard deviation; p* - p≤ 0.05, Student t test or chi-square test; DM: diabetes mellitus; BMI: body mass index; AMI: acute myocardial infarction; PCI: percutaneous coronary intervention; CABG: coronary artery bypass grafting. Clinical characteristics at the beginning of the study The clinical and socioeconomic characteristics, risk factors for CAD, previous medical history, clinical presentation of CAD and history of psychological diagnosis were assessed and included in a dedicated database. Hypertension was defined as previous diagnosis of the condition or use of anti‑hypertensives. Dyslipidemia was considered present in those previously diagnosed with the condition or on lipid-lowering drugs. Diabetes mellitus was defined as the previous use of insulin or oral hypoglycemic drugs, or the presence of documented fasting blood sugar > 126 mg/dL on two occasions. Previous history of depression was defined as the occurrence of at least one major depressive episode that required treatment with antidepressants. Arq Bras Cardiol. 2018; 111(3):410-416 411 Schmidt et al Anger and coronary artery disease in women Schmidt et al Anger and coronary artery disease in women Schmidt et al Anger and coronary artery disease in women Original Article Results al characteristics, medical history and STAXI scales according to the presence of coronary artery disease (CAD) Arq Bras Cardiol. 2018; 111(3):410-416 412 Original Article Schmidt et al Anger and coronary artery disease in women Table 2 – Relationship between coronary artery disease and baseline characteristics Characteristics Beta Coefficient 95% CI for Beta Coefficient Total: 255 p * Low control of anger 0.15 0.03 – 0.27 0.01 Married - 0.12 - 0.24 – - 0.01 0.03 Previous PCI 0.14 0.04 – 0.40 0.02 Previous CABG 0.16 0.26 – 0.90 < 0.001 p * - p ≤ 0.05, Wald test; CI: confidence interval; PCI: percutaneous coronary intervention; CABG: coronary artery bypass grafting. Schmidt et al Anger and coronary artery disease in women Table 2 – Relationship between coronary artery disease and baseline characteristics Characteristics Beta Coefficient 95% CI for Beta Coefficient Total: 255 p * Low control of anger 0.15 0.03 – 0.27 0.01 Married - 0.12 - 0.24 – - 0.01 0.03 Previous PCI 0.14 0.04 – 0.40 0.02 Previous CABG 0.16 0.26 – 0.90 < 0.001 p * - p ≤ 0.05, Wald test; CI: confidence interval; PCI: percutaneous coronary intervention; CABG: coronary artery bypass grafting. Results This study corroborates that by Haukalla et al.,20 who reported that individuals with lower anger control were at higher risk for the first incidence of fatal and nonfatal cardiovascular disease than those who scored higher. Results Table 2 – Relationship between coronary artery disease and baseline characteristics Table 3 – Patients’ clinical characteristics, medical history and STAXI scales according to anger control in a 48-month follow-up Characteristics Control of anger below average n = 89 Control of anger above average n = 156 Total: 245 p * Age (years), mean ± SD 58.1 ± 8.9 62.2 ± 10.9 0.001 White, n (%) 71 (79.8) 128 (83.1) 0.52 Married, n (%) 48 (53.9) 79 (50.6) 0.62 Schooling, years 6.1 ± 4.9 6.1 ± 4.9 0.97 Current job, n (%) 20 (22.5) 31 (19.9) 0.63 Living alone, n (%) 20.2 (18) 38 (24.4) 0.46 Risk factors Hypertension, n (%) 73 (82.0) 140 (89.7) 0.09 DM, n (%) 33 (37.1) 45 (28.8) 0.18 Dyslipidemia, n (%) 49 (55.1) 92 (59.0) 0.55 Smoking, n (%) 15 (16.9) 34 (21.8) 0.35 Family history of CAD, n (%) 48 (53.9) 46 (29.5) < 0.001 Depression, n (%) 35 (39.3) 55 (35.3) 0.53 BMI (kg/m2), mean ± SD 28.4 ± 5.4 27.9 ± 5.9 0.55 Previous medical history Previous AMI, n (%) 22 (24.7) 34 (21.8) 0.60 Previous PCI, n (%) 8 (9.0) 22 (14.1) 0.24 Previous CABG, n (%) 1 (1.1) 8 (5.1) 0.11 STAXI subscales Trait of anger (points), mean ± SD 23.67 ± 8.25 18.52 ± 7.53 < 0.001 Angry temperament (points), mean ± SD 10.20 ± 4.05 7.51 ± 3.74 < 0.001 Angry reaction (points), mean ± SD 9.19 ± 4.04 7.81 ± 3.70 0.007 Anger expression-In (points), mean ± SD 16.85 ± 5.08 16.06 ± 4.65 0.22 Anger expression-Out (points), mean ± SD 15.07 ± 4.97 11.81 ± 3.31 < 0.001 Control of anger (points), mean ± SD 22.19 ± 3.66 29.67 ± 1.72 < 0.001 Anger expression (points), mean ± SD 25.73 ± 9.79 14.21 ± 6.74 <0.001 p* - p ≤ 0.05, Student t test or chi-square test; DM: diabetes mellitus; CAD: coronary artery disease; BMI: body mass index; AMI: acute myocardial infarction; PCI: percutaneous coronary intervention; CABG: coronary artery bypass grafting. atients’ clinical characteristics, medical history and STAXI scales according to anger control in a 48-month follow-up dent t test or chi-square test; DM: diabetes mellitus; CAD: coronary artery disease; BMI: body mass index; AMI: acute myocardial infarction; s coronary intervention; CABG: coronary artery bypass grafting. poor control of anger had a positive family history for CAD and were younger. Arq Bras Cardiol. 2018; 111(3):410-416 Discussion The present study identified that poor control of anger associated with CAD angiographically detected. This study found a higher percentage of married patients in the group without CAD, indicating the importance of social support in treatment adhesion and healthcare.19 Women with Arq Bras Cardiol. 2018; 111(3):410-416 413 Original Article Schmidt et al Anger and coronary artery disease in women Figure 1 – Survival curve for major cardiovascular events in up to 48 months of follow-up of women with anger control below and above average. 1.0 0.9 0.8 0.7 0.6 0.5 5.00 10.00 15.00 20.00 25.00 35.00 40.00 50.00 45.00 30.00 CumSurvival p = 0.62 anger control: average < 27 ≥ 27 Time until 1st end-point The survival curves separate between 5 and 20 months of follow-up and approximate after 30 months of follow-up. showing that patients whose anger control was below average had survival around 60%. while those whose anger control was above average had survival around 65%. (p = 0.62) Survival curve according to anger control Table 4 – Association between control of anger and baseline characteristics Characteristics Beta Coefficient 95% CI for Beta Coefficient p* Age 0.15 0.002 - 0.013 0.01 Family history of CAD 0.22 0.100 - 0.340 < 0.001 Diabetes mellitus 0.007 - 0.039 - 0.170 0.21 Previous CABG - 0.06 - 0.480 - 0.140 0.27 p * - p ≤ 0.05, Wald test; CI: confidence interval; CAD: coronary artery disease; CABG: coronary artery bypass grafting. Original Article Table 4 – Association between control of anger and baseline characteristics Characteristics Beta Coefficient 95% CI for Beta Coefficient p* Age 0.15 0.002 - 0.013 0.01 Family history of CAD 0.22 0.100 - 0.340 < 0.001 Diabetes mellitus 0.007 - 0.039 - 0.170 0.21 Previous CABG - 0.06 - 0.480 - 0.140 0.27 p * - p ≤ 0.05, Wald test; CI: confidence interval; CAD: coronary artery disease; CABG: coronary artery bypass grafting. Table 4 – Association between control of anger and baseline characteristics Characteristics Beta Coefficient 95% CI for Beta Coefficient p* Age 0.15 0.002 - 0.013 0.01 Family history of CAD 0.22 0.100 - 0.340 < 0.001 Diabetes mellitus 0.007 - 0.039 - 0.170 0.21 Previous CABG - 0.06 - 0.480 - 0.140 0.27 p * - p ≤ 0.05, Wald test; CI: confidence interval; CAD: coronary artery disease; CABG: coronary artery bypass grafting. Discussion Table 4 – Association between control of anger and baseline characteristics Figure 1 – Survival curve for major cardiovascular events in up to 48 months of follow-up of women with anger control below and above average. 1.0 0.9 0.8 0.7 0.6 0.5 5.00 10.00 15.00 20.00 25.00 35.00 40.00 50.00 45.00 30.00 CumSurvival p = 0.62 anger control: average < 27 ≥ 27 Time until 1st end-point The survival curves separate between 5 and 20 months of follow-up and approximate after 30 months of follow-up. showing that patients whose anger control was below average had survival around 60%. while those whose anger control was above average had survival around 65%. (p = 0.62) Survival curve according to anger control 1.0 0.9 0.8 0.7 0.6 0.5 5.00 10.00 15.00 20.00 25.00 35.00 40.00 50.00 45.00 30.00 CumSurvival p = 0.62 anger control: average < 27 ≥ 27 Time until 1st end-point Survival curve according to anger control The survival curves separate between 5 and 20 months of follow-up and approximate after 30 months of follow-up. showing that patients whose anger control was below average had survival around 60%. while those whose anger control was above average had survival around 65%. (p = 0.62) Figure 1 – Survival curve for major cardiovascular events in up to 48 months of follow-up of women with anger control below and above average. run, similar to that observed in men. However, complications related to PCI and the mortality rates of women are three times higher as compared to those of men.24 Haukalla et al.20 have reported that low anger control in a 10- to 15-year clinical follow-up predicted ischemic myocardial disease in women, even after adjusting for sociodemographic variables, other cardiovascular risk factors and symptoms of depression. In the late clinical follow-up of this study, low control of anger was not associated with the occurrence of combined MCVE, myocardial revascularization included. In this sample, the women with low anger control were younger. Arq Bras Cardiol. 2018; 111(3):410-416 Discussion That characteristic can be interpreted in the sense that, as age advances, social relations are modulated through emotional regulation, which means that, as time advances and with aging, more appropriate forms of social behavior are learned, with more control over emotions and reactions.21 According to Cartensen’s socioemotional selectivity theory, as age advances, people become increasingly selective, tending to place a high value on positive contents and to avoid negative emotional states, because of adaptation and life changes experienced in social contexts.22,23 The literature available shows that anger is associated with several behavioral risk factors, such as tobacco use and inadequate dietary intake (hypercaloric and high sodium diets), and, in the long run, out of other cardiovascular risk factors, anger can cause LDL elevation, hypertension, diabetes mellitus, and obesity.13,25 In a study, Pérez-García et al.26 have reported According to Shirato et al.,24 gender differences are evident in the success rates of the interventions to improve coronary circulation (myocardial revascularization). After a well‑succeeded procedure, women submitted to coronary angioplasty had an excellent prognosis in the long Arq Bras Cardiol. 2018; 111(3):410-416 414 Schmidt et al Anger and coronary artery disease in women Schmidt et al Anger and coronary artery disease in women Original Article that emotional discomfort and symptoms were positively associated with higher inward expression of anger and lower control of anger. In addition, they have found that preventive practices were associated with lower supression and higher control of anger, with better channeling and regulation of anger feelings. The likelihood that patients with low anger control also have low control over other risk behaviors or use them as a comfort mechanism can be considered, because the effects of well-being through neuroendocrine mechanisms of hormone release, such as serotonin, producing well-being after energetic ingestion, have been described, which would be applicable to stress/anger situations.27 difference in the event-free time between patients with anger control scores above average range and those with anger control scores below it. Conclusions Women with CAD submitted to coronary angiography showed a trend towards lower control of anger, which was associated with age and the family history of CAD. The 48-month clinical follow-up showed no significant Author contributions Conception and design of the research: Moura MR, Gottschall CAM, Schmidt MM; Acquisition of data: Schmidt KES, Schmidt MM; Analysis and interpretation of the data: Schmidt KES, Quadros AS, Moura MR, Gottschall CAM, Schmidt MM; Statistical analysis: Schmidt MM; Writing of the manuscript: Schmidt KES, Quadros AS, Schmidt MM; Critical revision of the manuscript for intellectual content: Quadros AS, Moura MR, Gottschall CAM. The compensation, cognitive and affective value attributed to food overlaps the homeostatic control and the physiological signs of hunger and satiety that control food ingestion and body weight.27,28 However, if continuously evoked, that process would cause CAD as a factor associated not only with the feeling of anger, but with all the inappropriate coping mechanism that could accompany anger. Study’s forces and limitations There were no external funding sources for this study. This study’s force resides on its female sample, because women are usually under-represented in clinical trials. This is a segment of the real world, with few losses during a long follow‑up. The risk factors were assessed based on interviews with the participants, and there might have been bias of information. Assessing anger and its control, even with a tool developed for that purpose, is a hard task, considering that personality traits can be combined. Research in this area is a challenge. Potential Conflict of Interest No potential conflict of interest relevant to this article was reported. Ethics approval and consent to participate This study was approved by the Ethics Committee of the Instituto de Cardiologia / Fundação Universitária de Cardiologia under the protocol number 466/12. All the procedures in this study were in accordance with the 1975 Helsinki Declaration, updated in 2013. Informed consent was obtained from all participants included in the study. Study Association This article is part of the thesis of Doctoral submitted by Márcia Moura Schmidt, from Instituto de Cardiologia / Fundação Universitária de Cardiologia. Original Article 11. Claassen M, Sybrandy KC, Appelman YE, Asselbergs FW. Gender gap in acute coronary heart disease: myth or reality? World J Cardiol. 2012; 4(2):36–47. discontinuation of thienopyridine after coronary stent implantation. Am J Cardiol. 2011;107(5):685-9. discontinuation of thienopyridine after coronary stent implantation. Am J Cardiol. 2011;107(5):685-9. 20. Haukkala A, Konttinen H, Latikainen T, Kawachi I, Utela A. Hostility, anger control, and anger expression as predictors of cardiovascular disease. Psychosom Med. 2010;72(6):556–62. 12. Alam M, Bandeali SJ, Kayani WT, Ahmad W, Shahzad SA, Jneid H, et al. Comparison by meta-analysis of mortality after isolated coronary artery bypass grafting in women versus men. Am J Cardiol. 2013;112(3):309-17. 21. Carstensen LL, Pasupathi M, Mayr U, Nesselroade JR. Emotional experience in everyday life across the adult life span. J Pers Soc Psychol. 2000;79(4):644-55. 13. Williams JE, Couper DJ, Din-Dzietham R, Nieto FJ, Folsom AR. Race-gender differences in the association of trait anger with subclinical carotid artery atherosclerosis. Am J Epidemiol. 2007;165(11):1296-304. 22. Amado NM. Sucesso no envelhecimento e histórias de vida em idosos sócio- culturalmente muito e pouco diferenciados.Success in aging and life stories in elderly socio-culturally diferente; 2008. [Thesis] Lisboa: Universidade Nova de Lisboa,Instituto Superior de Psicologia Aplicada. 14. Puterman E, Epel ES, O’Donovan A, Prather AA, Aschbacher K, Dhabhar FS. Anger is associated with increased IL-6 stress reactivity in women, but only among those low in social support. Int J Behav Med. 2014; 21(6):936–45. 23. Otta E, Fiquer JT. Bem-estar subjetivo e regulação de emoções. (Subjective well- being and emotional regulation). Psicologia em Revista, 2004;10(15):144-9. 15. Markovitz JH. Hostility is associated with increased platelet activation in coronary heart disease. Psychosom Med. 1998;60(5):586-91. 24. Shirato S, Swan BA. Women and cardiovascular disease: an evidentiary review. MEDSURG Nursing. 2010;19(5):282-6. 16. Schmidt MM, Lopes RD, Newby LK, Moura MR, Stochero L, Gottschall CM, et al. Anger control and cardiovascular outcomes. Int J Cardiol. 2013;168(4):4338-9. 25. Glaser R, Herrmann HC, Murphy SA, Demopoulos LA, Di Battiste PM, Cannon CP, et al. Benefit of an early invasive management strategy in women with acute coronary syndromes. JAMA. 2002;288(24):3124-9. 17. Spielberger CD, Lushene RE, Gorsuch R, Jacobs GA. Manual for the state- trait anger inventory. STAXI.( translation Biaggio A, and brazilian validation of Spielberger CD). São Paulo: Vetor; 2003. 26. Pérez-García AM, Sanjuán P, Rueda B, Ruiz MA. Salud cardiovascular en la mujer: el papel de la ira y su expresión. Psicothema. 2011;23(4):593-8. 18. References 2018; 111(3):410-416 Schmidt et al Anger and coronary artery disease in women Schmidt et al Anger and coronary artery disease in women Original Article References 1. Fernandes CE, Pinto Neto JS, Gebara OCE, Santos Filho RD, Pinto Neto AM, Pereira Filho AS, et al. First brazilian guidelines on cardiovascular disease prevention in climacteric women and influence of hormone replacement therapy from Brazilian Cardiology Society and Brazilian Climacteric Association (SOBRAC). Arq Bras Cardiol. 2008;91(1 Suppl 1):1-23. 6. Gupta A, Wang Y, Spertus JA, Geda M, Lorenze N, Nkonde-Price C, et al. Trends in acute myocardial infarction in young patients and differences by sex and race, 2001 to 2010. J Am Coll Cardiol. 2014; 64(4):337–45. 1. Fernandes CE, Pinto Neto JS, Gebara OCE, Santos Filho RD, Pinto Neto AM, Pereira Filho AS, et al. First brazilian guidelines on cardiovascular disease prevention in climacteric women and influence of hormone replacement therapy from Brazilian Cardiology Society and Brazilian Climacteric Association (SOBRAC). Arq Bras Cardiol. 2008;91(1 Suppl 1):1-23. 7. Zhang Z, Fang J, Gillespie C, Wang G, Hong Y, Yoon PW. Age-specific gender differences in in-hospital mortality by type of acute myocardial infarction. Am J Cardiol. 2012;109(8):1097–103. 2. Shivpuri S, Gallo LC, Mills PJ, Matthews KA, Elder JP, Talavera GA. Trait anger, cynical hostility and inflammation in Latinas: variations by anger type? Brain Behav Immun. 2011; 25(6):1256–63. 8. Canto JG, Rogers WJ, Goldberg RJ, Peterson ED, Wenger NK, Vaccarino V, et al. Association of age and sex with myocardial infarction symptom presentation and in-hospital mortality. JAMA. 2012;307(8):813–22. 3. Mehta LS, Beckie TM, DeVon HA, Grines CL, Krumholz HM, Johnson MN, et al. Acute myocardial infarction in women. Circulation. 2016; 133(9):916-47. 3. Mehta LS, Beckie TM, DeVon HA, Grines CL, Krumholz HM, Johnson MN, et al. Acute myocardial infarction in women. Circulation. 2016; 133(9):916-47. 9. Egiziano G, Akhtari S, Pilote L, Daskalopoulou SS, GENESIS (GENdEr and Sex Determinants of Cardiovascular Disease), Investigators. Sex differences in young patients with acute myocardial infarction. Diabet Med. 2013;30(3):e108-14. 4. Chida Y, Steptoe A. The association of anger and hostility with future coronary heart disease: a meta-analytic review of prospective evidence. J Am Coll Cardiol. 2009; 53(11):936–46. 10. Champney KP, Frederick PD, Bueno H, Parashar S, Foody J, Merz CN, et al. The joint contribution of sex, age and type of myocardial infarction on hospital mortality following acute myocardial infarction. Heart. 2009; 95(11):895–9. 5. Mansur ADP, Favarato D. Tendências da taxa de mortalidade por doenças cardiovasculares no Brasil, 1980-2012. Arq Bras Cardiol. 2016;107(1):20-5. 415 Arq Bras Cardiol. Original Article Wong JM, Na B, Regan MC, Whooley MA. Hostility, health behaviors, and risk of recurrent events in patients with stable coronary heart disease: Findings From the Heart and Soul Study. J Am Heart Assoc. 2013;2(5):e000052. 27. Landeiro FM, Quarantini LC. Obesity: neuro-hormonal controlof food intake. R Ci Med Biol. Salvador. 2011;10(3)236-45. 19. Quadros AS, Welter DI, Camozzatto FO, Chaves Á, Mehta RH, Gottschall CA, et al. Identifying patients at risk for premature 28. Tataranni PA, Del Parigi A. Functional neuroimaging: a new generation of human brain studies in obesity research. Obes Rev. 2003;4(4):229-38. This is an open-access article distributed under the terms of the Creative Commons Attribution License This is an open-access article distributed under the terms of the Creative Commons Attribution License Arq Bras Cardiol. 2018; 111(3):410-416
W4311117174.txt	https://agdevresearch.org/index.php/aad/article/download/261/165	en		Best practices for mentoring: An exploratory study of cooperating teacher and student teacher perspectives	Advancements in agricultural development	2,022	cc-by	4,697	Nesbitt et al. Advancements in Agricultural Development Volume 3, Issue 4, 2022 agdevresearch.org Best Practices for Mentoring: An Exploratory Study of Cooperating Teacher and Student Teacher Perspectives H. R. Nesbitt1, D. M. Barry2, K. M. Lawson3, J. M. Diaz4 Abstract With nearly one-half of U.S. teachers leaving the profession within the first five years of their career, focusing on retention is an ongoing effort. Providing quality mentorship during the student teaching internship provides further support to new teachers preparing to enter the classroom. Cooperating teachers play a pivotal role in the success of these student teachers. However, little is known about the mentoring process between the cooperating teachers and their student teachers. This study compares the perspectives of the cooperating teacher and their student teacher on the frequency of 17 best practices employed by the cooperating teacher during the student teaching experience. The results suggest cooperating teachers rate themselves as utilizing 16 of the 17 best practices of a cooperating teacher more frequently than their paired student teacher observed. In addition, the student teachers rated their observed frequency for five of the 17 best practices employed by their mentor teacher between rarely and often, implying potential weaknesses in the preparation of the cooperating teacher. Differences between the perceived practices of the cooperating teachers and the observed frequencies of these practices by their student teachers warrants further research in the preparation and support of cooperating teachers in their roles as mentors. Keywords Agricultural education, internship, mentorship, retention, teaching 1. Heather R. Nesbitt, Graduate Assistant, University of Florida, 310 Rolfs Hall, PO Box 110540, Gainesville, FL 32611, 2. heather.nesbitt@ufl.edu, https://orcid.org/0000-0001-5339-8516 Debra M. Barry, Assistant Professor, University of Florida, 1200 N Park Rd, Plant City, FL 33563, dmbarry@ufl.edu, 3. https://orcid.org/0000-0001-9579-3872 Kati M. Lawson, Lecturer, University of Florida, 1200 N Park Rd, Plant City, FL 33563, katilawson@ufl.edu, 4. https://orcid.org/0000-0001-6271-8120 John M. Diaz, Associate Professor, University of Florida, 1200 N Park Rd, Plant City, FL 33563, john.diaz@ufl.edu, https://orcid.org/0000-0002-2787-8759 30 Nesbitt et al. Advancements in Agricultural Development Introduction and Problem Statement Attempts at teacher retention occurs through a variety of efforts and programs and is often focused on the first years of teaching because 44% of teachers leave the profession within the first five years of starting their teaching careers (Ingersoll et al., 2018). Increasing self-efficacy during the student teaching experience has been linked to a higher intention to teach, with previous research indicating the importance of an effective cooperating teacher (CT) relationship as a major component (Edgar et al., 2011; Kasperbauer & Roberts, 2007; Roberts, 2006; Rocca, 2005). Higher teacher self-efficacy can help to lower teacher burnout and increase retention (Swan et al., 2011). Roberts (2006) developed a model for CT effectiveness; however, confusion from cooperating teachers as to their exact role during the internship experience remains (Dunning et al., 2011; Ganser, 2002). To support the relationship of the CT and student teacher (ST), and provide guidance on how to utilize observation, feedback, and appropriate mentorship, CTs need be prepared and supported for their role (Young & MacPhail, 2005). To support the mentor-mentee relationship of the CT and their ST, Korte and Simonsen (2018) indicated the need to implement best practices of mentoring. Formalized mentoring should be structured during pre-service teacher experiences to support growth and development, as well as teacher self-efficacy (Korte & Simonsen, 2018). Previous research indicated that CTs tend to shy away from critical feedback and teaching observations overall (Zimpher et al., 1980). However, when CTs have been given guidance and training for their role, they are more likely to provide quality feedback to student teachers (McIntyre & Killian, 1987). Theoretical and Conceptual Framework The theoretical foundation for this study was rooted in constructivism, where assumptions are made that learners construct their own knowledge through experiences in a variety of social environments (Vijaya Kumari, 2014; Vygotsky, 1978). Constructivism posits that students who are learning a new skill are more likely to be successful if they are taught by someone advanced or experienced in that area. This is further realized by the description of instructional scaffolding, where the construction of new ideas can be strengthened and formed with a firmer foundation (Oyster & Bobbit, 2020; Seifert & Sutton, 2009). Higher education institutions are often criticized in this area as they typically elicit only a few means for evaluating student perspectives and outcomes within the broader purposes of the institution (Burke, 2005). Assessments will often extend beyond measuring knowledge acquisition to include behavioral evaluations, assessing the frequency for which students and teachers are carrying out certain practices. For example, it is common for assessments to ask students and teachers to report current or past participation, frequency, or duration in certain activities (Gonyea, 2005). According to Astin (1993), the main advantage and potential rationale for using self-reported data for evaluative purposes in educational context is feasibility. Astin (1993) claimed that selfhttps://doi.org/10.37433/aad.v3i4.261 31 Nesbitt et al. Advancements in Agricultural Development report questionnaires, while exhibiting lower fidelity, have a greater bandwidth to collect data. Many times, self-reported data from survey/questionnaires are often the only practical source of certain types of information because they are quicker and more economical to implement than objective testing or observational studies. One must consider that many desired outcomes are difficult to measure empirically, and in cases where measures are available, they are often costly and impractical (Gonyea, 2005). An issue that threatens the credibility of self-reported data is social desirability bias (SDB). SDB has the potential to present a challenge in conducting this line of research, including cooperating teacher assessments in the context of higher education (Chichekian & Shore, 2016). SDB is the desire of respondents to edit their responses before communicating such to researchers to present themselves in a more positive light (Beretvas et al., 2002). Respondents may feel a need to present themselves favorably to the interviewers or researchers to preserve their self-esteem (King & Bruner, 2000). SDB can compromise evaluations in one of two ways: (a) overreporting of socially desirable behavior (such as performing teaching best practices) and underreporting of socially undesirable behavior (e.g., extensive absence), and (b) attenuation, inflation, or moderation of relationships between variables (King & Bruner, 2000). This causes discrepancies in student and teacher responses and the researchers’ ability to generalize results. The development of effective mentoring programs and models can help to strengthen preservice teacher skills and assist in a successful start to a career in teaching (He, 2010). The areas of mentorship explored in this study support major tenets of successful mentorship characteristics and build on the work of previous research that has identified areas of focus for CT effectiveness (He, 2010). Roberts’ (2006) model of cooperating teacher effectiveness focused on the areas of teaching/instruction, professionalism, ST/CT relationship, and personal characteristics. The area of ST/CT relationship from Roberts’ 2006 study and findings from Stewart et al. (2017) make a case for mentoring and its imperative role in the development of student teachers. Further advancement of the nuanced understanding of the participation of CTs has the potential to help researchers better facilitate the significant role of the CT and the development of the next generation of school-based agricultural educators (Clarke et al., 2014). Purpose The purpose of this study was to compare the frequency of best practices for mentoring as selfreported by CTs with the perspectives of their paired STs. Three objectives guided this study: 1. Determine the frequency of use of best practices for mentoring behaviors from the perspective of CTs. 2. Determine the frequency of use of best practices for mentoring behaviors from the perspective of STs. 3. Compare the perspectives of CTs and STs for the frequency of best practice mentoring behaviors. https://doi.org/10.37433/aad.v3i4.261 32 Nesbitt et al. Advancements in Agricultural Development Methods This exploratory study compared the frequency of best practices for mentoring behaviors of CTs and the perspective of these behaviors from the viewpoint of their STs. This study utilized survey research methods. The data collected were part of a larger research project that explored CT needs for professional support and the use of best practices for mentoring. The University of Florida Department of Agricultural Education and Communication instituted a CT and ST workshop aimed at developing skillsets and increasing the understanding of expectations for both STs and the CTs. The population for this study included the University of Florida Department of Agricultural Education and Communication’s CTs who served in their role during the spring semester of 2021 (N = 7), as well as the University of Florida’s] STs who were under their supervision for that term (N = 7). Data were collected following the conclusion of the 14-week student teaching internship. The survey questionnaires were delivered using Qualtrics software. All participants were assigned a study identification number to complete paired analysis of the STs and CTs responses. Analysis of data was conducted using Statistical Package for Social Sciences (SPSS) version 26. Of the seven CTs, six responded to the survey; all seven STs responded to the survey. Because of the nature of this comparative study, the data for the unmatched ST were not analyzed. The questionnaire listed 17 best practices for mentoring behaviors in the areas of professional support, social support, and role modeling (Table 2). The 17 best practices for cooperating teachers used in this study were derived from the work of Alemdağ and Şimşek (2017) and their focus on practicum experiences of pre-service teachers, as well as Russell and Russell (2011) and the implications for more formal mentoring programs. To ensure content validity, the behaviors were vetted by three agricultural education faculty at the University of Florida. The construct used in this study was piloted in 2019 with 15 CTs who had served in that role during the spring 2019 semester. The construct measured the frequency of use of best practices for mentoring and had a Cronbach’s alpha of α =. 82. For the 2021 cohort, this same construct had a Cronbach’s alpha of α = .72 for the CT instrument and α =.94 for the ST instrument. The respondents were asked to rate the frequency of use of the behavior utilizing a frequency scale with response options of (1) always, (2) often, (3) sometimes, (4) rarely, and (5) never. The only variation in the instruments for the CTs and STs was the use of first-person pronouns for CTs. Cooperating teachers self-reported on frequency of use of best practices for mentoring behaviors that they implemented, while STs reported on their observation of best practices mentoring behaviors of their CTs. Researchers analyzed the overall mean scores for CTs and STs for each of the 17 mentoring behaviors. A paired samples t-test was used to analyze the set of matched pairs of data. Using histograms as suggested by Lomax and Hahs-Vaughn (2012), all dependent variables were distributed normally prior to analysis. The matched samples consisted of the student teacher https://doi.org/10.37433/aad.v3i4.261 33 Nesbitt et al. Advancements in Agricultural Development and the cooperating teacher, who worked together collaboratively during a 14-week student teaching spring 2021 internship. Findings Objective 1 Of the seven CT’s surveyed, six responded. Participants had an average rating of 1.52 (SD = .67). This shows that cooperating teachers perceived that they exhibited best practices for mentoring an average between often and always (see Table 1). Of the 17 best practices for mentoring behaviors, cooperating teachers rated themselves significantly lower for two behaviors, i.e., the use of observational data as the basis for feedback sessions (M = 2.17, SD = .75) and encouragement of the student teachers to maintain active memberships in Florida Association of Agricultural Educators (FAAE), National Association of Agricultural Educators (NAAE), and Florida Association for Career and Technical Education (FACTE) (M = 2.83, SD = 1.17) (see Table 2). It is important to note that the standard deviation for encouragement of the student teachers to maintain active memberships in FAAE, NAAE, and FACTE was larger than 1.0, which reflects a broader range of responses from participants. Table 1 Cooperating Teacher and Student Teacher Frequency of Best Practices for Mentoring Behaviors. Respondents n M SD Cooperating Teachers 6 1.52 .67 Student Teachers 6 1.79 .80 Note. The ratings on the scale were (1) always, (2) often, (3) sometimes, (4) rarely, and (5) never. https://doi.org/10.37433/aad.v3i4.261 34 Nesbitt et al. Advancements in Agricultural Development Table 2 Cooperating Teacher’s Frequency Scores for Perceived Demonstration of Best Practices for Mentoring Behaviors. Behavior n M SD Encouraged the student teacher to maintain active memberships in 6 2.83 1.17 FAAE, NAAE, and FACTE Used observational data as the basis for feedback sessions 6 2.17 .75 Communicated openly with my student teacher/intern 6 1.67 .82 Involved my student teacher in all of my roles as a teacher 6 1.67 .52 Discussed strategies for effectively managing time, 6 1.67 .82 priorities/projects, and email Provided weekly comprehensive feedback on performance in an 6 1.50 .55 uninterrupted setting Encouraged the student teacher to take the lead in evaluating their 6 1.50 .84 teaching Made an effort to introduce my student teacher to the school 6 1.50 .55 community Discussed effective student discipline strategies with my student 6 1.50 .55 teacher for maintaining a productive learning environment Shared approaches for effectively managing the administrative aspects of teaching, including building effective relationships 6 1.50 .55 with administrators and other teachers Communicated regularly with my student teacher/intern 6 1.33 .52 Supported my student teacher’s effort by staying attuned to their 6 1.33 .52 mindset, attitude, and well-being Talked to my student teacher about how to become an excellent 6 1.33 .82 teacher through all phases of their career Made an effort to help my student teacher develop positive views 6 1.17 .41 of teaching Coached my student teacher on strategies for developing a positive 6 1.17 .41 rapport with students Shared my approaches for SAE program development and 6 1.00 0 supervision Shared my philosophy for FFA advising 6 1.00 0 Note. The ratings on the scale were (1) always, (2) often, (3) sometimes, (4) rarely, and (5) never. Objective 2 All seven student teachers responded to the survey, but for the purposes of this study and alignment of responses for the paired t analysis, one student teacher was removed from the data set. Respondents had an average rating of 1.79 (SD = .80) for their observation of mentoring best practices. Overall, the frequency observed by student teachers was closest to often (see Table 1). When reviewing individual best practices, there were five that had a mean https://doi.org/10.37433/aad.v3i4.261 35 Nesbitt et al. Advancements in Agricultural Development score between 2.00-4.00 (see Table 3), indicating a frequency between rarely and often. The following behaviors were included in that range of 2-4: shared approaches for SAE program development and supervision; shared philosophy for FFA advising; shared approaches for effectively managing the administrative aspects of teaching, including building effective relationships with administrators and other teachers; encouraged the student teacher to maintain active memberships in FAAE, NAAE, and FACTE; and discussed strategies for effectively managing time, priorities/projects, and email. Three CT behaviors had standard deviations larger than 1.0, indicating a broader range of participant responses: encouragement to maintain active memberships in FAAE, NAAE, and FACTE, sharing approaches for SAE program development, and sharing philosophies for FFA advising. Table 3 Student Teachers’ Frequency Scores for Observed Best Practices of Mentoring Behaviors Demonstrated by their Cooperating Teachers. Behaviors n M SD Encouraged to maintain active memberships in FAAE, NAAE, and FACTE 6 3.83 1.48 Shared their approaches for SAE program development and supervision 6 2.00 1.10 Shared their philosophy for FFA advising 6 2.00 1.27 Shared their approaches for effectively managing the administrative aspects of teaching, including building effective relationships with 6 2.00 .89 administrators and other teachers. Shared strategies for effectively managing time, priorities/projects, and 6 2.00 .63 email. Involved me in all of my roles as a teacher 6 1.83 .75 Talked to me about how to become an excellent teacher through all 6 1.83 .98 phases of their career Discussed effective student discipline strategies with me for maintaining a 6 1.83 .75 productive learning environment Was attuned to my mindset, attitude, and well-being 6 1.67 .41 Encouraged to take the lead in evaluating my own teaching 6 1.67 .41 Made an effort to introduce me to the school community 6 1.67 .82 Coached me on strategies for developing a positive rapport with students 6 1.67 1.21 Communicated regularly with me 6 1.50 .55 Provided weekly comprehensive feedback on performance in an 6 1.50 .55 uninterrupted setting Made an effort to help me develop positive views of teaching 6 1.50 .55 Communicated openly with me 6 1.33 .52 Used observational data as the basis for feedback sessions 6 1.33 .52 Note. The ratings on the scale were (1) always, (2) often, (3) sometimes, (4) rarely, and (5) never. https://doi.org/10.37433/aad.v3i4.261 36 Nesbitt et al. Advancements in Agricultural Development Objective 3 When comparing the perspectives of the cooperating teachers and student teachers, the cooperating teachers had an overall slightly higher frequency for their self-reporting of best practice mentoring behaviors (M = 1.52, SD = .67) than the student teachers who would have received the mentoring and observed these behaviors (M = 1.79, SD = .80) (see Table 1). Only one instance occurred of an overlap in lower frequency behaviors from both groups, indicating agreement that the behavior was not implemented at a high frequency. The behavior at an equally low frequency included the encouragement of the student teacher to maintain active memberships in FAAE, NAAE, and FACTE (see Table 2 & 3). A paired samples t-test was conducted to determine if a difference existed in the mean scores of the cooperating teachers’ frequencies and the mean scores of the paired student teachers. Descriptive statistics in Tables 4 and 5 show missing data for Pair 4. This was due to the same reported responses by cooperating teacher and student teacher for both the self-reported and observed best practices mentoring behaviors. The t-test was run to determine if a significant difference existed within pairs (see Table 6). One paired sample was removed from the data set because of matching responses. The results indicated that the reported frequency for best practice mentoring behaviors were statistically significantly different for Pair 3 (t = -2.135, df = 16, p = .049), and Pair 5 (t = -3.246, df = 16, p = .005). Effect size was calculated as d = .37. Using Cohen’s (1988) guidelines, this is a small effect size. Table 4 Paired Samples Statistics for Cooperating Teachers’ and Student Teachers’ Frequency of Best Practices for Mentoring Behaviors. M n SD SE Pair 1 CT1 1.41 17 .51 .12 ST1 1.88 17 .99 .24 Pair 2 CT2 1.77 17 .66 .16 ST2 1.70 17 .99 .24 Pair 3 CT3 1.24 17 .56 .14 ST3 1.65 17 .86 .21 Pair 4 CT4 1.41 17 .51 .12 ST4 1.00 17 .00 .00 Pair 5 CT5 1.35 17 .61 .15 ST5 2.41 17 1.23 .30 Pair 6 CT6 1.94 17 1.14 .20 ST6 2.00 17 .75 .18 Note. CT = Cooperating Teacher; ST = Student Teacher https://doi.org/10.37433/aad.v3i4.261 37 Nesbitt et al. Advancements in Agricultural Development Table 5 Paired Samples Correlations for Cooperating Teachers’ and Student Teachers’ Frequency of Best Practice Mentoring Behaviors n Correlation P Pair 1 CT1 & ST1 17 .23 .38 Pair 2 CT2 & ST2 17 .56 .02 Pair 3 CT3 & ST3 17 .44 .08 Pair 4 CT4 & ST4 17 . . Pair 5 CT5 & ST5 17 .04 .87 Pair 6 CT6 & ST6 17 .44 .08 Note. CT = Cooperating Teacher; ST = Student Teacher Table 6 Paired Samples t-Test for Cooperating Teachers’ & Student Teachers’ Frequency of Best Practice Mentoring Behaviors Paired Differences Sig. (295% Confidence Interval t df tailed) M SD SE Lower Upper CT1 Pair 1 -.47 1.00 .24 .99 .05 -1.93 16 .07 ST1 CT2 Pair 2 .06 .83 .20 -.37 .48 .29 16 .77 ST2 CT3 Pair 3 -.41 .80 .19 -.82 .00 -2.13 16 .05 ST3 CT5 Pair 5 -1.06 1.34 .33 -1.75 -.37 -3.25 16 .01 ST5 CT6 Pair 6 -.12 1.05 .26 -.66 .42 -.46 16 .65 ST6 Note. CT = Cooperating Teacher; ST = Student Teacher. Conclusions, Discussion, and Recommendations This study begins to provide a better understanding into the mentor-mentee relationship between the cooperating teachers and student teachers. First, the cooperating teachers felt successful in their implementation of the behaviors after receiving training prior to the mentor experience as supported by the constructivist foundation of the study (Vijaya Kumari, 2014; Vygotsky, 1978). However, the cooperating teachers’ self-reported data could potentially be explained by their desirability to look successful when completing the survey questionnaire (i.e., SDB) (Beretvas et al., 2002). Based on literature about self-reported data, regarding the mentoring they received, student teachers’ perceptions may provide a more accurate representation of what has occurred (Beretvas et al., 2002; Goneya, 2005). https://doi.org/10.37433/aad.v3i4.261 38 Nesbitt et al. Advancements in Agricultural Development Our recommendation for cooperating teachers is to self-assess their use of the 17 best practice behaviors before, during, and after each experience with a student teacher. Cooperating teachers should ideally incorporate best practices at a high frequency when mentoring student teachers, striving to make the time for formal observations, as well as regular feedback and reflection time with their mentees. We recommend that the university faculty who partner with cooperating teachers take the time to assess potential gaps in the knowledge and understanding of best practices for mentoring student teachers. Once identified, these gaps could help provide teacher educators with guidance on how to adjust professional development to meet the needs for the preparation and support of cooperating teachers. We recommend that similar studies be conducted with a larger sample size and recognize the findings from this study are not generalizable due to the small sample size and lack of random selection of participants. The needs of the cooperating teachers may vary according to current practices being implemented as well as the experiences of the cooperating teachers. It would be helpful to partner with other institutions to implement similar professional development and support measures to gain a richer perspective. In addition to collecting survey data, we recommend the use of observational data to gain a more robust view of the mentoring experience from both perspectives. Exploring the perspectives of cooperating teachers through interviews would help researchers obtain additional insight into how teachers implement mentorship best practices. Gaining a deeper understanding on ways that cooperating teachers are actively mentoring student teachers could help researchers clarify best practices for future cooperating teachers. We are currently conducting interviews to better grasp how cooperating teachers specifically implement best practices for mentoring student teachers during the internship experience. Additional research should be conducted to explore additional ways to support the implementation of best practices that are being utilized less in the student teaching experience. Furthermore, research regarding the differences between cooperating teachers’ and student teachers’ perceptions is needed to better understand why student teachers observed less practices than the cooperating teachers perceived they used. This type of research could also provide insight on potential needs to modify current mentoring models in teacher education. Young & MacPhail (2005) stressed the importance of preparation and support of cooperating teachers, and results from this exploratory study may provide some guidance for teacher educators toward their needs regarding student teacher mentorship. Acknowledgements We would like to thank the Florida cooperating teachers and student teachers who participated in our study. Author Contributions: H. Nesbitt - analysis, writing-review & editing; D. Barry investigation, analysis, writing-original draft, review & editing; K. Lawson - writing-review and editing; J. 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https://openalex.org/W4393958174	https://jcs.greenpublisher.id/index.php/jcs/article/download/1/1	Indonesian	null	Peran Sikap Optimis Dan Dukungan Sosial Pada Subjective Well-Being Di Masa Pandemic Covid-19	Journal of Comprehensive Science	2,022	cc-by-sa	3,744	Keywords: Subjective Well- being, Optimism, Social Support, Covid-19 ABSTRACT This study aims to determine the role that influences Subjective Well-being during a pandemic through optimistic attitudes and social support factors. This research is qualitative with a library study approach. The data used by the researcher are secondary data that are magazines related to optimistic attitudes and social support for Subjective Well-being. The researchers analyzed several journals classifying them correctly for data collection so that they were new to this study. The results found by the researchers in the form of subjective well-being have supporting factors to face the pandemic period that hit various fields, both economic and social. This shows that an optimistic attitude must be presented to strengthen the body's resistance during a pandemic that hits a person's psyche and mentality. In addition, social support allows someone to get up and find temporary solutions during a pandemic with various activities to explore hobbies that generate income. ABSTRAK Kata Kunci: Subjective Well- Being, Optimis, Dukungan Sosial, Covid-19 , Penelitian ini bertujuan untuk mengetahui peran yang berdampak pada Subjective Well-Being di masa pandemic melalui factor sikap optimis dan dukungan sosial. Penelitian ini berjenis kualitatif dengan pendekatan studi Pustaka. Data yang di gunakan oleh peneliti merupakan data sekunder yang merupakan jurnal-jurnal yang berkaitan dengan sikap optimis dan dukungan sosial terhadap Subjective Well-Being. Peneliti menganalisis beberapa jurnal dengan menyortir dengan tepat untuk pengambilan data agar menjadi kebaharuan didalam penelitian ini. Hasil yang ditemukan oleh peneliti berupa kesejahteraan subjektif memiliki faktor pendukung untuk menghadapi masa pandemic yang menerjang berbeagai bidang baik ekonomi maupun sosial. Hal tersebut membuktikan bahwa sikap optimis harus dihadirkan untuk memperkuat ketahan tubuh saat pandemic yang menerpa psikis maupun mental seseorang. Selain itu dukungan sosial menjadikan seseorang untuk bangkit dan mencari solusi sementara saat pandemic dengan berbagai kegiatan untuk mengeksplorasi hobi yang menghasilkan pendapatan. PERAN SIKAP OPTIMIS DAN DUKUNGAN SOSIAL PADA SUBJECTIVE WELL-BEING DI MASA PANDEMIC COVID-19 Fizi Fauziya STKIP Asy-Syafi’iyah Internasional Medan email: fizifauziya@stembi.ac.id PENDAHULUAN Kesejahteraan subjektif atau bisa disebut dengan Subjective Well-Being adalah evaluasi yang dilakukan individu atas hidupnya sendiri, baik secara afektif maupun kognitif (Sari dan Maryatmi, 2019). Terkait kesejahteraan subjektif, di Indonesia, beberapa pekerjaan berpotensi menurunkan tingkat kesejahteraan subjektif seseorang, bahkan di masa pandemi saat ini. Masa pandemi terjadi karena ada virus corona yang sedang mewabah dan pemerintah membuat kebijakan untuk menekan penyebaran virus tersebut, sehingga dilakukan PSBB. Pandemi penyakit coronavirus 2019 (Covid-19) sedang dialami oleh beberapa negara di dunia (WHO, 2020). Upaya preventif terus dikampanyekan untuk meminimalisir risiko penyebaran Covid-19, salah satunya physical distancing (WHO, 2020). Aturan tersebut telah mengganggu beberapa rutinitas sehari-hari, salah satunya adalah pandemi coronavirus disease 2019 (Covid-19) yang dialami beberapa negara di dunia (WHO, 2020). Kegiatan pembatasan orang untuk bekerja ini dapat memicu peningkatan angka pengangguran. Pengangguran adalah orang yang tidak melakukan kegiatan yang menghasilkan uang. Pengangguran tidak hanya terbatas pada orang yang belum bekerja, 1 Volume 1, No 1, Agustus 2022, Page: 1-8 Volume 1, No 1, Agustus 2022, Page: 1-8 tetapi orang yang sedang mencari pekerjaan dan orang yang bekerja tetapi pekerjaannya tidak produktif dapat digolongkan sebagai pengangguran (Sukino, 2000). Termasuk masyarakat yang berdiam diri di rumah, semuanya untuk menghentikan penyebaran virus covid-19 ini (Kasnelly, 2020). y Masa pandemi Covid-19 juga melanda semua bidang, termasuk pengurangan pekerjaan yang dilakukan oleh perusahaan, organisasi dan instansi (Sunarti, 2021). Kurangnya pekerjaan menciptakan stres bagi tanggungan karena dampak ekonomi yang kuat, sehingga menciptakan masalah baru seperti pengangguran. Melihat semakin banyaknya PHK dan pengangguran, maka akan sering mempengaruhi berbagai sektor kehidupan di masyarakat, salah satunya adalah tingkat kesejahteraan subjektif yang bisa disebut dengan Subjective Well-Being. Salah satu faktor yang dapat meningkatkan kesejahteraan subjektif yang bisa disebut dengan Subjective Well-Being adalah optimisme. Optimisme adalah sifat berbasis pemikiran dari seorang individu yang dapat mengarah untuk bergerak maju dengan mempertahankan pola pikir yang dapat dikaitkan dengan kesuksesan seseorang dalam hidup. Oleh karena itu, orang yang optimis dapat mencapai kesuksesan dalam sebagian besar aktivitasnya (Veronica, 2021). Selain optimisme, faktor yang dapat meningkatkan kesejahteraan subjektif atau Subjective Well-Being adalah dukungan sosial. Dukungan sosial adalah informasi yang mengarah pada keyakinan bahwa seseorang dicintai, dihargai, dan termasuk dalam kewajiban bersama. Chunkai, Shan, & Xinwen, (2019) mendefinisikan dukungan sosial dengan mengambil salah satu jenis dukungan sosial yang paling berpengaruh yaitu dukungan sosial keluarga dan memberikan pengertian bahwa dukungan sosial adalah konsep multidimensi yang mencakup dukungan dari keluarga yang diterima secara subyektif dan praktis. PENDAHULUAN Karena telah terbukti bahwa optimis dan dukungan sosial merupakan pengaruh utama terhadap peningkatan kesejahteraan subjektif yang bisa disebut dengan Subjective Well-Being. Penelitian ini menganalisis beberapa penelitian yang nantinya mengambil aspek optimis dan dukungan sosial yang akan dianalisis pengaruhnya terhadap kesejahteraan subjektif yang bisa disebut dengan Subjective Well-Being dan ini merupakan salah satu hal baru dari penelitian ini. HASIL DAN PEMBAHASAN Penelitian yang dilakukan oleh Dewi dan Rahayu (2020) yang berjudul Optimisme dan keberfungsian keluarga hubungannya dengan Subjective Well-Being pekerja perempuan yang Work From Home di Kecamatan Tambun Utara Kabupaten Bekasi. Hasil penelitian menunjukan bahwa Analisis data menggunakan multivariate correlation antara variabel optimisme dan keberfungsian keluarga dengan subjective well-being diperoleh R square = 0,075 dengan nilai p = 0,002 (p < 0,05), artinya ada hubungan signifikan dengan arah positif antara variabel optimisme dan keberfungsian keluarga dengan Subjective Well- Being perempuan pekerja yang bekerja secara Work From Home di Kecamatan Tambun Utara Kabupaten Bekasi. Penelitian yang dilakukan oleh Dewanti dan Ayriza (2021) yang berjudul Pengaruh Optimisme Terhadap Kesejahteraan Subjektif atau Subjective Well-Being pada Mahasiswa yang Mengerjakan Tugas Akhir. Hasil penelitian menunjukkan bahwa terdapat pengaruh optimisme yang positif dan signifikan terhadap kesejahteraan subjektif atau Subjective Well-Being (0,000 <0,05) dengan garis regresi yang didapatkan yakni Y = 33,631 + 0,901X. Dengan demikian dapat dikatakan bahwa optimism mampu memprediksi kesejahteraan subjektif atau Subjective Well-Being individu yang mengerjakan tugas akhir. Penelitian yang dilakukan oleh Irianti (2020) yang berjudul Gambaran Optimisme Dan Kesejahteraan Subjektif Pada Ibu Tunggal di Usia Dewasa Madya. Hasil penelitian ini menunjukkan gambaran optimisme dan kesejahteraan subjektif atau Subjective Well-Being pada ibu tunggal paruh baya, dimana keempat subjek mampu bersyukur atas kehidupan yang mereka jalani sambil berfikir positif terhadap apapun yang akan terjadi. Hal tersebut dapat dicapai karena (1) dukungan dari masyarakat sekitar para ibu tunggal atau tetangganya. (2) Rutinitas atau aktivitas sehari-hari yang sangat mempengaruhi kondisi emosional subjek. (3) Anak-anak mereka yang menjadi sumber kebanggaan dan prestasi bagi para ibu tunggal. (4) Penghasilan atau pekerjaan tidak menjadi faktor utama kesejahteraan subjek atau Subjective Well-Being. Penelitian yang dilakukan oleh Pratiwi (2019) yang berjudul Peranan Optimisme Terhadap Kesejahteraan Subjektif pada Mahasiswa yang Sedang Mengerjakan Skripsi Serta Tinjauannya dalam Islam. Hasil yang didapatkan menunjukkan bahwa terdapat peran signifikan optimisme terhadap kesejahteraan subjektif mahasiswa yang mengerjakan skripsi (R2 = 0,071; p < 0,05). Optimisme yang baik akan membuat mahasiswa memiliki kesejahteraan subjektif atau Subjective Well-Being yang baik dalam dirinya. Hal ini sesuai dengan pandangan Islam bahwa optimisme dapat meningkatkan kesejahteraan subjektif atau Subjective Well-Being mahasiswa. Penelitian yang dilakukan oleh Situmorang (2018) yang berjudul Kesejahteraan Subjektif Perempuan Pemimpin Ditinjau dari Peran Optimisme dan Efikasi Diri. Hasil analisis regresi ganda menunjukkan adanya korelasi positif antara optimism dan efikasi diri terhadap Kesejahteraan subjektif atau Subjective Well-Being perempuan pemimpin. METODE PENELITIAN Penelitian ini ber jenis kualitatif-fenomenologi. Tahap terakhir dari penelitian ini adalah mengungkap atau menemukan makna dari apa yang ditemukan (Soeherman, 2019, pp. 12-13). Penelitian ini dalam mendapatkan data dengan melakukan studi pustaka yang bertujuan mendapatkan informasi berkaitan dengan Optimis dan Dukungan sosial terhadap Subjective Well-Being. Penelitian ini dirancang akan memerlukan beberapa tahapan sebagai berikut: g 1. Persiapan Peneliti melakukan studi pendahuluan untuk mengumpulkan informasi yang menguatkan tujuan penelitian ini. g j p 2. Pelaksanaan Selanjutnya peneliti menentukan penelitian-penelitian yang meneliti tentang Optimis dan Dukungan sosial terhadap Subjective Well-Being. 3. Analisis Data Peneliti menganalisis berupa penelitian yang berkiatan dengan Optimis dan Dukungan sosial terhadap Subjective Well-Being. Peneliti menganalisis berupa penelitian yang berkiatan dengan Optimis dan Dukungan sosial terhadap Subjective Well-Being. ukungan sosial terhadap Subjective Well-Being. 4. Penarikan Kesimpulan 2 Setelah peneliti menganalisis penelitian-penelitian yang berkaitan dengan Optimis dan Dukungan sosial terhadap Subjective Well-Being kemudian disimpulkan untuk mencapai tujuan penelitian. Volume 1, No 1, Agustus 2022, Page: 1-8 Volume 1, No 1, Agustus 2022, Page: 1-8 Volume 1, No 1, Agustus 2022, Page: 1-8 kesejahteraan subjektif atau Subjective Well-Being pada pekerja kontrak atau PKWT (Perjanjian Kerja Waktu Tertentu) berkaitan dengan internalisasi nilai religius dan budaya, yang melandasi keyakinan positif dan optimisme bahwa bekerja keras akan menghasilkan kesejahteraan dan ketercukupan, walaupun belum tercapai. Afek positif yang mendukung daya tahan pekerja kontrak antara lain karena kuatnya optimisme, adanya dukungan keluarga dan relasi positif dengan sesama pekerja. Sedangkan faktor penghambat pencapaian kesejahteraan subjektif atau Subjective Well-Being pada pekerja kontrak yaitu afek negatif (ketidakberdayaan, ketidakpastian dan rasa tidak aman) yang berkaitan dengan status kerjanya. Dengan demikian, peningkatan status kerja menjadi diperlukan untuk menghilangkan ketidakpastian, dan mendorong pencapaian kesejahteraan subjektif atau Subjective Well-Being pekerja. Penelitian yang dilakukan oleh Zalki dkk (2021) yang berjudul Optimisme Dan Kesejahteraan Subjektif Pada Pengangguran Dewasa Muda. Hasil analisis korelasi antara optimisme dan pengaruh menyenangkan menunjukkan bahwa p-value = 0,000 (p<0,05) dan r = 0,516 yang berarti terdapat hubungan positif yang signifikan. Optimisme dan afek tidak menyenangkan menunjukkan bahwa p-value = 0,000 (p<0,05) dan r = -0,559 yang berarti terdapat hubungan negatif yang signifikan. Optimisme dan kepuasan hidup menunjukkan bahwa p-value = 0,000 (p<0,05) dan r = 0,457 yang berarti terdapat hubungan positif yang signifikan. Penelitian yang dilakukan oleh Hartono & Soeharto (2021) yang berjudul Review Literatur: Prediktor Kesejahteraan Subyektif Pada Perawat Di Tinjau Dari Teori Bottom- Up Dan Top-Down Pada Masa Global Pandemi Corona Virus. Hasil penelitian menunjukan bahwa prediktor kesejahteraan subyektif atau Subjective Well-Being yang di jabarkan dalam dua pendekatan teori pendekatan teori top-down dan teori bottom-up memiliki Pengaruh baik langsung maupun tidak langsung terhadap kesejahteraan subyektif pada perawat. Beberapa prediktor dalam dua pendekatan teori tersebut telah dibuktikan berkorelasi dengan kesejahteraan subyektif atau Subjective Well-Being pada perawat. Prediktor tersebut antara lain; Job Satisfaction and Emotional Subjective Well-Being, Faktor demografis dan faktor pekerjaan, optimisme, dukungan sosial, Self-Efficacy and Hope, persepsi dukungan organisasi dan harga diri, regulasi emosi dan stress, strategi koping, dan spiritual leadership. Penelitian yang dilakukan oleh Wahyuni (2018) yang berjudul Pengaruh dukungan sosial dan religiusitas terhadap Subjective Well-Being pada pensiunan PNS. Hasil penelitian menggunakan analisis regresi berganda menunjukan bahwa seluruh variabel bebas yang digunakan berpenngaruh signifikan terhadap Subjective Well-Being dengan proporsi varian sebesar 50,6%, sedangkan sisanya 49,4% dipengaruhi oleh variabel lain diluar penelitian. HASIL DAN PEMBAHASAN Sumbangan efektif variable Optimism dan efikasi diri Kesejahteraan subjektifperempuan pemimpin.adalah R2= 33.30%, dengan demikian kontribusi optimism dan efikasi diri terhadap Kesejahteraan subjektif atau Subjective Well-Being perempuan pemimpin sebesar 33.30 %. Penelitian yang dilakukan oleh Sengkey dan Tiwa (2020) yang berjudul Kesejahteraan Subjektif Pekerja Kontrak (PKWT). Hasil penelitian menunjukan bahwa 3 Volume 1, No 1, Agustus 2022, Page: 1-8 Volume 1, No 1, Agustus 2022, Page: 1-8 Sementara, hasil analisis masing-masing variabel secara terpisah menunjukan bahwa variabel dukungan emosional, dukungan informasional, dukungan instrumental, dukungan persahabatan dan praktek agama berpengaruh signifikan terhadap Subjective Well-Being pada pensiunan PNS. Sedangkan empat variabel keyakinan agama, konsekuensi agama, pengalaman agama dan pengetahuan agama tidak signifikan berpengaruh terhadap Subjective Well-Being pada pensiunan PNS. Penelitian yang dilakukan oleh Thohiroh (2019) yang berjudul Peranan persepsi dukungan sosial terhadap kesejahteraan subjektif di sekolah pada siswa Pondok Pesantren Modern. Hasil penelitian menunjukkan bahwa persepsi dukungan sosial dari guru dan teman berperan secara signifikan terhadap kesejahteraan subjektif atau Subjective Well- Being di sekolah, sementara persepsi dukungan sosial dari orang tua tidak berhubungan signifikan. Persepsi dukungan sosial teman berhubungan signifikan dengan dimensi school satisfaction dan affect in school; persepsi dukungan sosial dari guru berhubungan signifikan dengan school satisfaction tetapi tidak dengan affect in school. 4 Penelitian yang dilakukan oleh Azhima dan Indrawati (2018) yang berjudul Hubungan Antara Dukungan Sosial Keluarga Dengan Subjective Well-Being Pada Narapidna Perempuan Di Lembaga Pemasyarakatan "X". hasil penelitian menunjukan bahwa Uji hipotesis dengan menggunakan analisis regresi sederhana menunjukkan koefisien korelasi sebesar 0,661 dengan signifikansi 0,000 (p < 0,05) yang berarti terdapat hubungan positif yang signifikan antara dukungan sosial keluarga dengan kesejahteraan subjektif atau Subjective Well-Being. Semakin positif dukungan sosial keluarga yang dirasakan, semakin besar kesejahteraan subjektif perempuan yang dirampas kebebasannya di Lembaga Pemasyarakatan “X”. Dukungan sosial keluarga memberikan kontribusi yang efektif yaitu R squared = 0,437 atau 43,7% terhadap kesejahteraan subjektif atau Subjective Well-Being. Sebanyak 61,7% subjek dalam penelitian ini merasakan dukungan sosial keluarga yang positif dan 81,7% memiliki kesejahteraan subjektif atau Subjective Well- Being yang tinggi. g y g gg Penelitian yang dilakukan oleh Wijayanti (2020) yang berjudul Peran dukungan sosial dan interaksi ibu-anak dalam meningkatkan kesejahteraan subjektif remaja pada keluarga orang tua bekerja. Hasil uji PLS menunjukkan bahwa dukungan sosial dan interaksi ibu-anak berpengaruh langsung terhadap kesejahteraan subjektif atau Subjective Well-Being remaja. Untuk itu orang tua hendaknya menyiapkan dukungan sosial yang sesuai bagi remaja dan membangun interaksi yang baik dengan anak diantara kesibukannya bekerja. Penelitian yang dilakukan oleh Fajar dan Lestari (2020) yang berjudul Peran Dukungan Sosial Dan Harga Diri Terhadap Kesejahteraan Subjektif Penyandang Disabilitas Fisik. Hasil analisis data diperoleh nilai koefisien korelasi R= 0.694 dan signifikansi 0,00 (p< 0,05) artinya ada hubungan yang sangat signifikan antara dukungan sosial, harga diri, dengan kesejahteraan subjektif atau Subjective Well-Being pada penyandang disabilitas fisik. Volume 1, No 1, Agustus 2022, Page: 1-8 Volume 1, No 1, Agustus 2022, Page: 1-8 Volume 1, No 1, Agustus 2022, Page: 1-8 Irianti (2020); Pratiwi (2019); Situmorang (2018); Zalki dkk (2021) dan Hartono & Soeharto (2021). Hasil tersebut menunjukan saat mulai bekerja dan mencari pekerjaan selama situasi pandemi, orang mulai beradaptasi, kemudian keputusasaan mulai mereda dan optimisme meningkat, sehingga minat pada pekerjaan mereka. Walaupun banyak literature yang menyatakan bahwa terdapat pengaruh signifikan optimis terhadap Subjective Well-Being terdapat juga temuan sebaliknya, Lai (2015); Lui dkk, (2016) dan Sengkey dan Tiwa (2020) menemukan bahwa optimis tidak berpengaruh signifikan terhadap Subjective Well-Being. Kesejahteraan subjektif sebelum masa pandemi dapat menunjukkan adanya tekanan di tempat kerja dan kurangnya waktu bersama keluarga, yang menyebabkan optimisme lebih besar, namun setelah masa pandemic yang diterjemahkan ke dalam pengurangan karyawan dan bekerja di rumah perasaan terkejut, cemas, ragu, kurang minat dan diterjemahkan ke dalam pengurangan optimisme. g g j p g g p Beberapa penelitian terdahulu menemukan bahwa dukungan sosial mampu berpengaruh signifikan terhadap subjective well-being seperti penelitian Wahyuni (2018); Azhima dan Indrawati (2018); Wijayanti (2020); Fajar dan Lestari (2020); Asirwada dan Utami (2022); Adzhani dkk (2020) dan Zahra (2021). Di sisi lain, dukungan sosial membuat masyarakat merasa bahwa selama masa pandemi ini, mereka memiliki pengalaman bekerja dengan perasaan bahagia dan sukses dengan menyalurkan hobi dan menghabiskan waktu bersama keluarga dan berinteraksi satu sama lain, yang pada akhirnya merasakan kepuasan hidup. Berbeda dengan beberapa penelitian terdahulu yang telah disebutkan dalam penelitian Thohiroh (2019) dan Gülaçti, (2010) menemukan bahwa dukungan sosial dari teman dan orang-orang spesial tidak dapat berpengaruh signifikan terhadap kesejahteraan subjektif yang bisa disebut dengan Subjective Well-Being. Kesejahteraan yang diperkirakan akan menurun akibat kurangnya dukungan sosial akibat pandemi akan menurunkan kepuasan karena merasa sia-sia dengan hidupnya dan pasrah dengan masa baktinya, runtuhnya cita-citanya yang tidak sesuai dengan yang diharapkan, memiliki tujuan yang berubah selama pandemi dan dukungan sosial yang tidak memadai untuk apa yang dibutuhkan. Selanjutnya, ketika mereka menganggur, mereka malu dengan hasil pekerjaannya dan tertekan oleh kenaikan biaya hidup dan pendapatan yang menurun. Volume 1, No 1, Agustus 2022, Page: 1-8 Sumbangan efektif untuk hubungan antara dukungan sosial dan harga diri dengan kesejahteraan subjektif atau Subjective Well-Being, berdasarkan koefisien R2 = 48,1 %. Berdasarkan hasil kategorisasi tingkat dukungan sosial, harga diri dan kesejahteraan subjektif atau Subjective Well-Being tergolong sedang. Penelitian yang dilakukan oleh Asirwada dan Utami (2022) yang berjudul Peran Harga Diri Dan Dukungan Sosial Terhadap Kesejahteraan Subjektif Pada Remaja. Hasil penelitian menunjukkan bahwa harga diri dan dukungan sosial memberikan peran sebesar 69,1% terhadap kesejahteraan subjektif atau Subjective Well-Being pada remaja. Penelitian yang dilakukan oleh Adzhani dkk (2020). Persepsi dukungan sosial sebagai mediator pengungkapan diri dan kesejahteraan subjektif pada pengguna instagram. Hasil penelitian menunjukkan bahwa: 1) pengungkapan diri berpengaruh secara signifikan terhadap kesejahteraan subjektif atau Subjective Well-Being, 2) pengungkapan diri berpengaruh secara signifikan terhadap persepsi dukungan sosial, 3) persepsi dukungan sosial berpengaruh secara signifikan terhadap kesejahteraan subjektif atau Subjective Well- Being, 4) persepsi dukungan sosial secara signifikan menjadi variabel mediator bagi peran pengungkapan diri terhadap kesejahteraan subjektif atau Subjective Well-Being. Penelitian yang dilakukan oleh Zahra (2021) yang berjudul Peran Dukungan Sosial Terhadap Kesejahteraan Subjektif Mahasiswa Dalam Masa Pandemi Covid-19. Hasil uji statistik Pearson Correlation menunjukkan bahwa dukungan sosial memiliki hubungan positif yang kuat dan signifikan dengan kesejahteraan subjektif atau Subjective Well- Being. Semakin besar dukungan sosial, maka semakin besar kesejahteraan subjektif. Berdasarkan hasil analisis penelitian diatas menunjukan bahwa Hubungan antara optimis terhadap Subjective Well-Being juga dibahas oleh beberapa penelitian terdahulu bahwa optimis mampu memberikan pengaruh terhadap Subjective Well-Being seperti penelitian yang dilakukan oleh Dewi dan Rahayu (2020); Dewanti dan Ayriza (2021); 5 Volume 1, No 1, Agustus 2022, Page: 1-8 BIBLIOGRAFI Adzhani, S. N., Baihaqi, M. I. F., & Kosasih, E. (2020). Persepsi dukungan sosial sebagai mediator pengungkapan diri dan kesejahteraan subjektif pada pengguna instagram. MEDIAPSI, 6(1), 60-70. Asirwada, Ikana Naifah Tahara dan Utami, Muhana Sofiati (2022). Peran Harga Diri Dan Dukungan Sosial Terhadap Kesejahteraan Subjektif Pada Remaja (Doctoral dissertation, Universitas Gadjah Mada). j Azhima, D. D., & Indrawati, E. S. (2018). Hubungan Antara Dukungan Sosial Keluarga Dengan Subjective Well-Being Pada Narapidna Perempuan Di Lembaga Pemasyarakatan" X" (Doctoral dissertation, Undip). Darusmin, D. F., & Himam, F. (2015). Subjective well being pada hakim yang bertugas di daerah terpencil. Jurnal Psikologi UGM, 1(3), 196618. Dewanti, A. D. P., & Ayriza, Y. (2021). Pengaruh Optimisme Terhadap Kesejahteraan Subjektif pada Mahasiswa yang Mengerjakan Tugas Akhir. Acta Psychologia, 3(2), 119-126. Dewi, A. K., & Rahayu, A. (2020). Optimisme dan keberfungsian keluarga hubungannya dengan subjective well-being pekerja perempuan yang work from home di Kecamatan Tambun Utara Kabupaten Bekasi. IKRA-ITH HUMANIORA: Jurnal Sosial dan Humaniora, 4(3), 1-8. Fajar, M., Lestari, R., & Psi, S. (2020). Peran Dukungan Sosial Dan Harga Diri Terhadap Kesejahteraan Subjektif Penyandang Disabilitas Fisik (Doctoral dissertation, Universitas Muhammadiyah Surakarta). Gülaçti, F. (2010). The effect of perceived social support on subjective well-being. Procedia Social and Behavioral Sciences, 2, 3844–3849. Hartono, S., & Soeharto, T. N. E. D. (2021). Review Literatur: Prediktor Kesejahteraan Subyektif Pada Perawat Di Tinjau Dari Teori Bottom-Up Dan Top-Down Pada Masa Global Pandemi Corona Virus. Mempersiapkan Generasi Digital Yang Berwatak Sociopreneur: Kreatif, Inisiatif, dan Peduli di Era Society 5.0. Irianti, S. (2020). Gambaran Optimisme Dan Kesejahteraan Subjektif Pada Ibu Tunggal di Usia Dewasa Madya. Psikoborneo: Jurnal Ilmiah Psikologi, 8(1), 107-116. Karni, A. (2018). Subjective well-being pada lansia. Jurnal Ilmiah Syi'ar, 18(2), 84-102. Kasnelly, F. A. J. S. (2020). Meningkatnya Angka Pengangguran Ditengah Pandemi (Covid-19). Al-Mizan: Jurnal Ekonomi Syariah, 3(1), 45-60. Lai, L. C.-H. (2015). Buddhism and Subjective Wellbeing: Do Self-Esteem, Optimism and Perceived Control Play a Role? Journal of Social Sciences, 3, 1-7. Lui, P., Rollock, D., Chang, E., Leong, F., & Zamboanga, B. (2016). Big 5 Personality and Subjective Well-Being in Asian Americans: Testing Optimism and Pessimism as Mediators. Asian American Journal of Psychology, 7(4), 274–286. Mujamiasih, M. (2013). Subjective well-being (SWB): Studi indigenous karyawan bersuku Jawa. Journal of Social and Industrial Psychology, 2(2). Ola`h. (2002). Positive traits: Flow and psychological immunity. Washington: Paper presented at the First. Panggagas, F. A. W., Imawati, D., & Sari, M. T. (2020). KESIMPULAN Pekerja yang memiliki pekerjaan dan mereka yang kehilangan pekerjaan selama pandemi merasa puas dengan kesejahteraan subjektif mereka saat ini, meskipun mereka masih harus menghadapi masalah ekonomi. Pasalnya, masyarakat saat ini harus menerapkan strategi agar mampu mengambil pelajaran dari peristiwa yang dialami adalah tetap optimis, berusaha mengendalikan diri, sabar dan mampu memahami kebersihan demi Kesehatan sekitar. Selain itu, dukungan sosial terutama dukungan keluarga membuat mereka meningkatkan strategi untuk tetap hidup agar sejahtera di masa pandemi. Berbagai faktor diduga mempengaruhi kesejahteraan subjektif, yaitu faktor agama seperti keyakinan akan adanya surga dan keyakinan akan takdir, serta insentif yang mempengaruhi kesejahteraan subjektif, seperti gaji. Selain itu, latar belakang pendidikan juga diyakini mempengaruhi kesejahteraan subjektif, seperti perasaan nyaman, senang, dan menikmati pekerjaan. Bagi mereka yang tidak memiliki pekerjaan, ada bantuan pemerintah yang dapat memperpanjang kesejahteraan subjektif sementara untuk mengeksplorasi hobi yang menghasilkan pendapatan. 6 6 Volume 1, No 1, Agustus 2022, Page: 1-8 Sari, F. I. P., & Maryatmi, A. S. (2019). Hubungan antara konsep diri (dimensi internal) dan optimisme dengan subjective well-being siswa sma marsudirini bekasi. IKRA- ITH humaniora: Jurnal Sosial Dan Humaniora, 3(1), 23-29. Seligman, M.E.P. (1991). Learned optimism. New York : A.A knopt. Inc. Sengkey, M. M., & Tiwa, T. M. (2020). Kesejahteraan Subjektif Pekerja Kontrak (PKWT). Seurune: Jurnal Psikologi Unsyiah, 3(2), 1-21. Situmorang, N. Z. (2018). Kesejahteraan Subjektif Perempuan Pemimpin Ditinjau dari Peran Optimisme dan Efikasi Diri. Fakultas Psikologi Universitas Ahmad Dahlan. Subadi, T. (2006). Metode penelitian kualitatif. Surakarta: Muhammadiyah University Pers Sunarti, E. (2021). Ketahanan Keluarga Indonesia di Masa Pandemi Covid 19. PT Penerbit Subadi, T. (2006). Metode penelitian kualitatif. Surakarta: Muhammadiyah University Pers Subadi, T. (2006). Metode penelitian kualitatif. Surakarta: Muhammadiyah University Pers Sunarti, E. (2021). Ketahanan Keluarga Indonesia di Masa Pandemi Covid 19. PT Penerbit IPB Press ( ) p y y Sunarti, E. (2021). Ketahanan Keluarga Indonesia di Masa Pandemi Covid 19. PT Penerbit IPB Press. Thohiroh, H., Novianti, L. E., & Yudiana, W. (2019). Peranan persepsi dukungan sosial terhadap kesejahteraan subjektif di sekolah pada siswa Pondok Pesantren Modern. Psympathic: Jurnal Ilmiah Psikologi, 6(2), 131-144. Veronica, D. (2021). Pengaruh Layanan Konseling Kelompok Pendekatan Cognitive Behavioral Therapy Terhadap Sikap Optimis Di Era Pandemi Covid-19 Pada Siswa Kelas Xi-Sos 1 Di Sma Negeri 1 Kabanjahe Ta 2020/2021 (Doctoral dissertation, Universitas Negeri Medan). Wahyuni, Z. I. (2018). Pengaruh dukungan sosial dan religiusitas terhadap subjective well- being pada pensiunan PNS (Bachelor's thesis, Fakultas Psikologi UIN Syarif Hidayatullah Jakarta). y WHO. (2020). Coronavirus disease (COVID-19) pandemic. Wijayanti, R., Sunarti, E., & Krisnatuti, D. (2020). Peran dukungan sosial dan interaksi ibu-anak dalam meningkatkan kesejahteraan subjektif remaja pada keluarga orang tua bekerja. Jurnal Ilmu Keluarga & Konsumen, 13(2), 125-136. Zahra, H. A. (2021). Peran Dukungan Sosial Terhadap Kesejahteraan Subjektifpe Mahasiswa Dalam Masa Pandemi Covid-19 (Doctoral dissertation, Universitas Gadjah Mada). Zalki, R., Juniarly, A., & Fikri, M. Z. (2021). Optimisme Dan Kesejahteraan Subjektif Pada Pengangguran Dewasa Muda (Doctoral dissertation, Sriwijaya University). Zimmerman, S. L. (1999). Self-Esteem, Personal Control, Optimism, Extraversion, and the Subjective Well-Being of Midwestern University Faculty. USA: Dissertations Graduate Research Andrews University This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License. BIBLIOGRAFI Pengaruh gratitude terhadap subjective well-being pada mahasiswa kuliah dan bekerja di Universitas 17 Agustus 1945 Samarinda. Motivasi, 7(1), 30-40. Pratiwi, A. E. (2019). Peranan Optimisme Terhadap Kesejahteraan Subjektif pada Mahasiswa yang Sedang Mengerjakan Skripsi Serta Tinjauannya dalam Islam (Doctoral dissertation, Universitas YARSI). Sadono sukino, Makro Ekonomi Modern, (Jakarta: PT Raja Grafindo Persada, 2000), hlm. 7 This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.
https://openalex.org/W4249555304	https://www.qeios.com/read/T60FZ3/pdf	English	null	Supraglottic Cancer pT1 TNM Finding v6 and v7	Definitions	2,020	cc-by	104	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Supraglottic Cancer pT1 TNM Finding v6 and v7 National Cancer Institute National Cancer Institute Qeios ID: T60FZ3 · https://doi.org/10.32388/T60FZ3 Source National Cancer Institute. Supraglottic Cancer pT1 TNM Finding v6 and v7. NCI Thesaurus. Code C64736. National Cancer Institute. Supraglottic Cancer pT1 TNM Finding v6 and v7. NCI Th C d C64736 National Cancer Institute. Supraglottic Cancer pT1 TNM Finding v6 and v7. NC Thesaurus. Code C64736. Supraglottic cancer with tumor limited to 1 subsite of supraglottis with normal vocal cord mobility. (from AJCC 6th and 7th Eds.) Qeios ID: T60FZ3 · https://doi.org/10.32388/T60FZ3 1/1
https://openalex.org/W4297857865	https://zenodo.org/records/1252214/files/2.pdf	English	null	AUTOMATED DETECTION OF HARD EXUDATES IN FUNDUS IMAGES USING IMPROVED OTSU THRESHOLDING AND SVM	Zenodo (CERN European Organization for Nuclear Research)	2,018	cc-by	5,963	International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 2016 International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 2016 KEYWORDS KEYWORDS Diabetic retinopathy, Fundus images, Hard exudates, Improved Otsu thresholding, SVM, Automated detection. Diabetic retinopathy, Fundus images, Hard exudates, Improved Otsu thresholding, SVM, Automated detection. 1College of Mechanical Engineering, Shanghai University of Engineering Science, Shanghai, 201620, China 2 2Department of Ophthalmology, Jiangsu province hospital of TCM, Nanjing, 210029, China ABSTRACT One common cause of visual impairment among people of working age in the industrialized countries is Diabetic Retinopathy (DR). Automatic recognition of hard exudates (EXs) which is one of DR lesions in fundus images can contribute to the diagnosis and screening of DR.The aim of this paper was to automatically detect those lesions from fundus images. At first,green channel of each original fundus image was segmented by improved Otsu thresholding based on minimum inner-cluster variance, and candidate regions of EXs were obtained. Then, we extracted features of candidate regions and selected a subset which best discriminates EXs from the retinal background by means of logistic regression (LR). The selected features were subsequently used as inputs to a SVM to get a final segmentation result of EXs in the image. Our database was composed of 120 images with variable color, brightness, and quality. 70 of them were used to train the SVM and the remaining 50 to assess the performance of the method. Using a lesion based criterion, we achieved a mean sensitivity of 95.05% and a mean positive predictive value of 95.37%. With an image-based criterion, our approach reached a 100% mean sensitivity, 90.9% mean specificity and 96.0% mean accuracy. Furthermore, the average time cost in processing an image is 8.31 seconds. These results suggest that the proposed method could be a diagnostic aid for ophthalmologists in the screening for DR. Weiwei Gao1 and Jing Zuo2 Weiwei Gao1 and Jing Zuo2 1College of Mechanical Engineering, Shanghai University of Engineering Science, Shanghai, 201620, China 2 1. INTRODUCTION At present, a main challenge of current health care in the world is fast progression of diabetes. The number of people with diabetics would increase to 4.4% of the global population by 2030 expected by the world health organization [1]. However, the fact is that only one half of the patients are aware of the disease. And in the medical perspective, diabetes will lead to severe late complications. These complications are macro and micro vascular changes which would result in heart disease, renal problems and retinopathy. Diabetic retinopathy (DR) which is one of the common complications and it remains the most common cause of blindness among adults greater than 65 years in the developed countries [2]. Although early detection and laser treatment of DR have proved effective in preventing visual loss [3], too many diabetic patients are not treated in time because of inadequacies of the currently available screening programmes [4]. It is now a recognised urgent worldwide priority that institutes an efficient screening programme for the detection of at-risk patients at a stage when they can still be effectively treated because 75% of the blindness due to diabetes can be preventable. International guidelines recommend annual 1 DOI:10.5121/ijcses.2016.7101 International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 2016 fundus examination for all diabetic patients to detect the early stage of DR [5]. Now, it is certain that the most effective treatment for DR is only in the first stages of the disease. So, early detection by regular screening is of paramount importance. Digital image capturing technology must be used because it can lower the cost of such screenings, and this technology enables us to employ state-of-the-art image processing techniques which automate the detection of abnormalities in fundus. DR clinical signs include red lesions and bright lesions. The former include intraretinal microvascular abnormalities (IRMAs) and hemorrhages (HEs), the latter include hard exudates (EXs) and soft exudates or cotton-wool spots (CWs) [6]. EXs are yellowish intraretinal deposits and usually located in the posterior pole of the human fundus which is shown in figure 1. Hard exudates are made up of serum lipoproteins which leak from the abnormally permeable blood vessels, especially across the walls of leaking MAs. It can represent the only visible sign of DR for some patients [6]. Because different lesions have different diagnostic importance and management implications, it is very important to distinguish among lesion types. 2. MATERIALS In this work, a total of 120 fundus images which came from department of Ophthalmology, Jiangsu province hospital of TCM were obtained from a non-mydriatic retinal camera with a 45° field of view. The image resolution was 800600 at 24 bit RGB. An ophthalmologist manually marked all EXs in these images. The results obtained by our automatic method were compared with these hand-labeled ones. According to the ophthalmologist, these images in the database were divided as follows: (1) 68 of these images belong to patients who suffered from mild to moderate nonproliferative DR. The images from DR patients all contained EXs. (1) 68 of these images belong to patients who suffered from mild to moderate nonproliferative DR. The images from DR patients all contained EXs. g p (2) The remaining 52 images belonged to healthy patients. (2) The remaining 52 images belonged to healthy patients. Drusens were not present in any of these images. The total 120 fundus images were divided at random into two subsets, one is a training set the other is a test set: Drusens were not present in any of these images. The total 120 fundus images were divided at random into two subsets, one is a training set the other is a test set: Drusens were not present in any of these images. The total 120 fundus images were divided at random into two subsets, one is a training set the other is a test set: (1) The training set contained 70 images of which 40 images were from DR patients. 2560 segmented regions were extracted from these images after the segmentation step of the method named improved Otsu threshold segmentation. They were labeled as EXs, or non-EXs according to the annotation of the ophthalmologist. Consequently, a fully labeled ground truth database was created which would be used to determine the parameters of SVM. The test set contained the remaining 50 images (22 from healthy retinas and 28 from DR patients). It was used to validate the effectiveness of the complete algorithm by comparing our result with the expert annotation. For this study we have used a Intel(R) Core(TM) Duo E7500 CPU PC with 6.0 GB RAM and the platform of MATLAB R2009a. 1. INTRODUCTION We did research on EXs detection and their differentiation from other bright areas in fundus images in this paper. EXs detection plays a very important role on DR screening tasks, as EXs are among the common early clinical signs of DR [7]. Additionally, EXs detection could act as the first step for a complete monitoring and grading of DR. Many attempts to detect EXs from fundus images can be found in the literature. Some of them use the high luminosity of EXs to distinguish the lesions from background by the method of thresholding.Ward [8] used shade correcting to reduce the shade variation in the color fundus image. EXs were detected by thresholding. It required the user to select the threshold manually according to the histogram. Phillips [9] detected the large EXs by a global threshold and segmented the smaller, lower intensity ones by local threshold. The thresholds were selected automatically, but the region of interest must be chosen manually. Sinthanayothin[10] applied a recursive region growing segmentation to detect EXs after the color image standardization. Li[11] modified the region growing method by introducing the Luv color space. Walter[12] proposed the approach on EXs detection by their high grey level variation and their contours determined by morphological reconstruction. S´anchez[13] raised the algorithm employing statistical recognition, Fisher’s linear discriminant analysis, colour information and the edge sharpness by applying a Kirsch operator to detect hard exudates and based on the detection to perform the classification. Jaafar[14] proposed the algorithm that included a coarse segmentation which is based on a local variation operation to outline the boundaries of all candidates with clear borders and a following fine segmentation which is based on an adaptive thresholding as well as a new split-and-merge technique to segment all bright candidates locally. Other approaches were based on neural network classifiers, such as multilayer perceptrons as well as support vector machines (SVM) [15-18]. In the following section 2, the characteristics of the image database under study is presented. Section 3 describes the detection method. We test the performance of the proposed method and compare our method with other methods in section 4. In section 5, discussion and conclusion of this paper is made. 2 2 International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 2016 Figure 1. Color fundus image and close-up of EXs EXs Optic disc Figure 1. Color fundus image and close-up of EXs 3.1. Improved Otsu thresholding segmentation stage Pigments contained in the fundus structures have different absorption characteristics, and different wavelength monochromatic lights also have different penetration performance through fundus, so spectral signatures of different layer of fundus structure are different. We found that EXs appear most contrasted in the green channel, and optic disc appears most continuous and most contrasted against the background in the red channel [19]. So we coarsely segmented EXs in the green channel as shown in Figure 2(a), and segmented optic disc in the red channel which was 3 International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 2016 shown in Figure 2(b). In color fundus images, EXs are yellow white lesions with relatively distinct margins, and in the green channel, they are of high luminosity. Consequently, the most direct and simple approach to identify candidate regions of EXs might be to extract the bright pixels from green channel using a threshold. But residual variability in the luminosity or in the pigmentation of the retinal background made the utilization of an approach based on a threshold impractical.So we opted for an improved approach which is called improved Otsu thresholding based on minimum inner-cluster variance [20]. This method combines inner-cluster variance and between-cluster variance of adaptive threshold segmentation, and when the threshold makes the inner-cluster variance minimum and between-cluster variance maximum, it is considered to be the optimal threshold. Optic disc is also masked out as shown in Figure 2(b) using the method developed by Walter T [12] to reduce the computational cost of the classifier because optic disc has similar attributes in terms of brightness, color and contrast. Figure 2(c) is the result after segmentation of the image in Figure 1. Figure 2. The detection of EX. (a) green channel of original fundus image. (b) segmentation result of optic disc. (c) candidate regions of EXs. (d) classification result of SVM. (e) detection result superimposed over the original image. (f) close-up of detection result Figure 2. The detection of EX. (a) green channel of original fundus image. (b) segmentation result of optic disc. (c) candidate regions of EXs. (d) classification result of SVM. (e) detection result superimposed over the original image. (f) close-up of detection result (1) Region size A (2-4) Mean RGB values inside the region R µ , G µ , B µ (8-10) Mean RGB values around the region N R µ , N G µ , N B µ (1) Region size A (2-4) Mean RGB values inside the region R µ , G µ , B µ (5-7) Standard deviation of the RGB values inside the region R σ , G σ , B σ (8-10) Mean RGB values around the region N R µ , N G µ , N B µ 3.3. Feature selection stage Many features could be used to design and train the given classifier. However, misclassification probability might be to increase with the number of features and the structure of classifier would much more difficult to interpret [21]. Feature selection tries to avoid these problems by picking out the subset of the extracted features which is most useful for a specific problem. Feature analysis for a specific classification task is based on the discriminatory power of the features. This is a measure of the usefulness of each feature in predicting the class of an object. Traditional feature selection methods are classifier-driven, i.e., which rely on the results obtained by a particular classifier for different subsets of features. In addition, it is more appropriate for medical image analysis is that classifier independent feature analysis which collects information concerning the structure of the data rather than the requirements for a particular classifier [21]. Logistic Regression (LR) is a classifier-independent method usually used for feature selection. LR can be used to describe the relationship between a response or dependent variable and one or more explanatory or independent variables [22]. In our study, there were 24 independent variables and the dependent variable was dichotomous. LR could be modelled by function (1) if he possible values of the dependent variable are 0 and 1 [23]. z z Y Prob e 1 e )1 ( + = = (1) (1) where Y is the dependent variable, 1 is the desired outcome, p p x b x b x b b z L + + + = 2 2 1 1 0 , p is the number of ix , T 0 0 ] , , [ p b b b B L = is regression coefficient which can be identified by the maximum likelihood method. Model selection of LR can be carried out by several strategies. A stepwise selection method was adopted because it can provide a fast and effective means to screen a large number of variables, and to simultaneously fit a number of LR equations. Certain criteria are needed to be decided whether an independent variable provides enough valuable information to enter the model or whether, on the other hand, it can be considered redundant. In our study, an independent variable would enter the model if the p-value associated with the result of the score test was lower than 0.05 which was a standard significance level. 3.3. Feature selection stage Accordingly, a variable would remove from the model if the p-value associated with the result of the likelihood ratio test was higher than 0.10. (11-13) Standard deviation of the RGB values around the region N R σ , N G σ , N B σ (11-13) Standard deviation of the RGB values around the region N R σ , N G σ , N B σ (11-13) Standard deviation of the RGB values around the region N R σ , N G σ , N B σ (14-16) Ratio of the mean RGB values between inside region area and surrounding area R P , G P , B P (17-19) Homogeneity of the region R H , G H , B H (20-22) RGB values of the region center R C , G C , B C (23) Region compactness CP (24) Region edge strength ES (14-16) Ratio of the mean RGB values between inside region area and surrounding area R P , G P , B P 7-19) Homogeneity of the region R H , G H , B H (20-22) RGB values of the region center R C , G C , B C (23) Region compactness CP (24) Region edge strength ES International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 2016 International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 2016 3.2. Feature extraction stage In order to classify the candidate regions identified in the previous stage as EXs or non-EXs, some significant features which help ophthalmologists to visually distinguish EXs from other types of lesions as well as the background needed to be extracted from each region and to be used as inputs of SVM. In this way, prior knowledge was used to the classification task. We extracted the following 24 features[16]: (1) Region size A (2-4) Mean RGB values inside the region R µ , G µ , B µ (5-7) Standard deviation of the RGB values inside the region R σ , G σ , B σ (8-10) Mean RGB values around the region N R µ , N G µ , N B µ 3.4. SVM classification stage SVM is a typically statistical learning method based on structural risk minimization [24], and it has been successfully applied to many pattern recognition problems and the reader is referred to for details [25]. 5 International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 2016 International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 2016 Consider a labelled two-class training set { } i i y x , , n i , ,1 L = , d i R x ∈ ， { } 1 ,1 − + ∈ iy is the associated “truth”. The separating hyperplane must satisfy the following constraint: 0 1 ] ) [( ≥ + − + • i i i b x y δ ω n i L 2,1 = , 0 ≥ iδ (2) (2) Where ω is the weight vector, b is the bias, iδ is the slack variable. In order to find the optimal separating hyperplane, function (3) should be minimized subjected to function (2): Where ω is the weight vector, b is the bias, iδ is the slack variable. In order to find the optimal separating hyperplane, function (3) should be minimized subjected to function (2): ( ) ( )         + • = ∑ = n i i C 1 2 1 , δ ω ω δ ω φ (3) (3) Where C is a parameter chosen by the user and controls the trade-off between maximizing the margin and minimizing the training error. The classifier can be constructed as: Where C is a parameter chosen by the user and controls the trade-off between maximizing the margin and minimizing the training error. The classifier can be constructed as: { } ( )       + • ∂ = + • = ∑ = n i i i i b x x y b x x f 1 * * * sgn sgn ) ( ω (4) (4) Where * ω and * b denote the optimum values of the weight vector and bias respectively, and * i∂ is the Lagrange multiplier. Where * ω and * b denote the optimum values of the weight vector and bias respectively, and * i∂ is the Lagrange multiplier. 3.5. Postprocessing stage [ In fundus images, there may be bright spots or bright points of physiological structures due to reflection in the human fundus. The biggest difference between such false positive regions as shown in figure 3 and EXs is that it usually exists in isolation, whereas EXs appear in clumps. In order to improve the performance of this system, we regarded those images in which less than 0.01% of the total number of pixels had been detected as EXs as normal fundus. An example of the false positive regions is shown in Fig. 3. Figure 3. False positive regions 4 RESULTS Figure 3. False positive regions Figure 3. False positive regions Figure 3. False positive regions Where ( ) y x K , is the kernel function. Gaussian radial basis function is commonly used. Where ( ) y x K , is the kernel function. Gaussian radial basis function is commonly used. 3.4. SVM classification stage SVM will map the input vector x into a high dimensional feature space by means of choosing a nonlinear mapping kernel if a linear boundary being inappropriate. The optimal separating hyperplane in the feature space can be given by function (5): ( )       + • ∂ = ∑ = n i i i i b x x K y x f 1 * * sgn ) ( (5) (5) (5) Where ( ) y x K , is the kernel function. Gaussian radial basis function is commonly used. 4.1. System performance evaluation 4.1. System performance evaluation Now, there are no publicly available databases which can be used to test the performance of automatic DR detection algorithm on fundus images [26]. The performance of the proposed 6 International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 2016 method was tested using our test set of 50 fundus images. This evaluation requires the parameter of the algorithm to be previously settled using the training set. We varied the parameters and used 10-fold cross validation to assess the generalization ability of SVM. For each combination of the parameters, we measured the mean sensitivity ( val SE ), specificity ( val SP ) and accuracy ( val AC ) obtained for the validation set, defined as follows: FN TP TP + = val SE (6) FP TN TN + = val SP (7) FN FP TN TP TN TP + + + + = val AC (8) (6) (7) (8) Where TP is the number of classified EXs correctly, TN represents the number of exactly classified non-EXs, FP is the number of non-EXs mistakenly classified as EXs and FN is the number of EXs classified as non-EXs incorrectly. Where TP is the number of classified EXs correctly, TN represents the number of exactly classified non-EXs, FP is the number of non-EXs mistakenly classified as EXs and FN is the number of EXs classified as non-EXs incorrectly. Once all the parameters of the algorithm were fixed, we assessed the diagnostic accuracy of it on the test set in terms of a lesion-based criterion and an image-based criterion which are defined as follows : (1) Using a lesion-based criterion, we measured the mean les SE and positive predict value ( les PPV ) which is given by (9) obtained for the test set. Specificity is not an informative measure for the lesion-based criterion. PPV accounts for the probability that a detected region is really an EX, and it was regarded as a more significant criterion to evaluate the system. (1) Using a lesion-based criterion, we measured the mean les SE and positive predict value ( les PPV ) which is given by (9) obtained for the test set. Specificity is not an informative measure for the lesion-based criterion. PPV accounts for the probability that a detected region is really an EX, and it was regarded as a more significant criterion to evaluate the system. 4.1. System performance evaluation FP TP TP + = les PPV (9) (9) (2) The image-based criterion accounted for the ability of the algorithm to detect pathological images and separate them from normal ones in the test set due to the presence of EXs. Using the image-based criterion, each images in the test set is classified as belong to a patient with DR or to healthy subject. We used the image-based statistics mean sensitivity ( im SE ), mean specificity ( im SP ) and mean accuracy ( im AC ) to present the final results. The statistics are measured as described in Eqs. (6)-(8). 4.2. Experimental results We coarsely segmented the training set of 70 fundus images with variable color, brightness and quality using Otsu thresholding based on minimum inner-cluster variance. The segmentation resulted in 2560 candidate regions consisting of 1150 EXs and 1410 non-EXs. We computed the 24 features for 2560 candidate regions and used SPSS 18.0 to perform LR on these data. The inputs were normalized (mean = 0, standard deviation = 1) to insure that LR was not influenced by the numerical strength of a feature rather than by its discriminatory power. The following 10 features shown in Table 1 were selected and them were with the highest discriminatory power for separating EXs from other non-EX yellowish objects. 7 International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 2016 Table 1. Result of feature selection Step Parameters Significance Parameters Significance 16 A 0.002 CP 0.000 R µ 0.000 ES 0.000 N R µ 0.000 R H 0.000 G C 0.001 R P 0.000 B C 0.000 G P 0.000 International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 2016 Table 1. Result of feature selection SVM was used to classify candidate regions of EXs. At first, it must be specified by the obtained candidate regions with 10 features. It is that specifying a SVM requires two parameters: the kernel function and the regularisation parameter C . For training the SVM classifier, the Kernel-Adatron technique using a Gaussian Radial Basis kernel was used. To obtain the optimal values for the RBF kernel ( g ) and C , we experimented with different SVM classifiers with a range of values. We apply genetic algorithm combined with 10-fold cross-validation to find the best classifier on the base of validation error. Table 2 shows the optimal parameters of ( g ) and C with the SVM classification structures. The specified SVM classification result of Fig. 2(c) is shown in Fig. 2(d) which superimposed over the original image is shown in Fig. 2(e), and the close-up of the detection result is shown in Fig. 2(f). Table 2. Validated parameters Parameters Result C g val SE /% val SP /% val AC /% 2.39 1.25 95.13 98.01 96.72 Table 2. Validated parameters The proposed method was tested using a new set of 50 unseen fundus images: 28 belonged to patients in early stages of DR and 22 corresponded to healthy retinas. 4.2. Experimental results Performances of different classifiers Classifier Image-based criterion Lesion-based criterion im SE (%) im SP (%) im AC (%) les SE (%) les PPV (%) SVM 100 90.9 96.0 95.05 95.37 RBF 100 90.9 96.0 93.9 95.5 Bayes 96.43 90.9 94.0 90.15 92.06 4.2. Experimental results The test set contained 50 images which were with variable color, brightness and quality. Using the specified SVM to classify the candidate regions segmented by improved Otsu thresholding based on minimum inner-cluster variance from 50 fundus images, the performance is shown in Table 3. And it is stated that post processing excluded 2 images of false positive. In addition, the detection results of these fundus images processed by the method proposed by Jaafar [14] are also shown in Table 3. It is found that the automated detection performance including accuracy and efficiency of the method proposed in this paper is obviously superior to the one proposed by Jaafar. To assess the performance of the SVM we also classified our segmented candidate regions using other classifiers including RBF classifier and Bayes classifier. In order to estimate the generalisation error of all classifiers, a 10-fold cross-validation technique was used again. The results were summarised in Table 4 which are the best results from a selection of configurations used for training the classifiers. These results indicate that the diagnostic accuracy of SVM is the best in RBF classifier and Bayes classifier. 8 International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 201 Table 3. Detection result Method Image-based criterion Lesion-based criterion Efficiency /s im SE (%) im SP (%) im AC (%) les SE (%) les PPV (%) Proposed method 100 90.9 96.0 95.05 95.37 8.31 Jaafar [14] 96.43 90.9 94.0 89.7 90.12 13.58 Table 4. Performances of different classifiers Classifier Image-based criterion Lesion-based criterion im SE (%) im SP (%) im AC (%) les SE (%) les PPV (%) SVM 100 90.9 96.0 95.05 95.37 RBF 100 90.9 96.0 93.9 95.5 Bayes 96.43 90.9 94.0 90.15 92.06 International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 2016 Table 3. Detection result International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 2016 Table 3. Detection result Method Image-based criterion Lesion-based criterion Efficiency /s im SE (%) im SP (%) im AC (%) les SE (%) les PPV (%) Proposed method 100 90.9 96.0 95.05 95.37 8.31 Jaafar [14] 96.43 90.9 94.0 89.7 90.12 13.58 Table 4. Performances of different classifiers Table 4. 5. CONCLUSIONS AND DISCUSSION However, it may not yet be appropriate for clinic. So it is significant that develop more efficient EXs automatic detection algorithm in the future. ACKNOWLEDGEMENTS We would like to thank Department of Ophthalmology, Jiangsu province hospital of TCM who supplied all the images used in this project for its great support for the project. 5. CONCLUSIONS AND DISCUSSION Digital imaging is becoming available as means of screening for diabetic retinopathy because it can provide a high quality permanent record of the retinal appearance which can be used for monitoring of progression or response to treatment, and can be reviewed by an ophthalmologist. In other words, digital images have the potential to be processed by automatic analysis system. In this study, we developed a totally automatic method to detect EXs in fundus images taken from diabetic patients or healthy people with non-dilated pupils. The fundus images were segmented by improved Otsu thresholding based on minimum inner-cluster variance to obtain candidate regions of EXs. In order to distinguish EXs from retinal background, the effectiveness of a set of 24 features of the candidate regions were examined and the subset with maximum discriminatory power were selected , according to a LR analysis of our data.The optimum subset was used to train a SVM classifier. The algorithm was tested on 22 images from healthy retinas and 28 images of retinas with DR, with variable colour, brightness and quality. Furthermore, the result was compared with the method proposed by Jaafar [14]. The im SE in Table 3 shows that we detected all images with signs of DR. We also detected some false positives in the images of healthy subjects, as the image-based im SP demonstrates. However, the impact of wrongly classifying a person suffering from DR as a healthy subject is more important than the converse. Javitt et al. suggested that a sensitivity of 60% or greater maximized cost-effectiveness in screening for diabetic retinopathy in their health policy model [27]. It means that increasing sensitivity of screening from 60% to 100% can not provided additional benefit because of the frequency of screening as well as the likelihood that retinopathy cases missed at one visit will be detected at the next. That means efficiency of automated detection is more important when detection accuracy is adequate. For DR patients, the retinal lesions will be influenced by treatment, control and progression of diabetes, i.e.. Therefore, only higher detection frequency is guaranteed, related lesions of fundus can be founded in time. Higher detection frequency is guaranteed by efficient 9 International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 2016 automatic detection algorithm. The Efficiency in Table 3 of the proposed method is obviously superior to the one proposed by Jaafar [14]. REFERENCES [1] Wild S(2004) Global prevalence of diabetes: estimates for the year 2000 and projections for 20 Diabetes Care (27): 1047-1053 [2] Wong TY(2006) Diabetic retinopathy in a multi-ethnic cohort in the United States. Am J Ophthalmol 141: 446-55. [2] Wong TY(2006) Diabetic retinopathy in a multi-ethnic cohort in the United States. Am J Ophthalmol 141: 446-55. [3] Kristinsson JK(1997) Diabetic retinopathy, screening and prevention of blindness. A doctoral thesis. Acta Ophthalmol Scand Suppl (223):1-76 [3] Kristinsson JK(1997) Diabetic retinopathy, screening and prevention of blindness. A doctoral thesis. Acta Ophthalmol Scand Suppl (223):1-76 p pp ( ) [4] Fagot-Campagna A(2007) Non-insulin treated diabetes: Relationship between disease management and quality of care. The Entred study, 2001 quality of care. 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Ophthalmology 94:1389-1400 [8] Ward N P(1989) The detection and measurement of exudates associated with diabetic retinopathy. Ophthalmology, 96(1): 80-86 [9] Phillips R P(1993) Automated detection and quantification of retinal exudates. Graefe’s Archive for Clinical and Experimental Ophthalmology 231: 90-94 [10] Sinthanayothin C(1999) Image analysis for automatic diagnosis of diabetic retinopathy. PhD thesis, King’s College London. [11] Li H(2002) A model based approach for automated feature extraction in color fundus images. PhD thesis, Nanyang Technological University [12] Walter T(2002) A Contribution of Image Processing to the Diagnosis of Diabetic Retinopathy-Detection of Exudates in Color Fundus Images of the Human Retina. IEEE Transactions on Medical Imaging 21:1236-1243 g g [13] S´anchez C I(2008) A novel automatic image processing algorithm for detection of hard exudates based on retinal image analysis. Medical Engineering and Physics, Elsevier, 30: 350-357 [14] Jaafar H F(2010) Automated detection of exudates in retinal images using a split-and-merge algorithm. 18th European Signal Processing Conference. REFERENCES Aalborg, Denmark：EUSIPCO, 1622-1626 [15] Gardner GG (1996) Automatic detection of diabetic retinopathy using an artificial neural network: a [14] Jaafar H F(2010) Automated detection of exudates in retinal images using a split-and-merge algorithm. 18th European Signal Processing Conference. Aalborg, Denmark：EUSIPCO, 1622-1626 [15] Gardner GG.(1996) Automatic detection of diabetic retinopathy using an artificial neural network: a screening tool. Br. J. Ophthalmol. 80: 940-944 [15] Gardner GG.(1996) Automatic detection of diabetic retinopathy using an artificial neural networ screening tool. Br. J. Ophthalmol. 80: 940-944 [16] Ege BM(2000) Screening for diabetic retinopathy using computer based image analysis and statistical classification. Comput. Methods Programs Biomed 62 165-175 [17] Osareh, A(2004) Automatic identification of diabetic retinal exudates and the optic disc. Ph. D thesis, Bristol [18] Mir HS(2011) Assessment of Retinopathy Severity Using Digital Fundus Images. The First Middle East Conference on Biomedical Engineering, Sharjah, UAE [19] Osareh A(2003) Automated identification of diabetic retinal exudates in digital colour images. Br J Ophthalmol 87(10):1220-1223 0] Zhou YY(2007) Improved Otsu thresholding based on minimum inner-cluster variance. J. Huazhong Univ. of Sci. & Tech. (Nature Science Edition) 35(2): 101-103 [21] Loew MH(2000) Feature Extraction. in Handbook of Medical Imaging. Bellingham, WA: SPIE Press, 273–341 [22] Hosmer, D W(2000) Applied Logistic Regression. New York: John Wiley, 307:1989. 10 International Journal of Computer Science & Engineering Survey (IJCSES) Vol.7, No.1, February 2016 [23] Xu L(2005) Comparisons of logistic regression and artificial neural network on power distribution systems fault cause identification. Proc. 2005 IEEE Mid-Summer Workshop on Soft Computing in Industrial Applications, Washington, DC 128-131 [24] Burges CJC(1998) A tutorial on support vector machines for pattern recognition. Data mining and knowledge discovery 2(2): 121-167 g y ( ) [25] Zhang YL (2005) Automated defect recognition of C-SAM images in IC packaing using Support Vector Machines. International Journal of Advanced Manufacturing Technology 25: 1191-1196 [26] Sa´nchez CI(2007) A novel automated image processing algorithm for detection of hard exudates based on retinal images analysis. Med. Eng. Phys 30(3): 350-357 [27] Javitt JC(1990) Detecting and treating retinopathy in patients with type I diabetes mellitus. A health policy model. Ophthalmology 97: 483-494 AUTHOR My name is Gao Weiwei, and I am a teacher in Shanghai University of Engineering Science. I received my Master degree and Doctor's degree in Nanjing University of Aeronautics and Astronautics. I major in the technology of digital medical equipment. My research interests include medical image processing, Biomedical information analysis and processing, pattern recognition and so on. Specifically, I do research on medical image segmentation technology which was applied to automated screening of diabetic retinopathy. 11
https://openalex.org/W4361833670	https://figshare.com/articles/journal_contribution/Supplementary_Figure_2_from_Prediction_and_Genetic_Demonstration_of_a_Role_for_Activator_E2Fs_in_Myc-Induced_Tumors/22390647/1/files/39836178.pdf	English	null	Supplementary Figure 3 from Prediction and Genetic Demonstration of a Role for Activator E2Fs in Myc-Induced Tumors	null	2,023	cc-by	39	E2F2 E2F1 E2F3 0.1 0.9 0.2 0.8 0.7 0.6 0.5 0.4 0.3 Pathway Probability E2F2 E2F1 E2F3 0.1 0.9 0.2 0.8 0.7 0.6 0.5 0.4 0.3 Pathway Probability 0.1 0.9 0.2 0.8 0.7 0.6 0.5 0.4 0.3 Pathway Probability
https://openalex.org/W3038039599	https://www.frontiersin.org/articles/10.3389/frobt.2020.00090/pdf	English	null	Collective Computation in Animal Fission-Fusion Dynamics	Frontiers in robotics and AI	2,020	cc-by	12,392	Gabriel Ramos-Fernandez 1,2, Sandra E. Smith Aguilar 3, David C. Krakauer 4 and Jessica C. Flack 4 1 Departamento de Modelación Matemática de Sistemas Sociales, Instituto de Investigaciones en Matemáticas Aplicadas y en Sistemas, Universidad Nacional Autónoma de México, Ciudad de México, Mexico, 2 Unidad Profesional Interdisciplinaria en Ingeniería y Tecnologías Avanzadas, Instituto Politécnico Nacional, Ciudad de México, Mexico, 3 Conservación Biológica y Desarrollo Social A.C., Ciudad de México, Mexico, 4 Santa Fe Institute, Santa Fe, NM, United States Recent work suggests that collective computation of social structure can minimize uncertainty about the social and physical environment, facilitating adaptation. We explore these ideas by studying how ﬁssion-fusion social structure arises in spider monkey (Ateles geoffroyi) groups, exploring whether monkeys use social knowledge to collectively compute subgroup size distributions adaptive for foraging in variable environments. We assess whether individual decisions to stay in or leave subgroups are conditioned on strategies based on the presence or absence of others. We search for this evidence in a time series of subgroup membership. We ﬁnd that individuals have multiple strategies, suggesting that the social knowledge of different individuals is important. These stay-leave strategies provide microscopic inputs to a stochastic model of collective computation encoded in a family of circuits. Each circuit represents an hypothesis for how collectives combine strategies to make decisions, and how these produce various subgroup size distributions. By running these circuits forward in simulation we generate new subgroup size distributions and measure how well they match food abundance in the environment using transfer entropies. We ﬁnd that spider monkeys decide to stay or go using information from multiple individuals and that they can collectively compute a distribution of subgroup size that makes efﬁcient use of ephemeral sources of nutrition. We are able to artiﬁcially tune circuits with subgroup size distributions that are a better ﬁt to the environment than the observed. This suggests that a combination of measurement error, constraint, and adaptive lag are diminishing the power of collective computation in this system. These results are relevant for a more general understanding of the emergence of ordered states in multi-scale social systems with adaptive properties–both natural and engineered. ORIGINAL RESEARCH published: 21 July 2020 doi: 10.3389/frobt.2020.00090 Keywords: social systems, distributed computing, inductive game theory, social information, animal foraging, collective intelligence Edited by: Daniel Polani, University of Hertfordshire, United Kingdom Reviewed by: Deborah M. Gordon, Stanford University, United States Matthew Lutz, Max Planck Institute of Animal Behaviour, Germany Heiko Hamann, University of Lübeck, Germany Reviewed by: Deborah M. Gordon, Stanford University, United States Matthew Lutz, Max Planck Institute of Animal Behaviour, Germany Heiko Hamann, University of Lübeck, Germany Correspondence: Gabriel Ramos-Fernandez ramosfer@alumni.upenn.edu Specialty section: This article was submitted to Computational Intelligence in Robotics, a section of the journal Frontiers in Robotics and AI Keywords: social systems, distributed computing, inductive game theory, social information, animal foraging, collective intelligence Received: 31 October 2019 Accepted: 05 June 2020 Published: 21 July 2020 Collective Computation in Animal Fission-Fusion Dynamics Gabriel Ramos-Fernandez 1,2*, Sandra E. Smith Aguilar 3, David C. Krakauer 4 and Jessica C. Flack 4 Citation: This can then function like an information bottleneck (Tishby et al., 2000; Tishby and Zaslavsky, 2015; Flack, 2017a): the strategies, as coarse-grainings, capture regularities individuals perceive in the physical or social environment. The way individuals combine strategies to make decisions in the collective captures the regularities they perceive as most important. Emergent from these slowly changing mesoscopic individual strategies and collective metastrategies is social structure. As a social structure consolidates and individuals start to “reference it” for decision- making, it feeds back through eﬀective downward causation (Flack, 2017a) to modulate the cost of social interaction or interaction with the environment. Once complete, this process can give rise to a new scale, and under suitable conditions, novel functions. Fission-fusion social dynamics, in which individuals ﬁssion and fuse into subgroups of varying size, is a collective pattern arising from individual decisions (Sueur et al., 2011; Ramos- Fernández et al., 2018). These dynamics are thought to be adaptive, as they allow individuals to forage more eﬃciently in heterogeneous environments, share information about the location of resources, and adjust the size of their subgroups to resource availability (Aureli et al., 2008; Sueur et al., 2011; Palacios-Romo et al., 2019). The individual, strategic decisions to leave or join subgroups, how these decisions inﬂuence subgroup size distributions, and whether these are a good ﬁt or even predicted by environmental states, are open questions. Previous work on spider monkeys suggests individuals change their strategies based on environmental states to include the rate at which they encounter fruit and the presence of knowledgeable individuals in social networks (Ramos-Fernández and Morales, 2014; Palacios-Romo et al., 2019). We study how individual spider monkeys use social knowledge (information accumulation) to collectively compute adaptive subgroup size distributions (information aggregation). We use inductive game theory (DeDeo et al., 2010; Krakauer et al., 2010) to extract stay-leave probabilistic strategies from a time series of subgroup composition. The strategies constitute the microscopic input to the collective computation. From the microscopic input we construct a family of circuits in which nodes correspond to individuals and edges, weighted by probabilities obtained from the data, specify probabilistic rules— strategies—for remaining in or leaving a subgroup. Circuits capture variation in the way individuals integrate over their strategies (see section 3) to decide to stay or go. To make this concrete, consider as an example the collective computation of power structure in macaque societies (reviewed in Flack, 2012, 2017a). Citation: Ramos-Fernandez G, Smith Aguilar SE, Krakauer DC and Flack JC (2020) Collective Computation in Animal Fission-Fusion Dynamics. Front. Robot. AI 7:90. doi: 10.3389/frobt.2020.00090 In an inﬂuential framework for studying animal social organization, Hinde (1976) stressed that both animal and human societies are multiscale. Short-term interactions between pairs of individuals lead to longer-term social relationships and social structures, with social relationships arising as individuals generalize from a history of social interactions. Hinde noted that individuals July 2020 \| Volume 7 \| Article 90 Frontiers in Robotics and AI \| www.frontiersin.org Collective Computation in Animal Fission-Fusion Dynamics Ramos-Fernandez et al. classify social relationships into types (kin, matriline, etc.) regardless of the individuals involved. The idea that primates use abstraction to make sense of their world has been shown in a number of studies subsequent to Hinde (1976) (e.g., Cheney and Seyfarth, 1990, 2008). classify social relationships into types (kin, matriline, etc.) regardless of the individuals involved. The idea that primates use abstraction to make sense of their world has been shown in a number of studies subsequent to Hinde (1976) (e.g., Cheney and Seyfarth, 1990, 2008). Among other examples in the animal behavior literature that might result from collective computation are coordinated foraging and predator avoidance in animal groups (Couzin et al., 2003; Gordon, 2016; Sosna et al., 2019), rapid direction changes during collective motion in ﬁsh schools and bird ﬂocks (Hein et al., 2015), and distributed foraging in social insects (Gordon, 2016). Over a series of papers, Flack et al. (Flack, 2012, 2017a,b; Flack et al., 2013; Daniels et al., 2017; Brush et al., 2018) have been developing a theory of collective computation (inspired in part by Hopﬁeld’s collective computation in neural networks Hopﬁeld, 1982, 1984; Tank and Hopﬁeld, 1988). In the context of animal behavior, this work links Hinde’s (1976) generalization and abstraction processes to the formation of collectives. In Flack and Krakauer’s formulation, components (for the purposes of this paper, individuals) reduce uncertainty about the environment or state of a system by coarse-graining fast microscopic behavior (Flack, 2017a). An example of uncertainty reduction would be over the cost of social interaction (Flack, 2012). When coarse- grainings converge (meaning the estimates of regularities are largely shared by individuals), this can produce a coherent mesoscale (e.g., a social network or circuit). Citation: Individuals summarize ﬁght histories using unidirectional signals. The sender emits the signal once it perceives it is likely to loose a ﬁght. The signal reduces uncertainty in the receiver that the sender agrees to subordination—willingness to yield in future interactions. Encoded in the consolidating network or circuit of signals between group members is information about the distribution of power. Hence the power structure is computed as individuals estimate regularities about ﬁghting abilities and share these opinions with the receiver and other group members via signals. Through this process, diﬀerent levels of organization arise at successively slower timescales: ﬁghts (fast), signaling (slow), and power structure (slowest). The process of generating coarse-grained, slow variables (the signals, properties of the circuits) is the outcome of individual strategic computations (interaction and signaling decisions) that aggregate into an output collectively estimated to ﬁt the state of the environment (Flack, 2017a,b). This two- part process of information accumulation and aggregation makes up collective computation (Daniels et al., 2017; Flack, 2017a). Each circuit serves as a mesoscopic hypothesis for how strategies combine to produce decisions and how decisions combine to compute subgroup size distributions. In a computational language, the inputs (individual strategies) combine to produce an output (a subgroup size distribution). We run the circuits forward in simulation to determine how individuals combine strategies and hence how many information sources they take into account to make decisions. We construct a food abundance index based on the size and abundance of fruiting trees and calculate the transfer entropy between this index and the distribution of subgroup size in order to determine whether the circuit that best recovers the observed subgroup size distribution is also optimally computing the state of the environment. Frontiers in Robotics and AI \| www.frontiersin.org 2. DATA Subgroup composition data were collected in Punta Laguna, Yucatan, Mexico, as part of a long-term study of social behavior Frontiers in Robotics and AI \| www.frontiersin.org July 2020 \| Volume 7 \| Article 90 2 Collective Computation in Animal Fission-Fusion Dynamics Ramos-Fernandez et al. FIGURE 1 \| Our dataset samples the process of ﬁssion-fusion dynamics in the entire group Each row with colored circles on the top of the ﬁgure represents how the FIGURE 1 \| Our dataset samples the process of ﬁssion-fusion dynamics in the entire group. Each row with colored circles on the top of the ﬁgure represents how the 47 individuals that conform the spider monkey group are sorted into subgroups at a given moment, with each color indicating subgroup membership. Thus, in the ﬁrst row or time step represented, the group is organized in 9 different subgroups and, throughout the remaining steps, subgroups change size and memberships by individuals leaving (ﬁssion) and joining (fusion). Our sample includes only one subgroup followed at any time, so we have information on the presence or absence of each group member on the observed subgroup. For example, the bottom part of the ﬁgure shows observations from one subgroup (turquoise dots on the top). Here, rows represent 5 instantaneous scan samples taken every 20’ on individuals 17 thru 23, where each individual can be either present (full circles) or absent (empty circles). In this case, the subgroup shows a ﬁssion of two individuals in the third scan and the fusion of three at the ﬁfth scan. For analysis, we coded data as binary vectors corresponding to each scan sample. Frontiers in Robotics and AI \| www.frontiersin.org 3. MICROSCOPIC STRATEGY EXTRACTION AND DISTRIBUTION using identiﬁed individuals (details about study site and subjects can be found in the Supplementary Information). Data consist of scan samples of subgroup composition, taken every 20’ during an average of 5 h. per day throughout 2 years (Jan. 2013–Dec. 2014), for a total of 5,780 scan samples. A total of 47 known adult, sub-adult and juvenile individuals were observed during this period (see Supplementary Table 1). Thus, each sample is a vector of 47 binary digits, with 0 corresponding to an absence of the individual in the ith position and 1 corresponding to a presence (Figure 1). Continuous series of scans, averaging 8.4 scan samples (± 3.9 SD), include uninterrupted follows of a subgroup in which at least one individual remained during the full series. Given that the typical duration of a subgroup is 1.5 h. (Pinacho-Guendulain and Ramos-Fernández, 2017), a subgroup may persist over multiple scans. The temporal resolution of this sampling regime was maintained in the analysis in order to obtain a suﬃcient number of continuous series of scans. Had we resampled the original dataset at a larger temporal scale, we would have lost an important number of continuous series. Also, the persistence of a subgroup over several scans implies that individuals in a subgroup are tolerating one another, which is informative about the weight of their mutual inﬂuence (see below). using identiﬁed individuals (details about study site and subjects can be found in the Supplementary Information). Data consist of scan samples of subgroup composition, taken every 20’ during an average of 5 h. per day throughout 2 years (Jan. 2013–Dec. 2014), for a total of 5,780 scan samples. A total of 47 known adult, sub-adult and juvenile individuals were observed during this period (see Supplementary Table 1). Thus, each sample is a vector of 47 binary digits, with 0 corresponding to an absence of the individual in the ith position and 1 corresponding to a presence (Figure 1). Continuous series of scans, averaging 8.4 scan samples (± 3.9 SD), include uninterrupted follows of a subgroup in which at least one individual remained during the full series. Given that the typical duration of a subgroup is 1.5 h. (Pinacho-Guendulain and Ramos-Fernández, 2017), a subgroup may persist over multiple scans. The temporal resolution of this sampling regime was maintained in the analysis in order to obtain a suﬃcient number of continuous series of scans. 3. MICROSCOPIC STRATEGY EXTRACTION AND DISTRIBUTION Had we resampled the original dataset at a larger temporal scale, we would have lost an important number of continuous series. Also, the persistence of a subgroup over several scans implies that individuals in a subgroup are tolerating one another, which is informative about the weight of their mutual inﬂuence (see below). We distinguish between strategies and decisions. A decision is binary: to leave or stay in a subgroup (in the original inductive game theory work, to join or avoid a ﬁght, DeDeo et al., 2010). Strategies (called 1P, as in previous work, DeDeo et al., 2010) are “above-null” probabilities (see below for calculation) describing the weight of individual A’s presence or absence in the current subgroup (as determined by scan sampling, see section 2) on individual B’s decision to stay or go from the subgroup in the subsequent sample. Here and in previous work (DeDeo et al., 2010), multiple individuals can inﬂuence individual B. Hence B will have multiple strategies and, in the limit, a strategy for every other group member. We address how B integrates strategies to reach a decision in section 4. Here we quantitatively describe how we deﬁne and extract strategies from the time series. We end up with a list of pair-wise strategies for which our extraction method indicates above-null support in the time series. We do not consider higher order strategies as in DeDeo et al. (2010). For all pairs of individuals {A:B, A:C, A:D,...}, we calculate the probability an individual B is present or absent in a sample if individual A was present in the previous sample within the same continuous series of scans: The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualiﬁed researcher. P(A →B) = N(Bt+1 \| At) N(A) , (1) (1) Frontiers in Robotics and AI \| www.frontiersin.org July 2020 \| Volume 7 \| Article 90 3 Collective Computation in Animal Fission-Fusion Dynamics Ramos-Fernandez et al. FIGURE 2 \| Frequency distribution of the values of 1P for the different combinations of dyadic weights, as deﬁned in Equations (2) and (4) (A,B) and for the total sum of the incoming weights that each individual receives (C,D; this is the in-strength of nodes in Figure 3). 3. MICROSCOPIC STRATEGY EXTRACTION AND DISTRIBUTION The values of 1P(A →B) have a wider distribution around zero, with correspondingly higher total values of in-strength, than in the case of 1P(!A →B). FIGURE 2 \| Frequency distribution of the values of 1P for the different combinations of dyadic weights, as deﬁned in Equations (2) and (4) (A,B) and for the total sum of the incoming weights that each individual receives (C,D; this is the in-strength of nodes in Figure 3). The values of 1P(A →B) have a wider distribution around zero, with correspondingly higher total values of in-strength, than in the case of 1P(!A →B). where N(Bt+1 \| At) is the total number of times B was present at time t+1 given that A was present at time t within a continuous series of scans and N(A) is the number of times A was present in all samples. where N(Bt+1 \|!At) is the number of times B is present in a sample when A is absent in the previous sample within a continuous series of scans, N(!A) is the number of times A is absent in all samples, and Nnull(Bt+1 \|!At) is the average of the same number for 1,000 bootstrapped versions of the original data. As with previous work (DeDeo et al., 2010), to remove time-independent eﬀects from the transition probabilities (for example, due to general diﬀerences in gregariousness), we calculate the diﬀerence between the probability inferred from the data and a null expectation: These 1P constitute the pair-wise weight of each group member on a given individual’s binary decision to leave or join a subgroup. Figure 2 shows the frequency distribution of the values of 1P as deﬁned in Equations (2) and (4). In all cases values are centered around zero, with the values of 1P(!A →B) closer to zero than in other cases. This is because the denominator in Equation (4) is larger than in Equation (2), as it includes all instances of individual A being absent from the observed scan. There are proportionally fewer cases in which B is present after an absence of A because there are many cases where A is absent. Thus, these values of 1P(!A →B) should be interpreted with care. It is also the case that most values of the total sum of weights received are positive. In other words, most individuals receive a total positive weight from the presence or absence of strategically connected individuals. 3. MICROSCOPIC STRATEGY EXTRACTION AND DISTRIBUTION Only a few cases show a total negative weight of the presence or absence of others. 1P(A →B) = N(Bt+1 \| At) −Nnull(Bt+1 \| At) N(A) , (2) (2) where Nnull(Bt+1 \| At) is the average number of times B is present at time t+1 given that A is present at time t within a continuous series of scans, calculated from 1,000 bootstrapped permutations of the data. Similarly, we consider the weight of A’s absence on the presence of another individual B in a subsequent sample: P(!A →B) = N(Bt+1 \|!At) N(!A) , (3) (3) We identiﬁed signiﬁcantly positive dyadic weights as values of 1P higher than the 95% percentile of the permuted values for each dyad. Accordingly, signiﬁcantly negative dyadic weights were values of 1P lower than the 5% percentile of the permuted values for each dyad. and and 1P(!A →B) = N(Bt+1 \|!At) −Nnull(Bt+1 \|!At) N(!A) , (4) (4) July 2020 \| Volume 7 \| Article 90 Frontiers in Robotics and AI \| www.frontiersin.org Frontiers in Robotics and AI \| www.frontiersin.org Collective Computation in Animal Fission-Fusion Dynamics Ramos-Fernandez et al. 4. MESOSCOPIC CIRCUIT CONSTRUCTION Only one individual (female BL) had a negative in-strength value, implying that it received a total negative 1P(A →B) higher than the total positive 1P(A →B). FIGURE 3 \| Circuits showing the strategies (signiﬁcant, pairwise negative and positive weights) extracted from the data and as deﬁned in Equations (2) (upper panel, 1P(A →B)) and (4) (lower panel, 1P(!A →B)). Nodes correspond to individuals indicated by two-letter codes and their shape represents females (circles) and males (squares). Only for the purposes of this visualization we removed the 11 juvenile individuals, who do not move independently of their mothers. However, they were included in the analyses of 1P values. Edges correspond to signiﬁcant 1P values, of a width proportional to their value. Each circuit employs a different range of 1P values, as 1P(A →B) values range from −0.00076 to 0.3 and 1P(!A →B)) values from −0.00033 to 0.00315 (see Figure 2). Node size is proportional to the in-strength of the node, i.e., the total signiﬁcant weight from others as deﬁned by the sum of the incoming 1P values. Node color corresponds to whether the node has a positive (blue) or negative (purple) in-strength. The color of edges corresponds to negative (red) and positive (gray) values. g p The circuit for 1P(!A → B) shows a diﬀerent picture (lower panel in Figure 3). Here values were skewed below zero, although overall they were much closer to zero than the values of 1P(A →B) (Figure 2). Even considering that the variation around zero is small, this circuit contains both positive and negative weights, corresponding to repulsion and attraction, respectively, but the most important links are negative or attractive. There is, as in the previous circuit, evidence of some degree of homophily, with individuals of the same sex inﬂuencing each other through negative links more than those of the opposite sex. Conversely, a high proportion of positive or repulsive links occur between the sexes. Both males and females have high values of in-strength, although those with a negative in-strength (receiving many negative, attractive weights) in this circuit were all females. Individuals with the highest values of positive in-strength (corresponding to a total sum of positive or repulsive weights in this network) were males. Each individual can have multiple strategies, and they can be in conﬂict (DeDeo et al., 2010), with some weights positive and others negative. Frontiers in Robotics and AI \| www.frontiersin.org 4. MESOSCOPIC CIRCUIT CONSTRUCTION We use the strategies obtained from the data to construct circuits (i.e. the set of all signiﬁcant 1P values as weights between all pairs of individuals; this is the mesoscopic level of our analysis) each of which is a hypothesis for (1) how individuals integrate over their strategies to arrive at a binary decision to join or leave a subgroup and, (2) specify how the resulting decisions combine to produce the distribution of subgroup size. The circuits in Figure 3 give a qualitative summary of signiﬁcant strategies. For each individual, there are 46 potential weights (signiﬁcant 1P values) from either the presence or absence of others at scan time t, which could determine its presence or absence at scan time t + 1. The circuits in Figure 3 show only 31 individual nodes for 1P(A →B) and 36 for 1P(!A →B), who were involved in signiﬁcant weights. On average, each individual in these circuits is linked to 20.25 (± 1.98 SE) other individuals in the 1P(A →B) and to 31.67 (± 1.40 SE) in the 1P(!A →B) circuit (Figure 3). Similarly, whereas each of the circuits in Figure 3 could have up to 1,260 links, the 1P(A →B) circuit has 314 and the 1P(!A →B) circuit 570 links. Supplementary Figure 1 shows the values of all signiﬁcant weights included in these circuits, as well as the individual instrength and outstrength. The circuit for 1P(A → B) (upper panel in Figure 3) represents signiﬁcant weights of the presence of individual A at scan t on the presence of individual B at scan t + 1. Most of the values of 1P(A →B) were positive or close to zero (see Figure 2A), therefore this circuit contains mostly positive weights (gray links), corresponding to weights of attraction. There is an apparent homophily by sex in this circuit, with individuals inﬂuencing other individuals of the same sex more than those of the other. Other attractive interactions are those between some pairs of adult females and their subadult daughters (e.g., females VE-VI and JA-LX in the upper panel of Figure 3, CH-LO and ME-KL in the lower panel). Individuals diﬀer in their in-strength values (as can be observed in Figure 2B) with the individuals with the highest values of in-strength receiving many diﬀerent weights, some with high values of 1P, both females and males. 4. MESOSCOPIC CIRCUIT CONSTRUCTION In the lower panel, each individual arrives to its own value of P 1P, which will determine its presence or absence from the subgroup at time t + 1, depending on the values of U and L. A sum of 1Ps greater than U or smaller than L could lead an individual to either maintain (e.g., B was absent in time t and its P 1P is below L, leading to its absence in t + 1) or change its previous state (e.g., C was present in time t and P 1P is below L, leading to its absence in t + 1). to join or leave the subgroup. Figures 2B,D show frequency histograms for these incoming values, corresponding to the in- strength of the nodes in Figure 3. These in-strength values can be understood as the likelihood that an individual will be inﬂuenced by others: an individual with a high in-strength is more likely to decide to be present due to another individual’s presence (in the case of 1P(A →B) values, upper panel in Figure 3) or absence (in the case of 1P(!A →B) values, lower panel in Figure 3) than another individual with a lower in-strength. and the 1P(!A →B) values, such that an individual would be integrating the weights it receives across both circuits shown in Figure 3. At higher values of U, the presence of an individual in a subgroup is less likely to be inﬂuenced by others. In that sense, high values of U imply less interdependence of individuals in their decisions to be present or not in a subgroup. Conversely, L controls the opposite end of the range of values of P 1P, such that at more negative values of L, an individual should be less likely to be absent from a subgroup due to the previous weight from others. We tested U = {0.0001, 0.001, 0.01, 0.1, 0.2, ..., 0.9} and L = {−0.9, −0.8, ..., −0.1, −0.01, ..., −0.00001}. We further assume that at any given time t, if the sum of signiﬁcant 1P values P 1P directed toward an individual B is positive and greater than a threshold U, B will be present on the sample at t+1 (irrespective of whether it was present or absent in the previous sample; Figure 4). Frontiers in Robotics and AI \| www.frontiersin.org 4. MESOSCOPIC CIRCUIT CONSTRUCTION In addition, the weight or importance (given by 1P) of each strategy varies. Hence individuals must integrate over their set of strategies to make a decision about whether July 2020 \| Volume 7 \| Article 90 5 Collective Computation in Animal Fission-Fusion Dynamics Ramos-Fernandez et al. FIGURE 4 \| Example of rules by which individuals integrate incoming weights to decide their state at scan t + 1. In the upper panel, an individual B integrates incoming weights using a decision rule. If B, who is not present in the focal subgroup at scan t, receives a signiﬁcant weight 1P from the presence of A on its presence at scan t + 1, while receiving another signiﬁcant weight from individual D’s absence on its presence, B will integrate both weights by a simple sum. If this sum P 1P is above a certain threshold U, B will decide to join a subgroup where it was not present at time t. In the lower panel, each individual arrives to its own value of P 1P, which will determine its presence or absence from the subgroup at time t + 1, depending on the values of U and L. A sum of 1Ps greater than U or smaller than L could lead an individual to either maintain (e.g., B was absent in time t and its P 1P is below L, leading to its absence in t + 1) or change its previous state (e.g., C was present in time t and P 1P is below L, leading to its absence in t + 1). FIGURE 4 \| Example of rules by which individuals integrate incoming weights to decide their state at scan t + 1. In the upper panel, an individual B integrates incoming weights using a decision rule. If B, who is not present in the focal subgroup at scan t, receives a signiﬁcant weight 1P from the presence of A on its presence at scan t + 1, while receiving another signiﬁcant weight from individual D’s absence on its presence, B will integrate both weights by a simple sum. If this sum P 1P is above a certain threshold U, B will decide to join a subgroup where it was not present at time t. 5. TESTING CIRCUITS IN SIMULATION analyses in that work suggest these triadic strategies are non- decomposible into two pair-wise strategies (i.e., not reducible to additive individual or pair-wise interactions; Daniels et al., 2016; Chen et al., 2019). Individuals typically had multiple higher- order strategies and so, as with pair-wise, higher-order strategies were pushed through gates to produce binary decisions. Here we allow for the possibility that individuals take into account multiple strategies and hence be under the inﬂuence of multiple individuals, but we do not explore whether the interactions are pair-wise or higher-order. Here we assess how individuals integrate strategies to make decisions 1P and how decisions combine to compute the subgroup size distribution. We do so by asking which circuit, given an integration threshold, produces a simulated data set with a distribution of subgroup size that best recovers the observed one. We used each set of 100 simulated datasets with diﬀerent values of U to evaluate the set of subgroup size distributions that is in closest correspondence to the observed. We only show the eﬀects of varying U at L = −0.00001, since the variation in L for any value of U does not have an eﬀect on the subgroup size distribution. This is likely because values of P 1P are mostly positive (Figures 2C,D), so very few values are below the L threshold. In other words, even the smallest negative value of L has no eﬀect on the tendency of individuals to modify their presence based on the presence or absence of others. We use these circuits to generate, by simulation, new datasets from the original dataset. In what follows, we restricted our analyses and simulations to a subset of the original dataset that included the same months for which food abundance data was available (Sep. 2013–Sep. 2014; see section 6), corresponding to 3,032 scan samples. We started by randomly choosing a scan sample (subgroup) that serves as the “seed” or ﬁrst scan of a sequence of n samples, where n is randomly drawn from the frequency distribution of the number of samples per continuous observation period in the original biweekly period. Thus, the seed establishes which of the 47 monkeys in the group are present or absent in the ﬁrst sample. Because the seed and the duration of continuous observation periods are selected within observation periods, simulated data contain information about the variation in subgroup size and composition between bi- weekly periods. 4. MESOSCOPIC CIRCUIT CONSTRUCTION Conversely, if P 1P is negative and smaller than a threshold L, individual B will be absent from the following sample (again, independently of whether it was present or absent in the previous sample). However, if L < P 1P < U, then there is no eﬀect from others and B remains in the same state as in the previous sample (i.e., present if it was present at time t, absent if it was absent; Figure 4). Thus, U is a threshold parameter controlling how likely it is for individuals to be present in a subgroup based on the weight of others. The value L controls the opposite, i.e., how likely it is that individuals will be absent in a subgroup based on the weight of others. Note that the total sum P 1P includes both the 1P(A →B) Diﬀerent individuals could actually be using a diﬀerent value of the U and L thresholds, or the values could change over time, depending on slower ecological variables such as the dry and wet seasons or even longer timescales related to the ecological succession of the forest in the spider monkey’s habitat. In this work we assume, as a ﬁrst approximation, a single value of the threshold parameters for all individuals and seasons. There are also subtle points here concerning how strategies are aggregated by individuals to produce binary decisions. In previous work (DeDeo et al., 2010), higher order (triadic—C only joins current ﬁght if both A and B were present in the previous ﬁght) as well as pair-wise strategies (A joins if B was previously present) were extracted from time series data and a circuit was constructed for each strategy class. Preliminary July 2020 \| Volume 7 \| Article 90 6 Collective Computation in Animal Fission-Fusion Dynamics Ramos-Fernandez et al. FIGURE 5 \| Subgroup size distribution for the original dataset (black thick line) and for the simulated datasets. Lines of a given color correspond to the resulting distribution from 100 repetitions using different values of U, with L= −0.00001. FIGURE 5 \| Subgroup size distribution for the original dataset (black thick line) and for the simulated datasets. Lines of a given color correspond to the resulting distribution from 100 repetitions using different values of U, with L= −0.00001. Frontiers in Robotics and AI \| www.frontiersin.org 5. TESTING CIRCUITS IN SIMULATION If an individual A is present in the ﬁrst scan, the simulation looks at values of 1P(A →B) and considers any signiﬁcant values or weights of A on others. If, on the contrary, A is not in the seed, then the simulation looks for signiﬁcant values of 1P(!A → B). This applies to all 47 individuals. For values of U = 0.4 and above the subgroup size distribution from simulated datasets is similar to the observed (Figure 5). Values of U < 0.4 generate distributions where small subgroups are underrepresented and larger subgroups are overrepresented. This is due to the fact that, at lower values of U, individuals are more likely to be inﬂuenced by others, both through the signiﬁcant values of 1P(A →B) and 1P(!A →B). The former dominate the dynamics of subgroup size change because they have higher and positive values overall (Figure 2). Thus, when U < 0.4, individuals are aggregating more frequently, deciding to join subgroups at higher frequency as in the observed data. Values of U < 0.4 give rise to subgroups converging at a single size for each value of U (Figure 5). This may be due to all individuals deciding to join subgroups, even those without signiﬁcant weights, as must be the case in subgroups larger than 36, the number of nodes in the largest network in Figure 3 that depicts all individuals that are involved in signiﬁcant weights. These rules are used to determine subgroup composition of the n samples in the continuous observation period. This is repeated for 633 sequences, corresponding to the number of continuous observation periods in the original dataset. In total, we generated 100 simulated datasets for each combination of thresholds U and L. We compared the observed subgroup size distribution and those obtained by simulation under diﬀerent values of U using Frontiers in Robotics and AI \| www.frontiersin.org July 2020 \| Volume 7 \| Article 90 7 Collective Computation in Animal Fission-Fusion Dynamics Ramos-Fernandez et al. FIGURE 6 \| Jensen-Shannon (JS) distance between the observed and simulated subgroup size distributions shown in Figure 5. Each dot corresponds to the JS distance between an instance of 100 simulations for each value of U. For all simulations, L = −0.00001. FIGURE 6 \| Jensen-Shannon (JS) distance between the observed and simulated subgroup size distributions shown in Figure 5. 5. TESTING CIRCUITS IN SIMULATION Each dot corresponds to the JS distance between an instance of 100 simulations for each value of U. For all simulations, L = −0.00001. FIGURE 6 \| Jensen-Shannon (JS) distance between the observed and simulated subgroup size distributions shown in Figure 5. Each dot corresponds to the JS distance between an instance of 100 simulations for each value of U. For all simulations, L = −0.00001. the Jensen-Shannon distance (Figure 6). This distance between two random variables x and y is deﬁned as: D rather than tree number. To do so we calculated the sum of the D values of all the trees with fruit (Df ) in period p divided by the sum of D values for all the trees in the plot (Di), giving an index of food abundance for a period p, IFAp = P Df / P Di. JS(x\|y) = H x + y 2 −1 2[H(x) + H(y)] (5) (5) p f Figure 7 shows the time series for the IFA and subgroup size during one year. As mentioned above, maintaining the temporal resolution of the subgroup size time series was important in order to maintain a suﬃcient number of continuous series of observations. Despite the diﬀerent temporal resolution of each time series, it seems that subgroup size increases together with IFA during the second wet season. where H is the entropy of each variable, p(x) 1 logp(x) and X and Y are, in this case, the observed subgroup size and the subgroup size obtained in one run of a simulation, respectively. Figure 6 corroborates what is apparent in Figure 5, that simulations run with U ≥0.4 yield subgroup size distributions that are closer and indistinguishable from the observed distribution, with JS values that are close to zero, while simulations run with U < 0.4 have an increasing JS with respect to the observed. Simulations run with all values of L for U=0.4 yield subgroup size distributions that are equally close to the observed (data not shown). In previous work, the match between the collective computation output and the environment was evaluated using mutual information (Brush et al., 2018). Here we use transfer entropy: Tx→y(t) = H(yt\|yt−1) −H(yt\|yt−1, xt−1) (6) (6) Frontiers in Robotics and AI \| www.frontiersin.org 6. FIT OF OUTPUT TO ENVIRONMENT The IFA measures the overall abundance of fruit in the spider monkey’s abitat, considering their most preferred species, their fruiting status and the abundance and relative size of trees (see section 6). The temporal resolution of the ubgroup size data is 20 min, whereas food abundance was monitored biweekly. Thus, the IFA series has the same value throughout a given biweekly period, while ubgroup size ﬂuctuates at a much ﬁner temporal scale. Noted above are the seasons (wet or dry) to which each sample belongs. Panel (C) presents a fragment of e subgroup size time series showing its variation between September 30 and October 31st 2013. Note that the time series was constructed with sets of scan amples taken every 20’ collected throughout 4–8 h periods and that subgroups followed in consecutive days were not necessarily the same. Therefore, the spikes nd drops observed in the curve do not always reﬂect ﬁssion or fusion events. f iti t hi h f i it ith diﬀ t f f ti i i l t d d t t ith U ≥ 0 4 FIGURE 7 \| Time series for the index of food abundance (IFA; A) and subgroup size (B). The IFA measures the overall abundance of fruit in the spider monkey’s habitat, considering their most preferred species, their fruiting status and the abundance and relative size of trees (see section 6). The temporal resolution of the subgroup size data is 20 min, whereas food abundance was monitored biweekly. Thus, the IFA series has the same value throughout a given biweekly period, while subgroup size ﬂuctuates at a much ﬁner temporal scale. Noted above are the seasons (wet or dry) to which each sample belongs. Panel (C) presents a fragment of the subgroup size time series showing its variation between September 30 and October 31st 2013. Note that the time series was constructed with sets of scan samples taken every 20’ collected throughout 4–8 h periods and that subgroups followed in consecutive days were not necessarily the same. Therefore, the spikes and drops observed in the curve do not always reﬂect ﬁssion or fusion events. of fruiting trees, we assess which of our circuits with diﬀerent strategy integration rules (described in section 4), computes a distribution of subgroup size that is a good ﬁt to the current abundance of fruiting trees. 6. FIT OF OUTPUT TO ENVIRONMENT This is a measure of how much uncertainty in a variable y is reduced given past states of both y and a variable x that is assumed to be independent of y. This dependence is over and above the uncertainty about y reduced by consideration of its own past state. Here transfer entropy is measuring how much subgroup size uncertainty is reduced by considering past states of subgroup size and IFA, conditioned on the uncertainty reduction by the past states of subgroup size alone. Given the diﬀerence in time resolution for the two time series (Figure 7), this implies that, within a given bi-weekly period, we are measuring the transfer entropy between a constant value of IFA and varying values of subgroup size. We used the JIDT package (Lizier, 2014) in R (R Core Team, 2017) to estimate the transfer entropy between time series, using the Kraskov estimator with the number of closest neighbors k = 4. The two observed time series have a TIFA→SGS(t)=0.036 nats. A central question is whether the collective computation output is adaptive (Flack, 2017a; Brush et al., 2018). Previous studies of spider monkeys suggest there is a weak relationship between subgroup size and food abundance (Symington, 1988; Pinacho- Guendulain and Ramos-Fernández, 2017). In general, subgroups tend to be larger during periods of high food abundance. This suggests that subgroup size can track the abundance of resources. Here, we investigate whether subgroup size distribution is predicted by the relative abundance of fruiting trees. We use data from a 1-ha plot where all the trees (diameter at breast height, D > 10 cm) from the 15 most consumed species by the monkeys, were monitored bi-weekly for a year from September 2013 to September 2014, comprising 25 monitoring periods. A total of 487 trees were identiﬁed, their D was recorded, and every 2 weeks they were assessed for the presence of fruit. The data obtained were used to calculate the proportion of trees with fruit available in a given period expressed in terms of the total tree To explore whether spider monkeys collectively compute a subgroup size distribution that is a good match to the distribution July 2020 \| Volume 7 \| Article 90 8 Collective Computation in Animal Fission-Fusion Dynamics Ramos-Fernandez et al. IGURE 7 \| Time series for the index of food abundance (IFA; A) and subgroup size (B). Frontiers in Robotics and AI \| www.frontiersin.org 6. FIT OF OUTPUT TO ENVIRONMENT FIGURE 9 \| Transfer entropy between simulated IFA and simulated subgroup size. Each gray circle corresponds to an instance of 100 simulations run with varying values of U and L = −0.00001. Red dots indicate the upper and lower limits of 99 percent conﬁdence intervals of the mean. The dotted line corresponds to the value of transfer entropy found for the observed IFA and subgroup size data in Figure 7. FIGURE 9 \| Transfer entropy between simulated IFA and simulated subgroup size. Each gray circle corresponds to an instance of 100 simulations run with varying values of U and L = −0.00001. Red dots indicate the upper and lower limits of 99 percent conﬁdence intervals of the mean. The dotted line corresponds to the value of transfer entropy found for the observed IFA and subgroup size data in Figure 7 FIGURE 9 \| Transfer entropy between simulated IFA and simulated subgroup size. Each gray circle corresponds to an instance of 100 simulations run with varying values of U and L = −0.00001. Red dots indicate the upper and lower limits of 99 percent conﬁdence intervals of the mean. The dotted line corresponds to the value of transfer entropy found for the observed IFA and subgroup size data in Figure 7. background against which individuals can tune their own strategies (Flack, 2017a). Hence there are two challenges for a group computing its social structure: that it changes slowly enough to remain informative for decision-making and that it adaptively tracks the environment. sampled from the distribution of observed n) there is a certain degree of variation around the observed data. Each simulated IFA series was compared to its corresponding subgroup size series. These values of TIFA→SGS(t) are presented in Figure 9, which also shows the value of TIFA→SGS(t) obtained for the observed IFA and subgroup size time series (Figure 7). The results suggest simulated subgroup size data sets with 0.01 < U < 0.4 match the temporal variation in IFA values better than the empirically observed subgroup size distribution and better than the simulated distributions computed with U ≥0.4. 6. FIT OF OUTPUT TO ENVIRONMENT Shown in Figure 8 is the time series for the subgroup size values together with the subgroup size time series of all simulated data sets generated for diﬀerent values of U. Figure 8 shows what was already apparent in the subgroup size distributions shown in Figure 5, but in the form of a time series: simulated data sets with U ≥ 0.4 generate a subgroup size distribution that is closest to the observed distribution. We calculated the transfer entropy between the IFA time series and its corresponding subgroup size time series. We generated simulated data sets that included the same values of IFA as in the original dataset, but because the observation period length could vary (as the length of each observation period, n, was July 2020 \| Volume 7 \| Article 90 Frontiers in Robotics and AI \| www.frontiersin.org 9 Collective Computation in Animal Fission-Fusion Dynamics Ramos-Fernandez et al. FIGURE 8 \| Time series for subgroup size as observed (black line) and simulated (lines of varying color). Each colored line corresponds to an instance of 100 simulations for different values of U and L = −0.00001. Wet and dry seasons are noted above. FIGURE 9 \| Transfer entropy between simulated IFA and simulated subgroup size. Each gray circle corresponds to an instance of 100 simulations run with varying values of U and L = −0.00001. Red dots indicate the upper and lower limits of 99 percent conﬁdence intervals of the mean. The dotted line corresponds to the value of transfer entropy found for the observed IFA and subgroup size data in Figure 7. FIGURE 8 \| Time series for subgroup size as observed (black line) and simulated (lines of varying color). Each colored line corresponds to an instance of 100 simulations for different values of U and L = −0.00001. Wet and dry seasons are noted above. FIGURE 8 \| Time series for subgroup size as observed (black line) and simulated (lines of varying color). Each colored line corresponds to an instance of 100 simulations for different values of U and L = −0.00001. Wet and dry seasons are noted above. FIGURE 8 \| Time series for subgroup size as observed (black line) and simulated (lines of varying color). Each colored line corres simulations for different values of U and L = −0.00001. Wet and dry seasons are noted above. 6. FIT OF OUTPUT TO ENVIRONMENT Frugivorous spider monkeys are faced with two signiﬁcant sources of uncertainty related to foraging—to discover the location of fruiting trees and to distribute themselves over these fruiting trees to minimize conﬂict (Aureli et al., 2008) and the costs associated with large groups (Asensio et al., 2009), as well as to maximize resource intake (Symington, 1988). We have used a theory of collective computation (see references in the introduction) to explore how ﬁssion-fusion dynamics arises in spider monkey groups and whether the resulting distribution of subgroup size is a good match to the environment. We found spider monkey collectives appear to be able to partially match subgroup size to resource abundance. Our results suggest Frontiers in Robotics and AI \| www.frontiersin.org 7. DISCUSSION Gil et al., 2018). Exchanging information about available patches when foragers disperse and learning about the location and availability of diﬀerent patches increases the foraging success of the whole group (Falcón-Cortés et al., 2019). The circuit of individual strategies that we infer here is, at least in part, a reﬂection of information sharing about available patches. Following another individual when ignorant is a simple mechanism of information sharing (Palacios-Romo et al., 2019), that could be reﬂected in the dyadic weights we have measured. This would lead to a fully connected circuit with information about food sources promoting a ﬂexible grouping pattern that matches heterogeneity in the environment. We should also be cautious in interpreting the power of the collective computation at small U values. In these limits subgroups converge to a constant size where food abundance is expected to be somewhat predictive of size simply because both values remain constant during each bi-weekly period. These caveats aside, whereas collective computation in this system is not optimal, it remains nonetheless predictive and able to capture information about the environment. Speciﬁcally, the circuits that capture subgroup joining strategies can aggregate information about the environment. Although we did not study longer timescales, the slowly changing structure of groups provides a means for storing information accumulated by individuals about food availability across years (Palacios-Romo et al., 2019). With individuals that are more than 30 years old (see Supplementary Information), who are using spatial memory for their foraging decisions (Valero and Byrne, 2007), the information made available to the group through their experience is likely an important element to track long-term changes in the foraging environment. It is interesting to compare our approach to that of optimal foraging theory, which would postulate an optimal subgroup size distribution, based on a set of constraints and the best compromise between costs and beneﬁts, which for most cases are unknown (Fretwell and Lucas, 1970; Stephens and Krebs, 1986). An empirical test of this postulate would consist of the match or lack thereof of the observed distribution to the food abundance and this would be interpreted in terms of the unknown mechanisms for how subgroup size comes about (e.g., Chapman et al., 1995). 7. DISCUSSION Social structure typically changes slowly compared to the interactions giving rise to it. As such, social structure, whether optimal for the environment or not, reduces uncertainty about the future state of the system and provides a relatively stable Frontiers in Robotics and AI \| www.frontiersin.org July 2020 \| Volume 7 \| Article 90 10 Collective Computation in Animal Fission-Fusion Dynamics Ramos-Fernandez et al. however that the collective computation of subgroup size is not optimal with respect to food availability as measured by our index. processing to be addressed through simulation in more complicated systems. Additional factors that could aﬀect decision-making, thereby shifting the subgroup distribution from optimal to suboptimal, are a variety of social variables like sex and age, the previous history of interactions, and kinship relationships (Ramos- Fernández et al., 2009; Busia et al., 2017). However, because we are extracting individual strategies directly from the data, these modulating factors are already included in the weights between individuals. Other factors that are currently implicit include the risk of predation or location within the group’s home range, which could also aﬀect the subgroup size. In simulating the circuits of subgroup-joining strategies we discover values of a sensitivity parameter U (a measure of the degree of consensus among the incoming weights required for an individual to make a decision about whether to stay or go) leading to a distribution of subgroup size that is a better match (than the observed distribution of subgroup size) to the observed abundance of fruiting trees. This suggests collective computation is under constraint and the system is experiencing adaptive lag—that is, still learning the best collective strategy to integrate information accumulated by group members. The deviation might instead be spurious–an outcome of (1) the way in which we calculate the food abundance index, (2) the fact that the data used to construct the two distributions are noisy and have diﬀerent time resolutions: food abundance was measured at a bi-weekly scale while subgroup size was observed every 20 min, or (3) other factors besides social knowledge and relationships contributing to subgroup size decision-making. g p Our results shed light on how a group can best acquire and share information about patchy and dynamic environments. While individual foraging strategies based on spatial knowledge have been well-documented (Janson and Byrne, 2007; Fagan et al., 2013), group foraging strategies are less well-known outside of social insects (Gordon, 2016; cf. Frontiers in Robotics and AI \| www.frontiersin.org 7. DISCUSSION Our approach is more mechanistic: we observe a series of stay-leave decisions resulting from the interactions between individuals and construct a circuit of strategies that serves as a hypothesis for how the subgroup size distribution could emerge. We measure how similar these emerging distributions are to the observed and then test how well the time series matches the environmental variation. That we ﬁnd alternative circuits that could produce a better match to the environment implies that the system is not necessarily constrained, as would be postulated by optimal foraging theory. Some means by which computations can be reﬁned maximizing the match between group behavior and the abundance of food, includes individuals changing the way they accumulate information and/or compute strategies for staying or leaving, tuning how individuals integrate over those strategies, and tuning how the strategies interact in the circuit to produce subgroup size distributions. For example, are some individuals’ strategies (perhaps because they inﬂuence many others) exerting a disproportionate eﬀect on the output or do many individuals contribute in small ways? The problem of how collectives achieve optimal information processing is an important one in biology (Tkaˇcik and Bialek, 2016), and near optimal information processing has been discovered in a number of biological systems (e.g., Petkova et al., 2019). However, these examples tend to be relatively simple developmental mechanisms such as segmentation during development of the fruit ﬂy larval body plan. The circuit approach allows the question of tuning to obtain optimal information Mutual information, as a measure of uncertainty reduction, has some nice properties. It provides a robust way to study how near optimal a collective behavior is, and this provides a proxy for adaptiveness. We can also study diﬀerent kinds of uncertainty reduction: an endogenous one, that involves collective computation of social structure that makes the world more predictable for individuals within a system (e.g., Brush et al., 2018); and an exogenous one, whereby collective computation produces social structure that encodes July 2020 \| Volume 7 \| Article 90 11 Collective Computation in Animal Fission-Fusion Dynamics Ramos-Fernandez et al. of microscopic dynamics has parallels in multiplex networks, which have proven to be a better representation of the dynamics of many systems than the simple aggregation of diﬀerent layers (De Domenico et al., 2015; Smith-Aguilar et al., 2019). 7. DISCUSSION Using longer time series, we could ask whether collective computation and ﬁt to the environment are being reﬁned and improved over time. With higher resolution data on strategies, and using methods from information theory (e.g., Rosas et al., 2019), it should be possible to quantify the degree to which the output is irreducibly encoded in the circuit as opposed to decomposeable. Is social knowledge processed in a pairwise manner or do individuals perceive synergistic interactions among group members (e.g., does individual’s A perception of individuals B and C contribute non-additively to its social knowledge)? g Understanding how a natural social system carries out adaptive computations could help to improve the performance of artiﬁcial systems. For instance, our results could provide insight into the mechanisms underlying learning through backpropagation in artiﬁcial neural networks. The way in which individuals adjust their strategic signaling in computing an appropriate power structure that feeds back to provide information about social interaction cost might be analogous to unsupervised learning (i.e., where the target is endogenous to the system) (Flack, 2017a; Brush et al., 2018). A system like the one we study here, with ﬁssion-fusion dynamics that can adjust to environmental conditions like the availability of fruiting trees, might be analogous to supervised learning (i.e., where the target is exogenous to the system). In both cases, feedback might share features with backpropagation in the strong and weak senses–the connection weights in the circuits/networks appear to be adjusted with a combination of vector (Brush et al., 2018) and scalar feedback (Flack et al., 2006) to minimize the network’s error function when learning a task (Rumelhart et al., 1986; Lillicrap et al., 2020). This is just one of many exciting comparisons that could be made to better understand how diﬀerent types of feedback, through tuning (Daniels et al., 2017) and downward causation (Flack, 2017a), shape the ability of the circuit to learn. And, as described in the Introduction, collective coarse-graining can produce a coherent mesoscale functioning as an information bottleneck, an ideal that is at least conceptually similar to the information bottleneck described by Tishby and colleagues to explain how deep neural networks We used the extracted strategies to construct a family of circuits that vary in how individuals integrate these strategies to produce binary decisions to join or leave a subgroup. Individuals can have both repulsion (leave) and attraction (join) strategies. 7. DISCUSSION Moreover, this way of compressing information may allow the social structure of spider monkeys to be ﬂexible enough to track a dynamic environment, and, at the same time, be robust to disturbances. This has parallels to neural processing (Bassett et al., 2011; Daniels et al., 2017). As we have discussed elsewhere (see Brush et al., 2013, 2018; Daniels et al., 2017; Flack, 2017a) compression and related principles of collective computation have implications for engineered systems, such as web search and swarm robotics (e.g., Bonabeau et al., 1999; Seth, 2001; Young et al., 2013), as well as pattern recognition by artiﬁcial neural networks and human reputation networks. knowledge about resource availability in the environment (this paper). Uncertainty reduction is consistent with a cost-beneﬁt framework without requiring costs and beneﬁts to be estimated. And quantiﬁcation of the quality of the output of collective computation in information theoretic terms builds a technical bridge to Boltzmann’s and von Neumann’s ideas about the role of entropy in generating ordered states (Krakauer et al., 2020) that can form the basis of new levels of individuality, even at the social level. In addition to assessing whether the output matches the environment, we studied the mechanics of collective computation. Previous work suggests spider monkeys preferentially follow food-aware individuals (Palacios-Romo et al., 2019). In the time series we ﬁnd evidence in support of this result: we are able to extract signiﬁcant (above-null) pair-wise probabilistic strategies used by individuals to decide to stay in or leave subgroups. Each individual had 20-30 strategies of varying strength (out of 46 possible). Generally the 1P were larger for “stay” strategies than “leave” strategies, suggesting possible food presence is a more important factor to spider monkeys than possible food absence. This emphasis on “attraction” might also be important for maintaining cohesion in ﬁssion-fusion dynamics in the context of a heterogeneous foraging environment with multiple alternative foraging options (Ramos-Fernández, 2005; Sueur et al., 2011). The strategies we ﬁnd also recover well-known social patterns for Ateles spp., in particular—same sex based homophily for joining and repulsive tendencies between individuals of diﬀerent sex (Fedigan and Baxter, 1984; Ramos-Fernández et al., 2009). It remains to be determined whether further, more ﬁne-grained patterns like the frequency of dyadic interactions are also recovered by these strategies. How spider monkeys collectively compute ﬁssion-fusion social structure and how these computations can be tuned to realize adaptive variants raises many questions. Frontiers in Robotics and AI \| www.frontiersin.org REFERENCES Cheney, D. L., and Seyfarth, R. M. (1990). The representation of social relations by monkeys. Cognition 37, 167–196. doi: 10.1016/0010-0277(90) 90022-C Asensio, N., Korstjens, A. H., and Aureli, F. (2009). Fissioning minimizes ranging costs in spider monkeys: a multiple-level approach. Behav. Ecol. Sociobiol. 63, 649–659. doi: 10.1007/s00265-008-0699-9 Cheney, D. L., and Seyfarth, R. M. (2008). Baboon Metaphysics: The Evolution of a Social Mind. Chicago, IL: University of Chicago Press. doi: 10.7208/chicago/9780226102429.001.0001 Aureli, F., Schaﬀner, C. M., Boesch, C., Bearder, S. K., Call, J., Chapman, C. A., et al. (2008). Fission-fusion dynamics: new research frameworks. Curr. Anthropol. 49, 627–654. doi: 10.1086/586708 Couzin, I. D., Krause, J., et al. (2003). Self-organization and collective behavior in vertebrates. Adv. Study Behav. 32, 10–1016. doi: 10.1016/S0065-3454(03)01001-5 Bassett, D. S., Wymbs, N. F., Porter, M. A., Mucha, P. J., Carlson, J. M., and Grafton, S. T. (2011). Dynamic reconﬁguration of human brain networks during learning. Proc. Natl. Acad. Sci. U.S.A. 108, 7641–7646. doi: 10.1073/pnas.1018985108 Daniels, B. C., Ellison, C. J., Krakauer, D. C., and Flack, J. C. (2016). Quantifying collectivity. Curr. Opin. Neurobiol. 37, 106–113. doi: 10.1016/j.conb.2016.01.012 Bonabeau, E., Dorigo, M., and Theraulaz, G. (1999). Swarm Intelligence: From Natural to Artiﬁcial Systems. Oxford: Oxford University Press. Daniels, B. C., Flack, J. C., and Krakauer, D. C. (2017). Dual coding theory explains biphasic collective computation in neural decision-making. Front. Neurosci. 11:313. doi: 10.3389/fnins.2017. 00313 Brush, E. R., Krakauer, D. C., and Flack, J. C. (2013). A family of algorithms for computing consensus about node state from network data. PLoS Comput. Biol. 9:e1003109. doi: 10.1371/journal.pcbi.1003109 De Domenico, M., Lancichinetti, A., Arenas, A., and Rosvall, M. (2015). Identifying modular ﬂows on multilayer networks reveals highly overlapping organization in interconnected systems. Phys. Rev. X 5:011027. doi: 10.1103/PhysRevX.5.011027 Brush, E. R., Krakauer, D. C., and Flack, J. C. (2018). Conﬂicts of interest improve collective computation of adaptive social structures. Sci. Adv. 4:e1603311. doi: 10.1126/sciadv.1603311 Busia, L., Schaﬀner, C. M., and Aureli, F. (2017). Relationship quality aﬀects ﬁssion decisions in wild spider monkeys (Ateles geoﬀroyi). Ethology 123, 405–411. doi: 10.1111/eth.12609 DeDeo, S., Krakauer, D. C., and Flack, J. C. (2010). Inductive game theory and the dynamics of animal conﬂict. PLoS Comput. Biol. 6:e1000782. doi: 10.1371/journal.pcbi.1000782 Fagan, W. F., Lewis, M. A., Auger-Méthé, M., Avgar, T., Benhamou, S., Breed, G., et al. (2013). Spatial memory and animal movement. Ecol. Lett. 16, 1316–1329. doi: 10.1111/ele.12165 Chapman, C. A., Chapman, L. J., and Wrangham, R. FUNDING Data collection was aided by a grant from the Mexican 1431 Council of Science and Technology (CONACYT CB157656). JF thanks the Proteus Foundation and the Bengier Family Foundation for support during the project. JF also acknowledges JTF 60501/St. Andrews sub award 13337 for support during the project. SUPPLEMENTARY MATERIAL All authors conceived the idea for this study. JF and DK developed the theory. GR-F and SS designed the data collection and performed the analysis and simulations. SS collected the data. All authors discussed the results and The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/frobt. 2020.00090/full#supplementary-material ETHICS STATEMENT The animal study was reviewed and approved by the corresponding authorities in Mexico: the Direccion General de Vida Silvestre, Secretaria de Medio Ambiente y Recursos Naturales. ACKNOWLEDGMENTS The datasets generated for this study are available on request to the corresponding author. We thank the following people and organizations: Augusto, Macedonio, and Eulogio Canul for their invaluable assistance with data collection; Heiko Hamann, Deborah M. Gordon, and Matthew Lutz for their thorough review of a previous version of this article; Filippo Aureli and Colleen Schaﬀner for sharing the management of the ﬁeld project. Instituto Politecnico Nacional and the Center for Complexity Science (C3-UNAM) for their logistical support. 7. DISCUSSION In previous work (DeDeo et al., 2010), strategies were passed through an AND or OR gate that captured conﬂict averse (all strategies have to say “go” to join a ﬁght) and conﬂict prone dispositions (one “go” strategy was suﬃcient to join). Here we use thresholds. To recover the observed subgroup size distribution in simulation requires sums over strategies (P 1P ≥U = 0.4) much larger than the strength of individual strategies (the majority of individual 1P values are below 0.05). This suggests individual-level decisions, as well as the aggregate output, require that individuals take into account relationships and social knowledge of many group members. If so, this would suggest that spider monkeys rely on social information from the wisdom of crowds (e.g., Jayles et al., 2017; Moreno- Gámez et al., 2017; Kao et al., 2018) to make decisions. These decisions are aggregated to collectively compute subgroup size distributions. Mesoscale strategic circuits are summaries or average tendencies and therefore provide an economical way to process information. Slow variables, encoded in individual strategies, are compressed summaries of noisy interactions (Flack, 2017b). The idea that the mesoscale circuit is a compressed representation July 2020 \| Volume 7 \| Article 90 12 Collective Computation in Animal Fission-Fusion Dynamics Ramos-Fernandez et al. contributed to the manuscript and giving approval to the ﬁnal version. encode information parsimoniously (Tishby et al., 2000; Tishby and Zaslavsky, 2015; Flack, 2017a). We have studied how a natural social system collectively computes. This is achieved through feedback among diﬀerent scales of social organization, as proposed by Hinde’s (1976) early paradigm and made explicit in Flack (2017a) and Flack (2017b). 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Frontiers in Robotics and AI \| www.frontiersin.org Collective Computation in Animal Fission-Fusion Dynamics July 2020 \| Volume 7 \| Article 90 REFERENCES doi: 10.3389/frobt.2014.00011 Valero, A., and Byrne, R. W. (2007). Spider monkey ranging patterns in mexican subtropical forest: do travel routes reﬂect planning? Anim. Cogn. 10, 305–315. doi: 10.1007/s10071-006-0066-z Moreno-Gámez, S., Sorg, R. A., Domenech, A., Kjos, M., Weissing, F. J., van Doorn, G. S., et al. (2017). Quorum sensing integrates environmental cues, cell July 2020 \| Volume 7 \| Article 90 Frontiers in Robotics and AI \| www.frontiersin.org 14 Collective Computation in Animal Fission-Fusion Dynamics Collective Computation in Animal Fission-Fusion Dynamics Ramos-Fernandez et al. Ramos-Fernandez et al. Copyright © 2020 Ramos-Fernandez, Smith Aguilar, Krakauer and Flack. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. July 2020 \| Volume 7 \| Article 90 Frontiers in Robotics and AI \| www.frontiersin.org 15
https://openalex.org/W4239641641	https://www.biogeosciences.net/15/703/2018/bg-15-703-2018.pdf	English	null	Peat decomposability in managed organic soils in relation to land-use, organic matter composition and temperature	null	2,017	cc-by	13,612	Correspondence: Jens Leifeld (jens.leifeld@agroscope.admin.ch) Correspondence: Jens Leifeld (jens.leifeld@agroscope.admin.ch) Received: 14 May 2017 – Discussion started: 14 June 2017 Revised: 26 October 2017 – Accepted: 13 December 2017 – Published: 5 February 2018 Abstract. Organic soils comprise a large yet fragile carbon (C) store in the global C cycle. Drainage, necessary for agri- culture and forestry, triggers rapid decomposition of soil or- ganic matter (SOM), typically increasing in the order for- est < grassland < cropland. However, there is also large vari- ation in decomposition due to differences in hydrological conditions, climate and speciﬁc management. Here we stud- ied the role of SOM composition on peat decomposability in a variety of differently managed drained organic soils. We collected a total of 560 samples from 21 organic crop- land, grassland and forest soils in Switzerland, monitored their CO2 emission rates in lab incubation experiments over 6 months at two temperatures (10 and 20 ◦C) and related them to various soil characteristics, including bulk density, pH, soil organic carbon (SOC) content and elemental ratios (C / N, H / C and O / C). CO2 release ranged from 6 to 195 mg CO2- C g−1 SOC at 10 ◦C and from 12 to 423 mg g−1 at 20 ◦C. This variation occurring under controlled conditions suggests that besides soil water regime, weather and management, SOM composition may be an underestimated factor that de- termines CO2 ﬂuxes measured in ﬁeld experiments. How- ever, correlations between the investigated chemical SOM characteristics and CO2 emissions were weak. The latter also did not show a dependence on land-use type, although peat under forest was decomposed the least. High CO2 emissions in some topsoils were probably related to the accrual of la- bile crop residues. A comparison with published CO2 rates from incubated mineral soils indicated no difference in SOM decomposability between these soil classes, suggesting that accumulation of recent, labile plant materials that presum- ably account for most of the evolved CO2 is not systemati- cally different between mineral and organic soils. In our data set, temperature sensitivity of decomposition (Q10 on aver- age 2.57 ± 0.05) was the same for all land uses but lowest below 60 cm in croplands and grasslands. This, in turn, indi- cates a relative accumulation of recalcitrant peat in topsoils. 1 Introduction Organic soils represent a major global sink for atmospheric carbon (C). Although they cover only 3 % of the earth’s ter- restrial surface (Tubiello et al., 2016), they store up to 30 % of the global soil organic carbon (SOC) pool (Parish et al., 2008). In Europe, more than 50 % of the former peatland area has been degraded by peat mining and conversion of land use, including drainage, to improve their suitability for agriculture or forestry (Joosten, 2010). Drainage aerates the soil so that plants of interest for agriculture and forestry can grow and make these soils manageable. The change from anaerobic to aerobic conditions, however, triggers rapid de- composition of peat that had accumulated under the con- ditions of waterlogging. This transforms the former C-sink into a major source of atmospheric carbon dioxide (CO2) and makes peatlands an important contributor to global cli- mate change (Freeman et al., 2004). Around 85 % of the global annual CO2 emission of 915 Mt CO2-C from drained peatlands are estimated to originate from organic soils now used as croplands (Tubiello et al., 2016). With rates of 6.5– 9.4 t C ha−1 a−1 net CO2 ﬂuxes from organic soils now used as croplands were on average found to be higher than from organic soils under grassland, which were estimated to vary Biogeosciences, 15, 703–719, 2018 https://doi.org/10.5194/bg-15-703-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 3.0 License. Biogeosciences, 15, 703–719, 2018 https://doi.org/10.5194/bg-15-703-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 3.0 License. C. Bader et al.: Peat decomposability in managed organic soils Drained organic soils under for- est can act as both net sinks or sources of atmospheric CO2 (Cannell et al., 1993; Minkkinen and Laine, 1998; Minkki- nen et al., 1999; Wüst-Galley et al., 2016), although they are in general considered to represent a source with aver- age net CO2 emissions of 2.0–3.3 t C ha−1 a−1 in the tem- perate zone (IPCC, 2014). Temperature and soil moisture regime, which depends on drainage depth, among other fac- tors, have the greatest inﬂuence on peat decay in drained organic soils (Hogg et al., 1992; Berglund, 1995; Scanlon and Moore, 2000; Chimner and Cooper, 2003; Couwenberg et al., 2010; Leifeld et al., 2012). However, there are sub- stantial differences in CO2 emissions from organic soils with similar drainage and cultivation properties. The protection of organic matter (OM) against decomposition by mechanisms such as occlusion in aggregates and binding to mineral sur- faces, which are important for the stabilization of OM in mineral soils (Six et al., 2002), are of minor importance in organic soils due to the lack or low abundance of minerals (Han et al., 2016). Therefore, the intrinsic decomposability of organic matter is considered another major factor inﬂuenc- ing the rate of peat decomposition and a major cause of sub- stantial variation in CO2 emissions at different sites (Chim- ner and Cooper, 2003; Byrne and Farrell, 2005; Höper, 2007; Wickland and Neff, 2008; Reiche et al., 2010). change with increasing depth towards a relative enrichment of compounds that are recalcitrant against decomposition un- der anoxic conditions, such as lignins and polyphenols (Free- man et al., 2004), while the contents of labile oxygen-rich compounds, such as polysaccharides, were found to decrease (Leifeld et al., 2012; Biester et al., 2014; Sjögersten et al., 2016). ) Elemental ratios of oxygen (O), hydrogen (H), and nitro- gen (N) to carbon are widely used as indicators of the relative abundance of different groups of compounds such as phe- nols, lipids and polysaccharides, and proteins. Lignins and polyphenols have molar O / C ratios in the range of 0.2–0.6 and H / C ratios between 0.9 and 1.5, while the respective ratios of carbohydrates range from 0.8 to 0.9 for O / C and from 1.4 to 1.8 for H / C (Kim et al., 2003). C. Bader et al.: Peat decomposability in managed organic soils In line with the molecular and spectroscopic analyses mentioned before, both ratios were found to decrease with increasing depth in peat (Klavins et al., 2008; Biester et al., 2014; Wüst-Galley et al., 2016). On the other hand, both fresh plant residues and undisturbed peat usually have high C / N ratios (Loisel et al., 2012). When peat becomes exposed to oxic conditions, mineralization seems to lead to relative enrichment of N, ex- plaining why decreased C / N ratios are found in organic top- soils compared to undrained peat layers or bottom layers of drained organic soils (Malmer and Holm, 1984; Kuhry and Vitt, 1996; Krueger et al., 2015). While undisturbed organic soils have a low bulk density, drainage leads to subsidence processes and increasing bulk densities in the topsoils (Ro- giers et al., 2008; Leifeld et al., 2011a, b). ) Although intrinsic decomposability of SOM cannot be ad- dressed directly, useful indicators of the latter are the relative abundances of labile and recalcitrant C moieties, which shift towards progressively higher proportions of the recalcitrant C with decomposition (Beer et al., 2008; Tfaily et al., 2014) and result in selective enrichment and depletion of speciﬁc functionalities (Leifeld et al., 2017; McAnallen et al., 2017). It is important to recognize that during peat formation, most of the net primary production contained in the initial mass of plant residues are lost due to mineralization, and only 10– 20 % is transformed and accumulated as peat in the water- saturated zone of a peat bog or fen (Clymo, 1984). Although decomposition acts slowly on accumulating peat of undis- turbed (i.e. water-saturated organic) soils, it is believed that primarily the most labile OM moieties are lost. Due to fresh peat layers accumulating on top of older ones, age and de- pletion in labile compounds increase with soil depth. Incu- bation studies of peat samples and carbon loss studies with undisturbed organic soils found smaller CO2 emission rates from deeper peat layers, which was related to the absence of labile compounds i.e. a lower intrinsic decomposability of soil organic matter (SOM) (Hogg et al., 1992; Scanlon and Moore, 2000; Wang et al., 2010; Hardie et al., 2011; Leifeld et al., 2012). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. C. Bader et al.: Peat decomposability in managed organic soils 704 between 1.8 and 7.3 t C ha−1 a−1 (IPCC, 2014). However, re- cent studies reported emission rates of 7.6 ± 2.0 t C ha−1 a−1 on organic soils managed as grassland in Germany and thus much higher rates than previously found for this type of land use (Tiemeyer et al., 2016). Drained organic soils under for- est can act as both net sinks or sources of atmospheric CO2 (Cannell et al., 1993; Minkkinen and Laine, 1998; Minkki- nen et al., 1999; Wüst-Galley et al., 2016), although they are in general considered to represent a source with aver- age net CO2 emissions of 2.0–3.3 t C ha−1 a−1 in the tem- perate zone (IPCC, 2014). Temperature and soil moisture regime, which depends on drainage depth, among other fac- tors, have the greatest inﬂuence on peat decay in drained organic soils (Hogg et al., 1992; Berglund, 1995; Scanlon and Moore, 2000; Chimner and Cooper, 2003; Couwenberg et al., 2010; Leifeld et al., 2012). However, there are sub- stantial differences in CO2 emissions from organic soils with similar drainage and cultivation properties. The protection of organic matter (OM) against decomposition by mechanisms such as occlusion in aggregates and binding to mineral sur- faces, which are important for the stabilization of OM in mineral soils (Six et al., 2002), are of minor importance in organic soils due to the lack or low abundance of minerals (Han et al., 2016). Therefore, the intrinsic decomposability of organic matter is considered another major factor inﬂuenc- ing the rate of peat decomposition and a major cause of sub- stantial variation in CO2 emissions at different sites (Chim- ner and Cooper, 2003; Byrne and Farrell, 2005; Höper, 2007; Wickland and Neff, 2008; Reiche et al., 2010). between 1.8 and 7.3 t C ha−1 a−1 (IPCC, 2014). However, re- cent studies reported emission rates of 7.6 ± 2.0 t C ha−1 a−1 on organic soils managed as grassland in Germany and thus much higher rates than previously found for this type of land use (Tiemeyer et al., 2016). www.biogeosciences.net/15/703/2018/ C. Bader et al.: Peat decomposability in managed organic soils 705 dations (Flisch et al., 2009). Forest sites were managed and their vegetation was not peat-forming. stantially vary with land use, site conditions and time since land use conversion. Schulze et al. (2009) reported that inputs of fresh organic matter residues were smaller in croplands than in grasslands or forests, suggesting that SOM might be on average more aged and thus less decomposable. In situ measurements of CO2 ﬂuxes from managed organic soils re- veal slower decomposition of peat under forest (IPCC, 2014). Together, smaller peat loss rates and higher residue input make us expect that SOM decomposition rates under con- trolled conditions are fastest in forest topsoils. 2.2 Soil sampling Between October 2013 and June 2015, we sampled a to- tal of 84 peat cores from all 21 sites (4 cores per site). All cores were taken to a maximum depth of 1 m. If the under- lying mineral layer was reached before 1 m depth, coring was discontinued. We used a Belarusian peat corer (cuts a half-cylindrical undisturbed core of diameter 4 cm) for soils with low bulk densities and a motorized Humax corer (cuts a cylindrical core of diameter 5 cm) for denser soils. The sam- ples were stored at 4 ◦C for up to 2 months until analysis. We applied the method of Rogiers et al. (2008) to account for soil compaction during sampling for any sample and divided the cores into segments corresponding to 5–10 cm depth in- crements. This corresponded, depending on the type of soil corer used and length of the increment, to sample volumes of between 31 and 196 cm3 per segment. In total this resulted in 1605 soil samples. Some cores had interlayers of mineral sediment identiﬁed by a different colour (grey), a high bulk density and their SOC content was lower than 150 g kg−1. These interlayers were excluded from the analysis. The soil of one site (BI_FL) had no limnic layer and therefore was classiﬁed as a murshic Histosol; all others were classiﬁed as murshic limnic Histosols (WRB, 2014). In this study, we analysed the relationship between SOM properties, speciﬁc decomposition rates (CO2-C mg−1 SOC) and their temperature sensitivities to peat samples taken from depths between 0 and 200 cm of 21 drained organic soils in Switzerland managed as cropland, perennial grassland or for- est. These sites embody three major uses for drained peat- lands as they occur in Europe (Joosten, 2010) and are also representative of the situation in Switzerland where most for- mer peatlands are drained and managed (Wüst-Galley et al., 2015). We measured decomposition rates in incubation ex- periments under standardized lab conditions and interpreted the current decomposition status of peat using SOM proper- ties such as (i) carbon stocks, bulk densities and the elemental ratios O / C, H / C and C / N as well as (ii) the temperature sensitivity towards decomposition, expecting that 1. speciﬁc decomposition rates of SOM decline with depth, 2.3 Soil analysis 2. speciﬁc decomposition rates of SOM in managed or- ganic soils correlate with its composition and are in- versely related to the temperature sensitivity of decom- position, Soil pH was measured for two to three samples of each core (307 samples in total) using a ﬂat surface electrode (pH 100, Extech Instruments, USA) calibrated at pH 7.00 and pH 4.01. Aliquots of fresh soil (10 g dry matter) were diluted in dis- tilled water (2.5 parts water to 1 part material by mass), shaken, left for 20 h and shaken again, before the pH mea- surements were carried out. 3. speciﬁc decomposition rates of topsoil SOM are largest in the forest and smallest in the croplands. Prior to further chemical analysis, the samples were oven-dried at 105 ◦C and weighed to determine bulk den- sity (g cm−3). The dried samples were ground for 2 min at 25 rotations s−1 in a ball mill (Retsch MM400) and subsam- pled to determine total carbon (Ctot), SOC, hydrogen (H), nitrogen (N) and oxygen (O) contents. Ctot, H and N were analysed after dry combustion of ground subsamples in an el- emental analyser (Hekatech, Germany). To determine SOC, we hydrolysed ground aliquots with 36 % HCl (acid fumi- gation) in a desiccator to remove any carbonates before the samples were analysed in the elemental analyser. A third set of ground subsamples were used to determine the O contents by means of the same analyser after pyrolysis at 1000 ◦C. We corrected O contents for inorganic O, assuming that all inor- ganic O was present in form of CaCO3. The O / C and H / C ratios given in this paper represent mole ratios, whereas the C / N ratios represent mass ratios. For analysis O / C ratios and H / C ratios of samples having a SOC content lower than C. Bader et al.: Peat decomposability in managed organic soils Using solid-state 13C-NMR, DRIFT/FTIR spec- troscopy and pyrolysis-GC/MS, various studies of OM com- position of undisturbed peat proﬁles have shown a gradual The temperature sensitivity of peat mineralization, as ex- pressed by its Q10 value, is a useful parameter for charac- terizing the intrinsic decomposability of SOM (Hogg et al., 1992; Biasi et al., 2005; Davidson and Janssens, 2006; Co- nant et al., 2008; Boddy et al., 2008; Karhu et al., 2010; Hi- lasvuori et al., 2013). In line with the biochemical and ele- mental evidence reviewed above, it was reported to increase with increasing resistance of peat soils against OM decom- position (Scanlon and Moore, 2000), soil depth and peat age (Hardie et al., 2011; Hilasvuori et al., 2013). Despite its likely important role in determining future C losses from drained peatland, the inﬂuence of SOM compo- sition on peat decomposition in managed organic soils is not well studied. While decomposition rates seem to decline with increasing peat age, i.e. proﬁle depth, the oxic conditions, occurring after drainage onset, lead to fast SOM decompo- sition. As for undisturbed organic soils, we expect that post drainage decomposition primarily acts on the most labile OM moieties. However, the much faster decomposition of labile SOM might alter the depth interaction found in undisturbed peat soils. Further, recent inputs from plant residues may sup- ply the topsoils with labile OM. Around 20 % of carbon in organic soils under agriculture is derived from crop residues and thus decomposes more rapidly (Bader et al., 2017). The fractions of OM derived from peat and recent inputs and their decomposability in drained organic soils may, however, sub- www.biogeosciences.net/15/703/2018/ Biogeosciences, 15, 703–719, 2018 C. Bader et al.: Peat decomposability in managed organic soils 2.1 Sampling sites The soil samples used for this study were taken from organic soils distributed across Switzerland that were identiﬁed us- ing the map of Wüst et al. (2015). Apart from current land use (grassland, cropland, forest), they differed in the type of drainage system (ditches in forest, pipes in crop- and grass- land), time since drainage onset and drainage intensity, alti- tude (MASL) and climate (Table 1). All sites were classiﬁed as fens, although we found bog-derived peat layers within the top 30 and 40 cm of the soil proﬁles at two sites (SK_FL, K_FL). Cropland management comprised rotations typical for Switzerland with maize, winter wheat, ley and rapeseed as major crops. Sites were conventionally tilled. Grasslands were used for cutting and haymaking, not grazing, and are fertilized according to the Swiss Fertilization Recommen- www.biogeosciences.net/15/703/2018/ www.biogeosciences.net/15/703/2018/ www.biogeosciences.net/15/703/2018/ Biogeosciences, 15, 703–719, 2018 706 C. Bader et al.: Peat decomposability in managed organic soils Table 1. List of sampling locations, including information on the land-use type, peat thickness, approximate time since drainage onset, elevation (MASL), mean annual temperature (MAT) and mean annual precipitation (MAP) of each site. Location name Abbreviation of Co-ordinates; MASL (m) Area size Peat thickness1 Drainage Drainage MAT4 MAP5 location with WGS (1984) (ha) (cm) history2 class3 (◦C) (mm) land use (CL: cropland, GL: grassland, FL: forest) Birmensdorf BI_FL 8.454, 47.357 560 2.6 95 Unclear; peat excavation nearby s 9.2 1122 Brüttelen B_CL 7.175, 47.033 438 3.1 290 Drained by 1864 d 9.9 1003 Chreienriet F_FL 8.486, 47.434 440 6.9 330 Unclear; peat excavation site d 9.4 1040 nearby until 1940 Cressier C_CL, C_GL 7.047, 47.041 430 1.6, 1.6 120 Drained by 1864 d 10.0 1145 Gals G_CL, G_GL, G_FL 7.065, 47.040 430 1.2, 0.8, 1.0 < 100 Drained by 1864 d 10.0 1145 Im Moos IM_CL 9.573, 47.379 414 5.6 400 Drained by 1860; intensive d 10.1 1297 drainage between 1942 and 1962 Katzensee K_FL 8.495, 47.433 440 1.9 230 Unclear; peat excavation s 9.4 1040 site nearby until 1940 Kirchenthurnen K_GL 7.523, 46.821 540 9.9 302 Drained after 1860 d 8.9 1136 Lüchingen L_CL 9.574, 47.378 414 4.4 400 Drained by 1860; intensive d 10.1 1297 drainage between 1942 and 1962 Mühlethurnen M_CL, M_GL 7.523, 46.821 540 8.2, 7.6 400 Drained after 1860; intensive d 8.9 1136 7.523, 46.817 drainage in 1942 Rüthi R_GL 9.536, 47.283 435 13.3 > 700 Drained by 1970 d 10.1 1533 Staatswald 1 + 2 SW1_FL, SW2_FL 7.092, 46.984 431 30.0, 48.4 142 drained by 1864; intensive d 10.1 990 drainage in 1942 Summerigchopf SK_GL, SK_FL 9.399, 47.212 1300 11.5, 2.2 147–202 Drain established between s 6.0 1731 1935 and 1960 Treiten T_CL 7.145, 47.010 439 29.3 238 Drained by 1864 d 9.9 1033 Vorderwengi VW_GL, VW_FL 9.098, 47.196 1070 1.1, 0.9 100–146 grassland drained by 1935 s 6.2 2240 1 Peat thickness was determined by excavation of an additional peat core down to the underlying sediment layer. 2 Information on drainage was gained by viewing Siegfried topographical maps (1870–1949), considering information on Swiss organic soils by Lüdi (1935) as well as aerial photographs. 3 Shallow drainage < 0.5 m s, deep drainage > 0.5 m d. 4 MAT is the average for the years 1981–2010. 2.4 Incubation experiment We selected at least two soil segments of each soil core from depths between 0–30, 30–60 and 60–100 cm for incuba- tion to determine SOM decomposability. Each segment was divided in two subsegments, whereas one subsegment was incubated at 10 ◦C and the other subsegment at 20 ◦C for between 6 and 13 months. From the one location (M_CL) where we had taken cores of > 100 cm length, we selected six additional samples from the depth below 100 cm for incuba- tion, resulting in a total of 560 incubated samples. Prior to in- cubation, we thoroughly mixed every segment, removed vis- ible roots and adjusted the water potential to −10 kPa using a hanging water column. The sample weight was 53.9 ± 0.7 g (mean ± standard error) at −10 kPa. Following the method of Chapman (1971), we measured CO2 emission rates by means of a Respicond VII analyser (Nordgren Innovation, Sweden) over three to four measurement cycles of several weeks be- tween November 2013 and March 2016. The measurement principle is based on the change in electrical conductivity of the NaOH solution with increasing uptake of CO2. In each cycle, we vented the alkali CO2 traps (NaOH 0.6 M) of the analyser regularly after 50–60 mg of CO2 had been emitted to prevent O2 deﬁciency. In addition, we exchanged the NaOH solution while the traps were vented. Between measurement cycles, we kept the soil samples at the same temperature and moisture level as during the cycles. While R is the gas constant (8.314 J K−1 mol−1) and T is the temperature used for incubation (K). Ea = R × ln(Q10) ( 1 T 1 −1 T 2 ) 1000 (2) (2) Mixed linear models were used to analyse the effects of the various soil parameters on SOM mineralization and their in- teractions with land use. The function lmer from the pack- age lme4 (Bates et al., 2015) was implemented using the software R (R core Team, 2015) to run mixed linear mod- els. Heteroscedasticity or departure from normality was as- sessed graphically. In order to avoid heteroscedasticity, we log-transformed topsoil C stocks and bulk density data. We tested whether the factor “land use” had a signiﬁcant in- ﬂuence on the variation of each of the analysed variables (α = 0.05). 2.4 Incubation experiment To do this, the following two mixed models, 2 and 3, were run for each dependent variable and compared using an ANOVA. C. Bader et al.: Peat decomposability in managed organic soils SOC content of the sample [g kg−1], and msample is the mass of the soil sample [kg]. 150 g kg−1 were excluded from analysis. Soil carbon stocks (t C ha−1) refer to the organic horizons summed over each proﬁle and thus do not include sediment layers that inter- spersed the proﬁles. To determine Q10 values we applied the method used by others (e.g. Hogg et al., 1992; Scanlon and Moore, 2000; Wang et al., 2010; Wetterstedt et al., 2010; Hardie et al., 2011), dividing the 10 000 h length of the incubation period at 10 ◦C by the time span over which samples incubated at 20 ◦C emitted the same amount of CO2-C per mg SOC as those incubated at 10 ◦C emitted during 10 000 h. Given that the same amount of SOC is lost at both temperatures, changes in OM composition during incubation are also as- sumed to be the same and thus differences in the rates are assumed to reﬂect only the inﬂuence of temperature and not that of differences in composition. Q10 values are known to depend on incubation temperatures. In order to compare our results with those of other studies we calculated the activa- tion energy (Ea in kJ mol−1) required for decomposition of SOC using Q10 values. www.biogeosciences.net/15/703/2018/ 5 MAP is the average of the years 1971–1991 derived from original data of MeteoSchweiz. es, 15, 703–719, 2018 www.biogeosciences.net/15/7 Biogeosciences, 15, 703–719, 2018 www.biogeosciences.net/15/703/2018/ 707 2.5 Data analysis We only used CO2 data taken after the ﬁrst 3 days of each measurement cycle for data analysis to avoid artefacts that might have resulted from moving the samples and adjust- ing their water content. Furthermore, we excluded all neg- ative emission rate values (0.45 % out of 1700 CO2 measure- ments taken on average per sample). Data gaps (83 % of the timeline) between measurement cycles were ﬁlled by means of interpolation using a robust linear regression on the log- transformed data. The speciﬁc amount of SOC which was emitted from a sample as CO2 during 10 000 h of incubation at 10 or 20 ◦C [mg CO2-C g−1 SOC], L, was calculated as variable ∼land.use + random effects (3) variable ∼random effects (4) variable ∼land.use + random effects (3) variable ∼random effects (4) variable ∼land.use + random effects (3) variable ∼random effects (4) (3) (4) (4) Sampling depth, sampling location and site repetition were included as random effects to account for the dependence among segments of the same core and among cores from the same sampling location, respectively. In addition, we in- cluded bulk density, SOC, nitrogen, hydrogen and oxygen contents as well as the emitted CO2 as further random effects, given that there was no collinearity with the tested variable and that the Akaikes criterion (AIC) of the models revealed smaller scores with additional random effects. The additional random effects used for each model are given in Tables 2 and 3. Further, we determined the signiﬁcance of land-use- speciﬁc differences (CL vs. FL, CL vs. GL, FL vs. GL) using a least square means test for linear models (lsmeans pack- age). L = (CO2 sample −CO2 blank) × 12.01 44.01 SOCsample × msample , (1) (1) where CO2 sample is the amount of CO2 emitted from the sample over 10 000 h of incubation [mg CO2-C g−1 SOC], CO2 blank is the median of ambient CO2 accumulation col- lected in six blank vessels over more than 6 months and ex- trapolated to 10 000 h (on average 27 mg), SOCsample is the www.biogeosciences.net/15/703/2018/ Biogeosciences, 15, 703–719, 2018 C. Bader et al.: Peat decomposability in managed organic soils 708 C. Bader et al.: Peat decomposability in managed organic soils . Soil pH, bulk density, SOC content, cumulated C stocks, C / N ratios, CO2 emissions and temperature sensitivity (Q10) disp hree land-use types (cropland, grassland and forest) in relation to the proﬁle depth (cm, y axis). CO2 emissions are displayed mbols) and 20 ◦C (black symbols), while the area between dashed lines and error bars represents the standard errors of the me ciences 15 703 719 2018 www biogeosciences net/15/703/ Figure 1. Soil pH, bulk density, SOC content, cumulated C stocks, C / N ratios, CO2 emissions and temperature sensitivity (Q10) displayed for the three land-use types (cropland, grassland and forest) in relation to the proﬁle depth (cm, y axis). CO2 emissions are displayed at 10 (open symbols) and 20 ◦C (black symbols), while the area between dashed lines and error bars represents the standard errors of the mean. Biogeosciences, 15, 703–719, 2018 C. Bader et al.: Peat decomposability in managed organic soils C. Bader et al.: Peat decomposability in managed organic soils C. Bader et al.: Peat decomposability in managed organic soils 709 Table 2. Results of land-use effect analysis for the whole soil proﬁle as well as speciﬁcally in the topsoil (0–30 cm) and bottom layers (> 30 cm), displayed for SOC concentration, C stocks, bulk density C / N, H / C and O / C ratios, CO2 emissions at 10 and 20 ◦C and the resulting Q10 values. Table 2. Results of land-use effect analysis for the whole soil proﬁle as well as speciﬁcally in the topsoil (0–30 cm) and bottom layers (> 30 cm), displayed for SOC concentration, C stocks, bulk density C / N, H / C and O / C ratios, CO2 emissions at 10 and 20 ◦C and the resulting Q10 values. Land-use interaction1 P values between speciﬁc land-uses2 Attribute χ2 value P value CL vs. FL CL vs. GL FL vs. C. Bader et al.: Peat decomposability in managed organic soils GL Soil pH χ2(2) = 3.7 0.16 Soil pH (0–30 cm) χ2(2) = 14.9 0.0006 0.0003 0.9 0.0021 Soil pH (> 30 cm) χ2(2) = 0.7 0.7 SOC χ2(2) = 10.7 0.005 0.14 0.28 0.002 SOC (0–30 cm) χ2(2) = 14.5 0.0001 0.0001 0.5 0.009 SOC (> 30 cm) χ2(2) = 3.0 0.2 Cumulative C stock C stock (0–30 cm) χ2(2) = 5.4 0.07 0.06 0.4 0.6 C stock (0–100 cm) χ2(2) = 5.4 0.06 0.2 0.06 0.8 Bulk density χ2(2) = 3.4 0.2 Bulk density (0–30 cm) χ2(2) = 10.3 0.06 0.02 0.09 0.4 Bulk density (> 30 cm) χ2(2) = 2.0 0.4 C / N ratio χ2(2) = 5.9 0.05 0.06 0.9 0.1 C / N ratio (0–30 cm) χ2(2) = 15.0 0.0005 0.0002 0.8 0.003 C / N ratio (> 30 cm) χ2(2) = 2.2 0.3 H / C ratio χ2(2) = 6.7 0.04 0.5 0.4 0.02 H / C ratio (0–30 cm) χ2(2) = 6.3 0.04 0.6 0.4 0.03 H / C ratio (> 30 cm) χ2(2) = 3.5 0.2 O / C ratio χ2(2) = 11.5 0.003 0.4 0.7 0.001 O / C ratio (0–30 cm) χ2(2) = 10.5 0.005 0.06 0.7 0.003 O / C ratio (> 30 cm) χ2(2) = 8.5 0.014 0.008 1.0 0.003 CO2 10◦C χ2(2) = 2.4 0.3 CO2 10 ◦C (0–30 cm) χ2(2) = 2.9 0.2 CO2 10 ◦C (30–60 cm) χ2(2) = 7.17 0.03 0.023 0.38 0.34 CO2 10 ◦C (> 60 cm) χ2(2) = 1.6 0.4 CO2 20◦C χ2(2) = 1.4 0.5 CO2 20 ◦C (0–30 cm) χ2(2) = 6.5 0.04 0.03 0.2 0.7 CO2 20 ◦C (30–60 cm) χ2(2) = 1.7 0.4 CO2 20 ◦C (> 60 cm) χ2(2) = 1.2 0.5 Q10 χ2(2) = 3.5 0.2 Q10 (0–30 cm) χ2(2) = 0.4 0.8 Q10 (30–60 cm) χ2(2) = 1.1 0.6 Q10 (> 60 cm) χ2(2) = 1.0 0.6 1 P value of ANOVA comparing linear mixed models with and without the factor “land-use type”. 2 P value emitted using least square means between land-use types. 1 P value of ANOVA comparing linear mixed models with and without the factor “land-use type”. 2 P value emitted using least square means between land-use types. in addition to Eq. (3). To determine the signiﬁcance of depth- speciﬁc differences, we used a least square means test as mentioned before. in addition to Eq. (3). To determine the signiﬁcance of depth- speciﬁc differences, we used a least square means test as mentioned before. C. Bader et al.: Peat decomposability in managed organic soils Table 3. Results of the depth inﬂuence analysis displayed for Q10 values, CO2 emissions at 10 and 20 ◦C, SOC contents, bulk densities, C / N ratios, H / C ratios and O / C ratios. Ea values (not shown) had similar signiﬁcance to Q10 values. Depth interaction P values between speciﬁc depth classes Attributes χ2 values P value 0–30 vs. 30–60 0–30 vs. > 60 30–60 vs. C. Bader et al.: Peat decomposability in managed organic soils We used the same approach to test the inﬂuence of the factor “soil depth” on the target variables with interactions between the three sampling depths (0–30, 30–60, > 60 cm) using the model (5) Biogeosciences, 15, 703–719, 2018 710 Biogeosciences, 15, 703–719, 2018 www.biogeosciences.net/15/703/2018/ C. Bader et al.: Peat decomposability in managed organic soils C. Bader et al.: Peat decomposability in managed organic soils 711 C. Bader et al.: Peat decomposability in managed organic soils 711 Table 4. Coefﬁcients of determination and correlation for CO2 emissions measured at 20 ◦C and different soil attributes as explanatory variables (proﬁle depth, SOC content, bulk density, C / N, O / C and H / C ratio). Ea values (not shown) behaved similarly to Q10 values. C. Bader et al.: Peat decomposability in managed organic soils 711 Table 4. Coefﬁcients of determination and correlation for CO2 emissions measured at 20 ◦C and different soil attributes as explanatory variables (proﬁle depth, SOC content, bulk density, C / N, O / C and H / C ratio). Ea values (not shown) behaved similarly to Q10 values. C. Bader et al.: Peat decomposability in managed organic soils Table 4. Coefﬁcients of determination and correlation for CO2 emissions measured at 20 ◦C and different soil attributes as explanatory variables (proﬁle depth, SOC content, bulk density, C / N, O / C and H / C ratio). Ea values (not shown) behaved similarly to Q10 values. Table 4. Coefﬁcients of determination and correlation for CO2 emissions measured at 20 ◦C and different soil attributes as explanatory variables (proﬁle depth, SOC content, bulk density, C / N, O / C and H / C ratio). Ea values (not shown) behaved similarly to Q10 values. Figure 2. Van Krevelen plots of samples from (a) the upper 30 cm and (b) depths below 30 cm. Symbols represent averages for relevant core segments from each site, black bars represent the standard error of the mean, grey surfaces represent the range of O / C and H / C for lignin, carbohydrates and lipids, adapted from Preston and Schmidt (2006). Figure 2. Van Krevelen plots of samples from (a) the upper 30 cm and (b) depths below 30 cm. Symbols represent averages for relevant core segments from each site, black bars represent the standard error of the mean, grey surfaces represent the range of O / C and H / C for lignin, carbohydrates and lipids, adapted from Preston and Schmidt (2006). www.biogeosciences.net/15/703/2018/ Biogeosciences, 15, 703–719, 2018 Biogeosciences, 15, 703–719, 2018 C. Bader et al.: Peat decomposability in managed organic soils Attribute 0–30 cm 30–100 cm CO2 at 20 ◦C Intercept cor P value R2 Intercept cor P value R2 Depth (cm) < 2.0 × 10−16 −0.23 0.01 0.05 0.0001 0.11 0.2 0.01 SOC (g kg−1) 8.06 × 10−6 0.31 0.001 0.09 7.6 × 10−6 −0.01 0.9 4.8 × 10−5 Bulk density (g kg−1) < 2.0 × 10−16 −0.27 0.003 0.07 < 2 × 10−16 −0.19 0.02 0.04 C / N ratio 0.002 0.28 0.002 0.08 3.5 × 10−6 −0.1 0.5 0.004 O / C ratio 1.33 × 10−6 −0.02 0.9 0.0002 2.1 × 10−5 0.06 0.4 0.01 H / C ratio 0.046 0.03 0.8 0.0005 0.292 0.07 0.4 0.01 pH 2.3 × 10−8 −0.29 0.001 0.09 2.3 × 10−8 −0.25 0.003 0.06 CO2 at 10 ◦C Depth (cm) 1.7 × 10−13 0.19 0.2 0.02 0.0002 0.10 0.2 0.02 SOC (g kg−1) 1.7 × 10−5 −0.13 0.04 0.03 4.8 × 10−16 −0.24 0.002 0.06 Bulk density (g kg−1) < 2.0 × 10−16 −0.22 0.02 0.04 < 2.0 × 10−16 −0.01 0.9 9.1 × 10−5 C / N ratio 0.0007 0.31 0.0007 0.09 1.05 × 10−5 −0.03 0.7 0.0008 O / C ratio 3.8 × 10−15 0.01 1.0 3.4 × 10−5 3.4 × 10−5 0.10 0.2 0.01 H / C ratio 0.5 0.14 0.1 0.2 0.3 0.24 0.002 0.06 pH 6.6 × 10−5 −0.14 0.1 0.02 0.0001 −0.09 0.3 0.007 Q10 values Depth (cm) < 2.0 × 10−16 0.18 0.12 0.01 0.0001 −0.30 0.0001 0.08 SOC (g kg−1) < 2.0 × 10−16 −0.16 0.03 0.03 < 2.0 × 10−16 0.12 0.06 0.02 Bulk density (g kg−1) < 2.0 × 10−16 −0.02 0.6 −0.006 < 2.0 × 10−16 −0.02 0.4 −0.002 C / N ratio 8.1 × 10−16 −0.10 0.15 0.01 1.2 × 10−15 −0.08 0.4 −0.002 O / C ratio 5.3 × 10−15 0.20 0.07 0.02 < 2.0 × 10−16 −0.13 0.3 0.0002 H / C ratio 0.0001 0.06 0.2 0.004 2.9 × 10−8 −0.13 0.06 0.02 pH 1.02 × 10−9 −0.03 0.8 0.0006 2.7 × 10−8 −0.02 0.8 0.0003 Figure 2. Van Krevelen plots of samples from (a) the upper 30 cm and (b) depths below 30 cm. Symbols represent averages for relevant core segments from each site, black bars represent the standard error of the mean, grey surfaces represent the range of O / C and H / C for lignin carbohydrates and lipids, adapted from Preston and Schmidt (2006). C. Bader et al.: Peat decomposability in managed organic soils > 60 Q10 values χ2(2) = 46.2 9.56 × 10−11 0.05 < 0.0001 < 0.0001 Q10 cropland χ2(2) = 16.1 0.0003 0.8 0.0002 0.002 Q10 forest χ2(2) = 5.2 0.08 Q10 grassland χ2(2) = 29.5 3.9 × 10−7 0.06 < 0.0001 0.009 CO2 emission (10 ◦C) χ2(2) = 6.1 < 0.05 0.03 0.7 0.2 Cropland (10 ◦C) χ2(2) = 1.5 0.5 Forest (10 ◦C) χ2(2) = 17.3 0.0001 0.0001 0.01 0.5 Grassland (10 ◦C) χ2(2) = 7.9 0.02 0.01 0.3 0.5 CO2 emission (20 ◦C) χ2(2) = 0.9 0.6 Cropland (20 ◦C) χ2(2) = 8.4 0.015 0.02 1.0 0.09 Forest (20 ◦C) χ2(2) = 13.2 0.0001 0.0007 < 0.05 0.6 Grassland (20 ◦C) χ2(2) = 3.5 0.17 pH χ2(2) = 6.0 Cropland χ2(2) = 19.4 6.2 × 10−5 < 0.02 < 0.0001 0.09 Forest χ2(2) = 36.8 1 × 10−8 0.004 < 0.0001 0.001 Grassland χ2(2) = 27.4 1.1 × 10−6 0.0001 < 0.0001 0.9 SOC χ2(2) = 157.7 < 2.2 × 10−16 < 0.0001 < 0.0001 0.0001 Cropland χ2(2) = 158.2 < 2.2 × 10−16 < 0.0001 < 0.0001 < 0.0001 Forest χ2(2) = 3.8 0.15 0.2 1.0 0.3 Grassland χ2(2) = 143.2 < 2.2 × 10−16 < 0.0001 < 0.0001 < 0.0001 Bulk density χ2(2) = 57.6 < 3.1 × 10−13 < 0.0001 < 0.0001 0.9 Cropland χ2(2) = 312.6 < 2.2 × 10−16 < 0.0001 < 0.0001 < 0.0001 Forest χ2(2) = 31.6 1.4 × 10−7 0.7 < 0.0001 < 0.0001 Grassland χ2(2) = 49.9 1.4 × 10−11 < 0.0001 < 0.0001 0.08 C / N ratio χ2(2) = 325.5 < 2.2 × 10−16 < 0.0001 < 0.001 < 0.0001 Cropland χ2(2) = 199 < 2.2 × 10−16 < 0.0001 < 0.001 < 0.0001 Forest χ2(2) = 41.2 1.5 × 10−9 0.4 < 0.001 < 0.0001 Grassland χ2(2) = 152.8 < 2.2 × 10−16 < 0.0001 < 0.001 < 0.0001 H / C ratio χ2(2) = 19.9 < 4.7 × 10−5 < 0.0001 < 0.0002 0.7 Cropland χ2(2) = 46.7 7.3 × 10−11 < 0.0001 < 0.0001 0.3 Forest χ2(2) = 1.9 0.38 Grassland χ2(2) = 52.6 3.7 × 10−12 < 0.0001 < 0.0001 0.07 O / C ratio χ2(2) = 22.0 0.0005 < 0.06 0.03 0.9 Cropland χ2(2) = 0.01 1.0 Forest χ2(2) = 2.6 0.3 Grassland χ2(2) = 6.3 0.04 0.03 0.5 0.5 Biogeosciences, 15, 703–719, 2018 www.biogeosciences.net/15/703/2018/ Biogeosciences, 15, 703–719, 2018 www.biogeosciences.net/15/703/2018/ 3.1 SOM characteristics The samples incubated at 10 ◦C emitted 32.56 ± 1.39 mg CO2-C g−1 SOC, while samples incubated at 20 ◦C emit- ted 74.06 ± 2.98 mg CO2-C g−1 SOC (Fig. 1). At 10 ◦C we did not observe a land-use effect on CO2 emission (Fig. 1, Ta- ble 2), but at 20 ◦C the topsoil samples from croplands emit- ted less CO2 than those from forests. This effect occurred due to extraordinarily high emissions of the samples from two grassland and two forest sites (VW_GL, VW_F, SK_GL, SK_F) (Fig. 3). Those four sites experienced the least inten- sive drainage. Furthermore, these sampling sites were situ- ated at high altitude in a pre-alpine environment with lower mean annual temperatures and higher precipitation than at the other sites (Table 1). In pairwise comparisons between adjacent sites of different land use (i.e. VW_GL vs. VW_FL, SK_GL vs. SK_FL, C_CL vs. C_GL and G_CL vs. G_GL and G_FL; Table 1), land-use effects were only found for the last site (Fig. 3). Soil pH, SOC content, C / N ratio and bulk density showed signiﬁcant land-use effects (Fig. 1, Table 2, Table S1). The lowest soil pH values were found in the forest topsoil sam- ples, whereas SOC content and C / N ratio were the highest in these samples. Bulk density was highest in the cropland topsoils. Below 30 cm depth, soil pH, SOC content, C / N ra- tio and bulk density showed no land-use effect. In the forest soil proﬁles, soil pH overall increased with depth, whereas it decreased in the grassland and cropland soils (Table 3). Also bulk density decreased with depth in the grassland and cropland soils, while SOC content and C / N ratio increased. In the forest soils, SOC content, bulk density and C / N ratio did not differ between topsoil (0– 30 cm depth) and subsoil samples (30–60 cm depth); how- ever, below 60 cm depth SOC was slightly lower than above, while bulk density and C / N ratio were higher than above 60 cm depth (Fig. 1, Table 3). The cumulated topsoil C stocks showed no land-use effects but tended to be larger in crop- land and forest than in grassland soils over the entire proﬁle (Fig. 1, Table 2). C. Bader et al.: Peat decomposability in managed organic soils 712 Figure 3. CO2 emissions at 10 (open symbols) and 20 ◦C (grey symbols) displayed for upper soil layers (0–30 cm) and bottom layers (30– 100 cm) of all sampling locations. Sampling locations are sorted from the lowest to highest CO2 emissions in (a). Same order of sites was taken for (b). Error bars represent the standard error of the mean. If a symbol lacks error bars, the standard error was smaller than the symbol size or, as in the cases of upper soil layers SW1 and SW2, n = 2. www.biogeosciences.net/15/703/2018/ 712 C. Bader et al.: Peat decomposability in managed organic soils Figure 3. CO2 emissions at 10 (open symbols) and 20 ◦C (grey symbols) displayed for upper soil layers (0–30 cm) and bottom layers (30– 100 cm) of all sampling locations. Sampling locations are sorted from the lowest to highest CO2 emissions in (a). Same order of sites was taken for (b). Error bars represent the standard error of the mean. If a symbol lacks error bars, the standard error was smaller than the symbol size or, as in the cases of upper soil layers SW1 and SW2, n = 2. C. Bader et al.: Peat decomposability in managed organic soils C. Bader et al.: Peat decomposability in managed organic soils 713 Table 5. Incubation studies with organic and mineral soils at different moisture levels, soil depths and temperatures. If moisture level stays moist, samples were incubated directly after being retrieved from the ﬁeld, while saturated samples were incubated under wetter, i.e. anaerobic conditions. Similar stands for samples whose water content was similar to our samples. Soil Region Moisture ◦C Days C / N Depths CO2 emissions Q10 Ea level (cm) (mg CO2-C g−1 SOC d−1) (kJ mol−1) This study Drained Fens Switzerland −10 kPa 10 416 17.7 5–150 0.078 2.57 69.5 20 0.18 Chapman and Thurlow (1998) Drained/undrained UK (Scotland) Moist 10 unclear 0–20 0.051∗ 3.2 80.0 Bogs 20 0.030∗ Grover and Baldock (2012) Bog Australia Moist 20 38 15–25 5–110 0.13–0.78 Hahn-Schöﬂet al. (2011) Fen Germany Saturated 20 346 15.3 0.013 Hardie et al. (2011) Bog UK Drier 5 6 30 0-30 0.027 3.66 86.4 10 0.049 15 0.093 Hartley and Ineson (2008) Mineral soil UK Drier 10 124 unclear 0.046 3.25 81.3 20 0.074 Hilasvuori et al. (2013) Bog Finland Moist 10 short 83 0–44 0.016∗ 2 22.7 20 0.061∗ Hogg et al. (1992) Fen Canada Similar 8 120 40.6 5–40 0.083 1.9–2.2 62.0 16 0.282 24 0.381 Karhu et al. (2014) Organic soil UK (Scotland) Similar 11.4 174 28.6 0–10 0.065 Organic soil UK (Scotland) 7.6 174 36.5 0-10 0.105 Organic soil UK 13.3 174 18.7 0–10 0.201 Mineral soil UK 11.4 174 13.3 0–10 0.101 Mineral soil Spain 21.5 174 14.3 0–10 0.293 Mineral soil Spain 19 174 13.0 0–10 0.345 Mineral soil Spain 19 174 18.6 0–10 0.448 Mineral soil Italy 18.4 174 13.2 0–10 0.129 Karhu et al. (2010) Mineral soil Finland 8–25 540 – 0–30 – 3.0 45.0 Koch et al. (2007) Organic soil Austria Moist 0–30 25 21.6 0–5 – 2.0 31.9 Leifeld and Fuhrer (2005) Mineral soil Switzerland Similar 25 707 7.85 5–35 0.12 4.6 110.8 Neff and Hooper (2002) Organic soil USA (Alaska) Unclear 10 352 34.6 0–10 0.32 1.9 22.9 30 0.75 Plante et al. (2010) Mineral soil USA Similar 15 56 0–20 0.28 1.36–1.79 31.7 Reiche et al. (2010) Fen Germany Saturated 15 31 30.1 0–40 0.0022 Reichstein et al. (2000) Mineral soil Switzerland Similar 5 104 30.3 0.05 2.5–2.7 65.9 15 0.14 25 0.22 Scanlon and Moore (2000) Fen Canada Moist 4 12 43 5–45 0.227 2.0 45.8 14 0.109 Wang et al. 3.1 SOM characteristics At 10 ◦C, CO2 emissions of the topsoil samples from all sites together were higher than from samples taken at 30 to 60 cm depth, independent of land use (Table 3). When analysing the inﬂuence of depth separately by land-use type, this effect was found to only manifest in grassland and forest but not in cropland soils. We found no overall depth effect at 20 ◦C, but CO2 emissions of topsoil samples from forests were higher than those of samples taken at lower depths, whereas we found the opposite case for the cropland soils. Despite these depth effects, the general relationship between emissions and soil depth was weak and not consistent in its sign (Table 4). The molar H / C and O / C ratios of the organic matter fell between the typical values of the ratios for carbohydrates and lignin, which is displayed in a Van Krevelen plot (Fig. 2). The lowest values of both ratios were found in the forest soils, the highest in the grassland and cropland topsoils. Both ratios were lower in the topsoils than in the subsoils of the cropland and grassland sites, while there was no difference between the two depths in the forest soils (Table 3). At depths below 30 cm, the O / C ratio was lower in the forest soils than in the other soils but without a land-use effect in the H / C ratio. Over the course of the incubation, CO2 emissions in- creased for 40 % of the samples, as revealed in Table S1 by positive slopes of the regression lines. These increases were independent of land use. In total, the CO2 emissions from Biogeosciences, 15, 703–719, 2018 www.biogeosciences.net/15/703/2018/ C. Bader et al.: Peat decomposability in managed organic soils 4.1 SOM characteristics The SOC contents, bulk densities and C / N ratios found in the deeper parts of soil proﬁles presented here were close to values that are typical for undisturbed peat (Grover and Baldock, 2012; Loisel et al., 2014). They also indicate that soils of our study sites were characteristic of European fens and resembled typical properties of managed organic soils (Berglund, 1995; Kechavarzi et al., 2010; Eickenscheidt et al., 2015; Krueger et al., 2015; Wüst-Galley et al., 2016; Brouns et al., 2016). Several studies assume that deeper layer peat of managed organic soils is less decomposed (Ewing and Vepraskas, 2006; Rogiers et al., 2008; Leifeld et al., 2011a, b; Krueger et al., 2015; Wüst-Galley et al., 2016). We therefore interpret the different SOM characteristics found in the top- soils of our samples as indicators of advanced decomposition triggered by drainage. The land-use-speciﬁc differences manifested in different topsoil SOC contents and C / N ratios (highest under forest) and topsoil bulk densities (lowest under forest). The higher forest C / N ratios might be explained by absence of the use of N fertilizers and lower bulk densities by lower trafﬁc with ﬁeld machinery. In addition, differences in C / N between land-use types may also suggest that peat decomposition was less advanced in forests compared to croplands and grass- lands. Further, depth effects are lowest in forest soils, indi- cating a lower impact of soil management that could also result in a lower decomposition of forest topsoils. The rela- tively high carbon stocks found in cropland top soils are most likely the result of subsidence after drainage and compaction from ﬁeld trafﬁc, leading to increased soil bulk density in the uppermost layers. This effect, with respect to C stocks, overrides the overall much smaller C concentration in agri- culturally managed organic soils. 4 Discussion The H / C and O / C ratios reﬂect the mixing ratio of these two SOM sources. The H / C and O / C ratios in forest top- soils were lower than of those under cropland and grassland and did not change with depth. Interpreting these lower H / C and O / C ratios in the forest topsoils as indicators of more advanced peat decomposition (Klavins et al., 2008; Leifeld et al., 2012; Biester et al., 2014; Wüst-Galley et al., 2016) would be in contradiction to our conjecture that land manage- ment effects on peat decomposition, revealed by SOC, bulk density and C / N ratio, are less pronounced for forests. We rather argue that the reason for the low H / C and O / C ratio in the forest soils is a higher abundance of lignin rich (wood derived) plant residues. A second mechanism for comparably higher O / C and H / C ratios in cropland and grassland soils could be that peat loss in the uppermost layers was higher under agriculture than under forest, resulting in a relatively higher share of SOM from recent inputs. Considering all the available evidence of SOM characteristics, we conclude that peat decomposition is less advanced in forest soils than in agricultural soils and also in line with ﬁeld ﬂux measure- ments on managed organic soils that typically show faster decomposition in croplands and grasslands than in forests (IPCC, 2014). C. Bader et al.: Peat decomposability in managed organic soils 714 these samples were almost 50 % higher than those from the other samples that instead showed a trend of decreasing emis- sions. of polyphenols and aromatic carbon with depth, which is in line with the current understanding of peat development in peatlands (Cocozza et al., 2003; Zaccone et al., 2007; Klavins et al., 2008; Delarue et al., 2011; Leifeld et al., 2012, 2017). The increased H / C and O / C ratios in the grass- and crop- land topsoils can be attributed to inputs of fresh plant litter to the topsoil via above- and belowground residues, as such residues are rich in carbohydrates (Koegel-Knabner, 2002). In a previous study, in which we used stable and radiocarbon isotopes to label the SOC of two of the studied soils (C_CL and C_GL in Table 1), at least 20 % of topsoil organic mat- ter was not peat but derived from recent plant litter (Bader et al., 2017). The results further indicated that the OM derived from these fresh plant residues was a source of labile C that contributed more to decomposition than the old, peat-derived SOM. Mean Q10 values were 2.57 ± 0.05. The Q10 did not differ between the three land use types. It was lower below 60 cm depth in the cropland and grassland but not in the forest soils (Fig. 1, Table 3). Activation energies (Ea) calculated from Q10 values ranged around 48.1 and 123.5 kJ mol−1 and like Q10 values decreased with depth. There were signiﬁcant re- lationships between CO2 emission and SOC content, bulk density and C / N ratio in general but they were weak (Ta- ble 4). The Q10 values showed similar relationships to these soil variables as CO2 emission. C. Bader et al.: Peat decomposability in managed organic soils (2010) Organic soil China Similar 5–20 40 28.5 10–30 0.31 2.2 53.3 Wickland and Neff (2008) Organic soil Canada Similar 10 57 24.7 2–30 0.35 1.7 36.6 20 0.79 Yavitt et al. (2000) Bog Canada Moist 12–22 2 0–54 – 1.4 32.5 ∗Study authors are not speciﬁc about the SOC content of peat; therefore we assumed it to be 400 g kg−1, according to the ﬁndings of undisturbed bog peat (Loisel et al., 2014). www.biogeosciences.net/15/703/2018/ Biogeosciences, 15, 703–719, 201 www.biogeosciences.net/15/703/2018/ C. Bader et al.: Peat decomposability in managed organic soils C. Bader et al.: Peat decomposability in managed organic soils 715 C. Bader et al.: Peat decomposability in managed organic soils Figure 4. Comparison of daily CO2 emission rates from this study (box plots) with rates found during other incubation studies (organic soils and mineral soils). The curves represent the modelled CO2 emission rates for organic soils from other studies (solid thick line) rate = 0.06 × 100.08t and mineral soils (dashed thin line) rate = 0.09 × 100.05t for temperatures between 0 and 30 ◦C. sition rates in the top 30 to 50 cm (Hogg et al., 1992; Scanlon and Moore, 2000; Wang et al., 2010; Hardie et al., 2011), we found a decrease of speciﬁc CO2 release with depth. How- ever, the relationship between CO2 emissions and depth was rather weak in our case and not consistent for both incuba- tion temperatures and the different land uses. Compared to the studies on unmanaged organic soils, reporting declines of a factor 2 to 30 (Hogg et al., 1992; Scanlon and Moore, 2000; Wang et al., 2010; Hardie et al., 2011), our differ- ences were substantially smaller. Drainage and decadal agri- cultural use of the studied soils led to more intense decom- position processes in the topsoil, resulting in little depth in- teraction or, for croplands, sometimes maybe even a reversal of decomposability. Further, the abundance and decompos- ability of crop residues have to be considered as a substan- tial CO2 source. For two topsoils (C_GL and C_CL), Bader et al. (2017) showed that at least 20 % of the SOM is crop residue derived and responsible for 40 % of the emitted CO2. Assuming that the abundance of crop and plant residues is highest in topsoils, it might be possible that decomposabil- ity of peat-derived SOM either does not depend on depth or topsoil peat decomposes at smaller rates. Therefore, we can- not conﬁrm our ﬁrst hypothesis of decreasing decomposition rates with depth. of the regression line that is plotted through results from studies from other, mostly intact or little degraded organic soils. Hence, the pronounced oxidative decomposition after long periods of drainage might result in a relatively smaller labile SOC pool, but the large variability between experi- mental set-ups, incubation lengths and water contents among incubation studies prevents a stronger line of interpretation. 4.2 CO2 emissions and temperature sensitivity of decomposition The studied soils emitted, on average, ca. 5–10 % of their SOC (20 ◦C) as CO2, calculated for an incubation duration of 1 year. The advanced decomposition state of many of the samples might give reason to expect that CO2 rates are be- low that of more intact peat or mineral topsoils that con- tain a larger fraction of recent plant residues. To understand whether SOM in the studied organic soils is particularly sta- ble, we compared its average daily carbon loss with data from studies that used undisturbed to extensively managed organic soils or mineral soils (Table 5, Fig. 4). Indeed, our values are on average below those from other organic soil studies. However, their range overlaps with the uncertainty The H / C and O / C ratios in the deeper layers of the stud- ied soils were similar to those found in undisturbed bogs and drained bogs used for forestry in Switzerland (Zaccone et al., 2007; Wüst-Galley et al., 2016). They indicate an enrichment Biogeosciences, 15, 703–719, 2018 www.biogeosciences.net/15/703/2018/ C. Bader et al.: Peat decomposability in managed organic soils 715 Figure 4. Comparison of daily CO2 emission rates from this study (box plots) with rates found during other incubation studies (organic soils and mineral soils). The curves represent the modelled CO2 emission rates for organic soils from other studies (solid thick line) rate = 0.06 × 100.08t and mineral soils (dashed thin line) rate = 0.09 × 100.05t for temperatures between 0 and 30 ◦C. www.biogeosciences.net/15/703/2018/ C. Bader et al.: Peat decomposability in managed organic soils Interestingly, the regression lines modelled for organic and mineral soils did not deviate signiﬁcantly from each other. Therefore, the pools size of labile carbon, indicated by the decomposition rates, seem not to differ between these soil classes. This comparison suggests that accumulation of re- cent, labile plant materials that presumably account for most of the evolved CO2 is not systematically different between mineral and organic soils. Samples showing an increase in CO2 emission rate over time were predominantly of subsoil origin, where SOC con- tents and C / N ratios indicate a lower decomposition than in the topsoil. Furthermore, based on the information we have on land use and drainage depths, it appears that most of these samples were taken from soil layers that were protected from intensive decomposition by water saturation. The long incu- bation period in our study might have given aerobic decom- poser communities time to develop and grow, whereas time might not have been sufﬁcient in shorter studies. As Table 5 shows, the Q10 values found in our study (2.74 ± 0.06) were higher than Q10 values found elsewhere for similar sampling depths but in unmanaged organic soils Like other studies on extensively managed or undisturbed organic soils that investigated depth interaction of decompo- C. Bader et al.: Peat decomposability in managed organic soils 716 (Chapman and Thurlow, 1998; Hamdi et al., 2013; Hardie et al., 2011; Hilasvuori et al., 2013; Hogg et al., 1992; Scanlon and Moore, 2000; Yavitt et al., 2000). Also the temperature-independent Ea was higher in the studied sam- ples (69.4 ± 3 kJ mol−1) than in most other studies on undis- turbed organic soils (47.4 ± 7.2 kJ mol−1) (Table 5). How- ever, three studies (Chapman, 1971; Hardie et al., 2011; Hogg et al., 1992) found similar or higher Ea values in northern organic soils. In the case of Chapman and Thur- low (1998) they were also managed as grassland or forest, whereas the other studies used peat from undisturbed or- ganic soils. Nevertheless, the high Ea of the studied samples might reﬂect the change in chemical peat composition with decomposition after drainage towards higher recalcitrance. In contrast to other studies on unmanaged organic soils re- porting no trend or increasing Q10 values with depth (Scan- lon and Moore, 2000; Wang et al., 2010; Hardie et al., 2011; Hilasvuori et al., 2013), the cropland and grassland proﬁles in our study had a lower Q10 below the 60 cm depth. Var- ious studies on SOM decomposition used Q10 values as an indicator of SOM recalcitrance (Hogg et al., 1992; Biasi et al., 2005; Davidson and Janssens, 2006; Conant et al., 2008, 2011; Hartley and Ineson, 2008; Hilasvuori et al., 2013). Considering that the presence of labile crop residues would decrease Q10 in the topsoil rather than in the subsoil, the higher topsoil Q10 may be explained by an extended accu- mulation of recalcitrant moieties. This proposed a high abun- dance of recalcitrant moieties in topsoils of degrading or- ganic soils is in line with the reported differences in SOM composition in different layers as well with the pattern of CO2 emissions. The latter show no substantial difference with depth and indicate that a higher fraction of recent and labile plant residues in topsoils is counterbalanced by a high recalcitrance of the highly degraded peat. Comparing radio- carbon concentrations in SOC and emitted CO2 of two sites also used for this study (C_CL, C_GL), Bader et al. (2017) estimated that SOC from plant residue inputs is more labile than peat. The measured radiocarbon contents for SOC were 75 to 80 pMC and therefore indicated that peat of the topsoil must have experienced a substantial decomposition. al., 2012). C. Bader et al.: Peat decomposability in managed organic soils However, these studies focused mainly on single proﬁles of undisturbed or extensively used organic soils. A recent study investigated relationships between SOM param- eters and decomposition rates of German organic soils under controlled conditions (Säurich et al., 2017). These authors mostly studied strongly disturbed fens with similar proper- ties to the soils in our study. Besides SOC contents, soil pH and C / N ratios, Säurich et al. (2017) focused on other soil nutrients, stable isotopes and microbial biomass. In line with our results, they could not identify strong proxies for SOC decomposition by means of simple chemical attributes. In order to explain the weak relationships between SOM composition and CO2 release it should be considered that, in our case, the emitted CO2 comprised, on average, only 3.2– 7.4 % of the total SOC, while the analysed SOM parameters in this and other studies represent bulk SOM. Our methods allowed us to gain a broad overview of the chemical compo- sition of SOM, while decomposition might more tightly be bound to the abundance of speciﬁc OM moieties. Although land-use-affected SOM characteristics, such as elemental contents and their ratios, the amount of CO2 emit- ted from the soils did not differ among the three types of land use. We therefore have to also reject our third hypothesis of a higher SOM decomposition rate in forest topsoils. We as- sume that long-lasting drainage and management might have resulted in an equivalent decomposition of most of the labile OM, along with its intrinsic decomposability. www.biogeosciences.net/15/703/2018/ Biogeosciences, 15, 703–719, 2018 www.biogeosciences.net/15/703/2018/ References Bader, C., Müller, M., Schulin, R., and Leifeld, J.: Amount and sta- bility of recent and aged plant residues in degrading peatland soils, Soil Biol. Biochem., 109, 167–175, 2017. Couwenberg, J., Dommain, R., and Joosten, H.: Greenhouse gas ﬂuxes from tropical peatlands in south-east Asia, Global Change Biol., 16, 1715–1732, 2010. Bates, D., Maechler, M., Bolker, B. M., and Walker, S. C.: Fitting Linear Mixed-Effects Models Using lme4, J. Stat. Softw., 67, 1– 48, 2015. Davidson, E. A. and Janssens, I. A.: Temperature sensitivity of soil carbon decomposition and feedbacks to climate change, Nature, 440, 165–173, 2006. 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L.: Turnover of low molecular weight dissolved organic C (DOC) and microbial C exhibit different temperature sensitivities in Arc- tic tundra soils, Soil Biol. Biochem., 40, 1557–1566, 2008. Freeman, C., Ostle, N. J., Fenner, N., and Kang, H.: A regulatory role for phenol oxidase during decomposition in peatlands, Soil Biol. Biochem., 36, 1663–1667, 2004. Brouns, K., Keuskamp, J. C. Bader et al.: Peat decomposability in managed organic soils 717 Cannell, M., Dewar, R., and Pyatt, D.: Conifer Plantations on Drained Peatlands in Britain – a Net Gain or Loss, Forestry, 66, 353–369, 1993. Data availability. The underlying research data are provided in the supplement. Chapman, S. B.: A simple conductimetric soil respirometer for ﬁeld use, Oikos, 22, 348–353, 1971. Competing interests. The authors declare that they have no conﬂict of interest. Competing interests. The authors declare that they have no conﬂict of interest. Cocozza, C., D’Orazio, V., Miano, T. M., and Shotyk, W.: Char- acterization of solid and aqueous phases of a peat bog proﬁle using molecular ﬂuorescence spectroscopy, ESR and FT-IR, and comparison with physical properties, Org. Geochem., 34, 49–60, 2003. Acknowledgements. This study was funded by the Swiss National Science Foundation in the framework of the National Research Programme “Sustainable Use of Soil as a Resource”, grant number 406840_143145. We are grateful to Chloé Wüst-Galley for help with site selection and her statistical support, and to Robin Giger for analysing the soil samples. Conant, R. T., Drijber, R. A., Haddix, M. L., Parton, W. J., Paul, E. A., Plante, A. F., Six, J., and Steinweg, J. M.: Sensitivity of organic matter decomposition to warming varies with its quality, Global Change Biol., 14, 868–877, 2008. Conant, R. T., Ryan, M. G., Agren, G. I., Birge, H. E., Davidson, E. A., Eliasson, P. E., Evans, S. E., Frey, S. D., Giardina, C. P., Hopkins, F. M., Hyvönen, R., Kirschbaum, M. U. F., Lavallee, J. M., Leifeld, J., Parton, W. J., Steinweg, M., Wallenstein, M. D., Wetterstedt, J. Å. M., and Bradford, M. A.: Temperature and soil organic matter decomposition rates–synthesis of current knowl- edge and a way forward, Global Change Biol., 17, 3392–3404, 2011. Edited by: Jens-Arne Subke Edited by: Jens-Arne Subke Reviewed by: three anonymous referees Reviewed by: three anonymous referees 5 Conclusions Chemical characteristics of SOM indicated advanced peat decomposition in the uppermost layers of drained organic soils used as cropland or grasslands. Under controlled mois- ture and temperature conditions, CO2 emissions from peat samples had a similar variability, as found for in situ CO2 ﬂux experiments on drained organic soils. Therefore, car- bon loss from drained organic soils cannot be explained en- tirely by climate or drainage depth. However, simple chem- ical characteristics of SOM, as used in this study, were not speciﬁc enough to explain the variability in CO2 emissions or the temperature sensitivity of decomposition under con- trolled conditions. Despite CO2 emissions being occasion- ally higher in topsoils, probably derived from accrual of la- bile plant residues, the remarkable decrease of Q10 values with depth suggested that the relative content of recalcitrant peat-derived SOM was high in topsoils of managed organic soils, indicating advanced degradation in these uppermost layers. It is therefore necessary to quantify the fraction of peat-derived SOM throughout a drained soil proﬁle in order to verify this assumption. Yet we understand from the simi- lar magnitude of CO2 emission rates found above and below 30 cm depth that future peat loss will occur at similar or even faster rates, assuming an increasing mean annual tempera- ture. p p It is remarkable that despite the controlled conditions in our incubation experiment the variation in cumulative loss of initial SOC of between 0.6 and 42.3 % (Fig. 4) was similar to or even larger than that observed in ﬁeld ﬂux measure- ments (IPCC, 2014). This large variability suggests that the composition of SOM is of similar importance to drainage, climate and other site factors in controlling CO2 emissions from drained organic soils. Nevertheless, the relationships between the measured SOM parameters used to assess the biochemical decomposability, CO2 emissions and Q10 values were rather weak and thus do not support our second hypoth- esis. 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https://openalex.org/W1937603339	https://europeanjournaloftaxonomy.eu/index.php/ejt/article/download/265/508	English	null	Two new species of Leptanilloides Mann, 1823 (Formicidae: Dorylinae) from the Andes of southern Ecuador	European Journal of Taxonomy	2,015	cc-by	15,325	Two new species of Leptanilloides Mann, 1823 (Formicidae: Dorylinae) from the Andes of southern Ecuador Thibaut DELSINNE 1,4,, Gontran SONET 2 & David A. DONOSO 1,3 Thibaut DELSINNE 1,4, , Gontran SONET 2 & David A. DONOSO 1,3 1 Universidad Técnica Particular de Loja (UTPL), Departamento de Ciencias Naturales, Museo de Colecciones Biológicas (MUTPL), Loja, Ecuador. 2 Royal Belgian Institute of Natural Sciences, Joint Experimental Molecular Unit (JEMU) – OD Taxonomy and Phylogeny, Vautierstraat 29, B-1000 Brussels, Belgium. 3 Universidad de Cuenca, Facultad de Ciencias, Agropecuarias, Ave. 12 de Abril s/n, Cuenca, Ecuador Corresponding author: delsinnethibaut@yahoo.fr 2 E-mail: gontran.sonet@naturalsciences.be 3 E-mail: david.donosov@gmail.com 4 urn:lsid:zoobank.org:author:BA819CA7-A1BC-43AC-94A3-9DBB2D89D634 2 urn:lsid:zoobank.org:author:BA7353F8-5296-4968-9D2D-D474EF8FCEB6 3 urn:lsid:zoobank.org:author:BD09BA33-A9F8-4F77-A5B3-F277062FB286 Abstract. Two new species of Leptanilloides are described: L. copalinga Delsinne & Donoso sp. nov. and L. prometea Delsinne & Donoso sp. nov., based on workers collected in the leaf litter and soil of the Andes of southern Ecuador. Both species belong to the L. biconstricta species-group (formally diagnosed here). The metatibial gland, considered a synapomorphy for Dorylinae, is observed in L. prometea sp. nov. but seems absent in L. copalinga sp. nov. We provide a COI DNA barcode for both species and a revised key for the worker caste of all known species in the genus. We also describe a single male identified as a potential new Leptanilloides species on the basis of morphology. Furthermore, its mitochondrial COI gene sequence does not match any previously barcoded species. However, we refrain from giving it a specific name because of our lack of knowledge about the worker caste. So far, half of the 14 Leptanilloides species have been discovered above 1500 m in the mountain forests or páramos of the Ecuadorian Andes, confirming, if needed, the biological significance of these threatened habitats. words. Army ant syndrome, DNA barcoding, male description, metatibial gland, subterranean ant Key words. Army ant syndrome, DNA barcoding, male description, metatibial gland, sub Delsinne T., Sonet G. & Donoso D.A. 2015. Two new species of Leptanilloides Mann, 1823 (Formicidae: Dorylinae) from the Andes of southern Ecuador. European Journal of Taxonomy 143: 1–35. http://dx.doi. org/10.5852/ejt.2015.143 R e s e a r c h a r t i c l e urn:lsid:zoobank.org:pub:CA2B7F29-C4C3-4BCC-A65B-40733C856652 Delsinne T., Sonet G. & Donoso D.A. 2015. Two new species of Leptanilloides Mann, 1823 (Formicidae: Dorylinae) from the Andes of southern Ecuador. European Journal of Taxonomy 143: 1–35. http://dx.doi. org/10.5852/ejt.2015.143 The ant genus Leptanilloides Mann, 1923 belongs with Amyrmex Kusnezov, 1953 to a monophyletic clade on the basis of the molecular phylogeny of the recently reassessed subfamily Dorylinae Leach, 1815 (Brady et al. 2014). This clade was formerly recognized as a distinct subfamily, Leptanilloidinae Baroni Urbani et al., 1992, and also comprises a third genus, Asphinctanilloides Brandão et al., 1999, for which no DNA sequences are yet available. European Journal of Taxonomy 143: 1–35 ISSN 2118-9773 http://dx.doi.org/10.5852/ejt.2015.143 www.europeanjournaloftaxonomy.eu 2015 · Delsinne T., Sonet G. & Donoso D.A. This work is licensed under a Creative Commons Attribution 3.0 License. European Journal of Taxonomy 143: 1–35 ISSN 2118-9773 http://dx.doi.org/10.5852/ejt.2015.143 www.europeanjournaloftaxonomy.eu 2015 · Delsinne T., Sonet G. & Donoso D.A. This work is licensed under a Creative Commons Attribution 3.0 License. ISSN 2118-9773 www.europeanjournaloftaxonomy.eu 2015 · Delsinne T., Sonet G. & Donoso D.A. Introduction The ant genus Leptanilloides Mann, 1923 belongs with Amyrmex Kusnezov, 1953 to a monophyletic clade on the basis of the molecular phylogeny of the recently reassessed subfamily Dorylinae Leach, 1815 (Brady et al. 2014). This clade was formerly recognized as a distinct subfamily, Leptanilloidinae Baroni Urbani et al., 1992, and also comprises a third genus, Asphinctanilloides Brandão et al., 1999, for which no DNA sequences are yet available. 1 European Journal of Taxonomy 143: 1–35 (2015) Leptanilloides comprises 14 species of subterranean ants, including two species described as new in this paper, with morphological and behavioral traits distinctive of the army ant syndrome, including synchronization of larva production, brood carrying by workers under their mesosoma, nomadic habits, and a subdichthadiiform gyne (Brandão et al. 1999; Donoso et al. 2006; Borowiec & Longino 2011). They present an apparent disjunctive distribution with the majority of species infrequently collected from Mexico to Bolivia, at altitudes of between 440 and 3350 m (Brandão et al. 1999; Longino 2003; Donoso et al. 2006; Ward 2007; Ward & Brady 2009; Borowiec & Longino 2011), and a single species, L. atlantica Silva et al., 2013, only known from its type locality at 800 m in south-eastern Brazil, more than 2500 km away from the other species (Silva et al. 2013). However, this distribution pattern is suspected to be the result of the insufficient or inadequate collecting effort applied to the soil environment (Silva et al. 2013). Our knowledge concerning the diversity and distribution of this genus is indeed very low: most species are only known from their type locality, one species from just two sites (L. gracilis Borowiec & Longino, 2011 from Mexico and Guatemala; Borowiec & Longino 2011), and two species from three localities (L. mckennae Longino, 2003 in Costa Rica (AntWeb 2015a); L. biconstricta Mann, 1923 from Bolivia (Mann 1923), Colombia (Zabala et al. 2006) and Venezuela (AntWeb 2015b)). Borowiec & Longino (2011) updated the diagnosis of the genus based on the worker caste. They described the state of 38 morphological variables and discussed the variability observed for some of them within the genus. In particular, they noted the presence, albeit reduced, of a metatibial gland in two species (L. erinys Borowiec & Longino, 2011 and L. nubecula Donoso et al., 2006). Molecular analyses One hindleg was sampled from 4 specimens (specimen codes of L. prometea sp. nov. workers: 4052302, 4052314; code of L. copalinga sp. nov. worker: 4006302; code of the unassociated male: 4870101), and DNA was extracted with the NucleoSpin Tissue Kit (Macherey-Nagel, Germany) following the manufacturer’s protocol. Moreover, the total genomic DNA was isolated from the complete body of 4 workers of L. prometea sp. nov. (specimen codes 4052311, 4052312, 4060602, and 4060603) also using the NucleoSpin Tissue Kit. After DNA extraction, these 4 specimens were preserved as vouchers in 96% ethanol. PCR amplification of the 5’ end of the cytochrome c oxidase subunit I (COI) marker (standard DNA barcode region) was performed with the primers LCO1490 and HCO2198 (Folmer et al. 1994). The wingless wnt-1 gene (Wg) was amplified using primers Wg578F (Ward & Downie 2005) and Wg1032R (Abouheif & Wray 2002). PCR conditions were the same as in Delsinne et al. (2012). Amplicons were directly sequenced in both directions and aligned with all COI and Wg sequences of Leptanilloides and Cylindromyrmex Mayr, 1870 (as outgroup) available in GenBank and in BOLD, the Barcode of Life Data System (Ratnasingham & Hebert 2007). We obtained COI sequences for 6 specimens of L. prometea sp. nov., 1 of L. copalinga sp. nov., and 1 of the unnamed Leptanilloides male. Unfortunately, we obtained only one Wg sequence for L. prometea sp. nov. (specimen code 4052311; Appendix 1). Mega ver. 6.06 (Tamura et al. 2013) was used to perform alignments with the Clustal W algorithm (Thompson et al. 1994), calculate pairwise uncorrected p-distances and construct neighbor-joining trees with bootstrapping (1000 replicates). All COI and Wg sequences were deposited in GenBank with accession numbers KT601697– KT601704 (COI) and KT750331 (Wg). Process IDs of specimens in BOLD are from LEPEC001-15 to LEPEC007-15 and LEPEC009-15. Further phylogenetic analyses were performed on the basis of the Wg DNA dataset (Appendix 1), which included sequences representing more species than the COI dataset. Parsimony trees were calculated in R using the packages ape (Paradis et al. 2004) and phangorn (Schliep 2011) with 1000 bootstrap replicates (non-parametric bootstrapping) (Appendix 2). Best partitioning schemes and best-fit substitution models were estimated using PartitionFinder ver. 1.0 (Lanfear et al. 2012) in order to perform maximum likelihood analysis and Bayesian inference of phylogeny. Introduction This observation was interesting because this gland is considered to be a synapomorphy of Dorylinae in relation to Formicidae (Bolton 1990; 2003) but was estimated to be secondarily absent in Leptanilloides (Brandão et al. 1999; Bolton 2003; Longino 2003; Brady & Ward 2005; Donoso et al. 2006). Morphological data seem to support the segregation of Leptanilloides into two natural species-groups (Borowiec & Longino 2011): the Leptanilloides biconstricta species-group, easily recognizable for the completely unfused promesonotal connection and the postpetiole nearly as high as the following abdominal segment, and the Leptanilloides legionaria species-group, with ants that have a partially to completely fused promesonotal connection and a reduced and isolated postpetiole. Importantly, the phylogeny of Brady et al. (2014) inferred from 11 nuclear DNA markers also supports this distinction (but only five Leptanilloides species were included in their work). In this paper, we provide a formal diagnosis of both species-groups. The male caste has only been described for two species of the L. legionaria species-group, namely L. mckennae (Ward 2007) and L. nubecula (Donoso et al. 2006). In addition, Borowiec & Longino (2011) described three males without associating them with workers or giving them specific names. The first male (called ‘Male 1’ in Borowiec & Longino 2011) was suspected to belong to L. gracilis, a species from the L. biconstricta species-group, because of sympatric distribution, similar abundance, small size and the presence of two simple spurs on mid and hind tibia in both ‘Male 1’ and workers of L. gracilis. However, because several Leptanilloides species can occur in sympatry (Donoso et al. 2006; Borowiec & Longino 2011), the association was cautiously not formalized without further evidence. No hypotheses were proposed concerning the identities of the two other males. It is noteworthy that males are also known for Amyrmex (Ward & Brady 2009), but not for Asphictanilloides. Boudinot (2015) indicated that most males of Dorylinae are uniquely identified by the bidentate or pronged ninth abdominal sternite (subgenital plate), the lack of pygostyles, and the poorly developed clypeus. Males of Leptanilloides and Amyrmex are highly derived and are identifiable by the following combination of characters: antennal torulus abutting or very nearly abutting anterior clypeal margin, oblique mesopleural sulcus absent, four closed cells present on forewing, and cinctus between abdominal pre- and postsclerites IV absent (Boudinot 2015; see also Ward & Brady 2009). Moreover, the posterior 2 DELSINNE T., SONET G. Introduction & DONOSO D.A., Two new Leptanilloides from Ecuador margin of abdominal sternite IX is broad and deeply concave but not bifurcate (Ward 2007; Borowiec & Longino 2011). Here we describe the worker caste of two new Leptanilloides species belonging to the L. biconstricta species-group. A male with a unique mandible shape is also described, although we do not intend to formally name it until further information regarding its associated worker caste has been acquired. We also provide COI barcodes for the two new species and for this male. The key of Borowiec & Longino (2011) to the worker caste is updated to include our two new species as well as the recently described L. atlantica Silva et al., 2013. Finally, we provide evidence of a metatibial gland in one of our species and note the hitherto unnoticed occurrence of a pectinate spur on the midtibia in some species. Images High resolution digital images were taken using either a Leica DFC290 camera attached to a Leica Z6APO stereo microscope or a Leica MC170 camera attached to a Leica S8APO stereo microscope. A series of images was taken by focusing the sharpness on different levels of the specimen, using the Leica Application Suite ver. 38 (2003–2011) and combined with the stacking software Combine ZP (Hadley 2010). Final editing of the images was done in Adobe Photoshop CS5. SEM photographs of the specimens (spm 4006301 and spm 4052301 for L. copalinga sp. nov. and L. prometea sp. nov., respectively) were taken at the Royal Belgian Institute of Natural Sciences using a FEI Quanta 200 scanning electron microscope (SEM). Molecular analyses Two partitions (one partition for the third codon position and one partition for the two other positions) were selected, with best-fit substitution models TIM+Gamma (Posada 2003) and JC (Jukes & Cantor 1969), respectively (using the Bayesian information criterion). Maximum likelihood analysis was conducted using GARLI ver. 2.0 (Zwickl 2006) with a bootstrapping test (500 replicates) (Appendix 3). Bayesian inference was performed with MrBayes ver. 3.2.3 (Ronquist et al. 2012; Appendix 1). Two parallel runs, with four chains each, were run for ten million generations, with unlinked nucleotide substitution parameters for each data partition. Every 1000th generation was sampled, convergence of the Markov chains was monitored, average standard deviation of split frequencies decreased below 0.01, and traces of the parameters were checked using Tracer ver. 1.6 (Rambaut et al. 2014) following 3 European Journal of Taxonomy 143: 1–35 (2015) the recommendations of Ronquist et al. (2011), Lesaffre & Lawson (2012) and SAS Institute (2009). Finally, the first 25% of the trees were discarded (“burn-in”) to summarize the tree samples. the recommendations of Ronquist et al. (2011), Lesaffre & Lawson (2012) and SAS Institute (2009). Finally, the first 25% of the trees were discarded (“burn-in”) to summarize the tree samples. Measurements (2013) MH = Mesosoma height: in lateral view, maximum height measured from lowermost point of mesopleuron (in front of middle coxa) to dorsal edge of mesosoma, measured perpendicularly to long axis of mesosoma PL = Petiole length, maximum length of the petiole measured in dorsal view, starting at base of MH = Mesosoma height: in lateral view, maximum height measured from lowermost point of mesopleuron (in front of middle coxa) to dorsal edge of mesosoma, measured perpendicularly to long axis of mesosoma PL = Petiole length, maximum length of the petiole measured in dorsal view, starting at base of anterior face and ending at base of posterior edge PPL = Postpetiole (= third abdominal segment) length, maximum length of node measured in dorsal view PPW = Postpetiole width, maximum width of node measured in dorsal view AivW = Maximum width of fourth abdominal segment, in dorsal view FFeW = Maximum width in lateral view of fore femur HTL = Length of hind tibia, measured along extensor (outer) surface SI = Scape index, SL/HL × 100 PI = Petiolar index, PW/PL × 100 4 DELSINNE T., SONET G. & DONOSO D.A., Two new Leptanilloides from Ecuador MI = Mesosomal index, MH/WL × 100 , sc = specimen code(s) following the Maurice Leponce database at the Royal Belgian Institute of Natural Sciences , sc = specimen code(s) following the Maurice Leponce database at the Royal Belgian Institute of Natural Sciences For consistency, we follow Borowiec & Longino (2011) for terminology, in particular for wing venation and genitalia. Results Class Hexapoda Blainville, 1816 Order Hymenoptera Linnaeus, 1758 Suborder Apocrita Latreille, 1810 Infraorder Aculeata Latreille, 1802 Superfamily Vespoidea Latreille, 1802 Family Formicidae Latreille, 1809 Subfamily Dorylinae Leach, 1815 Genus Leptanilloides Mann, 1823 Class Hexapoda Blainville, 1816 Order Hymenoptera Linnaeus, 1758 Suborder Apocrita Latreille, 1810 Infraorder Aculeata Latreille, 1802 Superfamily Vespoidea Latreille, 1802 Family Formicidae Latreille, 1809 Subfamily Dorylinae Leach, 1815 Genus Leptanilloides Mann 1823 Measurements Measurements were made using an Olympus SZ61 stereo microscope at a magnification of 45× with a micrometer. All the measurements are presented in millimeters. The following abbreviations are used: Measurements were made using an Olympus SZ61 stereo microscope at a magnification of 45× with a micrometer. All the measurements are presented in millimeters. The following abbreviations are used: HL = Head length, measured in full face view, from anterior edge of frontal lobes (therefore without including clypeal lamella) to posterior border of head g yp ) p HW = Head width, maximum width of head measured in full face view; HW for males includes eyes (workers eyeless) SL = Scape length, excluding basal condyle and neck p g , g y LAII, LAIII, LAIV, LAXIII = Length of second, third, fourth and terminal (13th) antennal segments, respectively (male only) EL = Eye length, measured in full face view, maximum length of eye parallel to midline (male only) MaL = Mandible length, measured in full face view, length of line running medially from clypeal anterior margin to mandible apex (male only) WL = Weber’s length, measured from anterior edge of pronotum to posterior edge of metapleural lobe; this measure called Mesosoma Length (ML) in Borowiec & Longino (2011) and Silva et al. (2013) et al. L. biconstricta Mann, 1923 Deposition of material Holotypes and paratypes have been deposited at: BMNH = British Museum of Natural History (Natural History Museum), London, UK BMNH = British Museum of Natural History (Natural History Museum), London, UK CASC = California Academy of Sciences, San Francisco, CA, USA ICN = Insect Collection, Instituto de Ciencias Naturales, Universidad Nacional de Colombia, Bogotá D.C., Colombia JTLC = John T. Longino, personal collection, University of Utah, Salt Lake City, UT, USA MCZC = Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts, USA MUTPL = Museo de Colecciones Biológicas de la Universidad Técnica Particular de Loja, Loja, Ecuador MZSP = Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil QCAZ = Museum of Zoology of the Pontificia Universidad Católica del Ecuador, Quito, Ecuador RBINS = Royal Belgian Institute of Natural Sciences, Brussels, Belgium Results CASC = California Academy of Sciences, San Francisco, CA, USA CASC = California Academy of Sciences, San Francisco, CA, USA ICN = Insect Collection, Instituto de Ciencias Naturales, Universidad Nacional de Colombia, Bogotá D.C., Colombia ICN = Insect Collection, Instituto de Ciencias Naturales, Universidad Nacional de Colombia, Bogotá D.C., Colombia JTLC = John T. Longino, personal collection, University of Utah, Salt Lake City, UT, US MCZC = Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts, USA MUTPL = Museo de Colecciones Biológicas de la Universidad Técnica Particular de Loja, Loja, Ecuador MZSP = Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazili QCAZ = Museum of Zoology of the Pontificia Universidad Católica del Ecuador, Quito, Ecuador RBINS = Royal Belgian Institute of Natural Sciences Brussels Belgium This group includes the ten following species: L. atlantica Silva et al., 2013 L. biconstricta Mann, 1923 European Journal of Taxonomy 143: 1–35 (2015) L. caracola Donoso et al., 2006 L. caracola Donoso et al., 2006 L. caracola Donoso et al., 2006 L. copalinga Delsinne & Donoso sp. nov L. erinys Borowiec & Longino, 2011 L. femoralis Borowiec & Longino, 2011 L. gracilis Borowiec & Longino, 2011 L. improvisa Brandão et al., 1999 L. prometea Delsinne & Donoso sp. nov. L. copalinga Delsinne & Donoso sp. nov. L. erinys Borowiec & Longino, 2011 L. femoralis Borowiec & Longino, 2011 L. gracilis Borowiec & Longino, 2011 L. improvisa Brandão et al., 1999 L. prometea Delsinne & Donoso sp. nov. L. sculpturata Brandão et al., 1999 L. sculpturata Brandão et al., 1999 Diagnosis of the Leptanilloides biconstricta species-group Species from the Leptanilloides biconstricta species-group possess the typical characters of the genus (see diagnosis in Borowiec & Longino 2011) but can be grouped by the fact that they share the following criteria: promesonotal connection completely unfused and flexible; lateroclypeal tooth (called genal tooth in Brandão et al. 1999, Longino 2003 and Donoso et al. 2006) well-developed (although apparently lacking in L. caracola Donoso et al., 2006, a species known only by its holotype; because this criterion may be difficult to observe with a stereo microscope, even at high magnification, the absence of a lateroclypeal tooth in L. caracola should be confirmed in SEM); abdominal segment III (postpetiole) nearly as high as abdominal segment IV; and postpetiolar spiracle situated proximately to the anterior margin of the tergite. Moreover, species from this group are generally smaller than species of the L. legionaria species-group (Fig. 1). This group includes the ten following species: L. atlantica Silva et al., 2013 L. biconstricta Mann, 1923 5 5 European Journal of Taxonomy 143: 1–35 (2015) Diagnosis of the Leptanilloides legionaria species-group Species from the Leptanilloides legionaria species-group possess the characters of the genus (see diagnosis in Borowiec & Longino 2011) but are grouped because they share the following criteria: promesonotal connection at least partially fused; lateroclypeal tooth absent or reduced; postpetiole reduced and isolated; and postpetiolar spiracle shifted posteriad on anteromedian side of the tergite. This group includes the following four species: This group includes the following four species: L. legionaria Brandão et al., 1999 L. mckennae Longino, 2003 Fig. 1. Relationship between HL and WL among workers in species of Leptanilloides and Asphinct- anilloides. Specimens measured are holotypes or paratypes. Measurements are in mm. WL is called Mesosoma Length (ML) in Borowiec & Longino (2011) and Silva et al. (2013). Triangles: the two Leptanilloides described in this paper (L. copalinga Delsinne & Donoso sp. nov. and L. prometea Delsinne & Donoso sp. nov.); diamonds: Leptanilloides biconstricta species-group (La: L. atlantica, Lb: L. biconstricta, Lc: L. caracola, Le: L. erinys, Lf: L. femoralis, Lg: L. gracilis, Li: L. improvisa, Ls: L. sculpturata); dots: Leptanilloides legionaria species-group (Ll: L. legionaria, Lm: L. mckennae, Lno: L. nomada, Lnu: L. nubecula); stars: Asphinctanilloides species (Aa: A. amazon Brandão et al., 1999, Aan: A. anae Brandão et al., 1999, Am: A. manauara Brandão et al., 1999). Fig. 1. Relationship between HL and WL among workers in species of Leptanilloides and Asphinct- anilloides. Specimens measured are holotypes or paratypes. Measurements are in mm. WL is called Mesosoma Length (ML) in Borowiec & Longino (2011) and Silva et al. (2013). Triangles: the two Leptanilloides described in this paper (L. copalinga Delsinne & Donoso sp. nov. and L. prometea Delsinne & Donoso sp. nov.); diamonds: Leptanilloides biconstricta species-group (La: L. atlantica, Lb: L. biconstricta, Lc: L. caracola, Le: L. erinys, Lf: L. femoralis, Lg: L. gracilis, Li: L. improvisa, Ls: L. sculpturata); dots: Leptanilloides legionaria species-group (Ll: L. legionaria, Lm: L. mckennae, Lno: L. nomada, Lnu: L. nubecula); stars: Asphinctanilloides species (Aa: A. amazon Brandão et al., 1999, Aan: A. anae Brandão et al., 1999, Am: A. manauara Brandão et al., 1999). 6 DELSINNE T., SONET G. & DONOSO D.A., Two new Leptanilloides from Ecuador L. nomada Donoso et al., 2006 L. nubecula Donoso et al., 2006 Key to workers of Leptanilloides 1. Abdominal segment III (postpetiole) in lateral view much smaller than adjoining fourth abdominal segment. Spiracle of segment III shifted posteriad on anteromedian side of tergite. Diagnosis of the Leptanilloides legionaria species-group Body size relatively large, HL 0.68–0.75 (Fig. 1) ………………2 (L. legionaria species-group) – Abdominal segment III (postpetiole) in lateral view nearly as high as abdominal segment IV. Spiracle of segment III situated forward on the tergite. Body size relatively small, HL 0.31–0.62 (Fig. 1) ……………………………………………………………5 (L. biconstricta species-group) 2. Head subquadrate, CI 85–88; lateral margin nearly straight and parallel. Propodeal declivity short and vertical, propodeum with dorsal and posterior faces clearly differentiated (Ecuador) ……… ………………………………………………………………………L. nomada Donoso et al., 2006 – Head subrectangular, CI 75–83; lateral margin convex. Propodeal declivity usually rounded without clear distinction between dorsal and posterior faces ……………………………………3 3. Head sculpture less dense, at most 10–12 shallow foveolae across face at midlength. Lateral margin of head distinctly convex. Lateroclypeal tooth present. Posterior margin of head slightly concave (Colombia) ……………………………………………………L. legionaria Brandão et al., 1999 – Head sculpture denser, with at least 15 foveolae across face at midlength. Lateral margin of head slightly convex. Lateroclypeal tooth absent. Posterior margin of head deeply concave …………4 4. Legs shorter, HW/HTL×100 > 78. Hypostomal tooth present (Ecuador) ………………………… …………………………………………………………………L. nubecula Donoso et al., 2006 – Legs longer, HW/HTL×100 < 78. Hypostomal tooth absent (Costa Rica) ……………………… …………………………………………………………………………L. mckennae Longino, 2003 5. Lateroclypeal tooth absent. Masticatory margin of mandible edentate (Ecuador) ………………… ……………………………………………………………………L. caracola Donoso et al., 2006 – Lateroclypeal tooth present. Masticatory margin of mandible dentate (at least minute denticles present) ……………………………………………………………………………………………6 6. In lateral view, abdominal segment IV narrowly attached to preceding segment III and broadly to succeeding segment V, with contrast between widths of anterior and posterior articulations of segment IV in lateral view …………………………………………………………………7 – In lateral view, abdominal segment IV relatively broadly attached to preceding segment III, with little contrast between widths of anterior and posterior articulations of segment IV in lateral view ………………………………………………………………………………………8 7. In lateral view, sternite of abdominal segment III evenly rounded, making sternal and tergal portions subequal. Petiole as long as postpetiole. Reticulation on mesopleuron and metapleuron uninterrupted. Head densely foveolate, with c. 20–30 foveolae covering a straight transverse line at head midlength. HL ≤ 0.36 (Brazil) ………………………………L. atlantica Silva et al., 2013 – In lateral view, sternite of abdominal segment III (postpetiole) distinctly bulging anteriorly, making sternal portion of segment deeper than tergite (Borowiec & Longino 2011: fig. 2d). Petiole longer than postpetiole (Borowiec & Longino 2011: fig. 2d). Reticulation on mesopleuron and metapleuron superficial and interrupted. Diagnosis of the Leptanilloides legionaria species-group Head sculpture less dense, with only 10–20 foveolae covering a straight transverse line at head midlength. HL = 0.49 in only measured specimen (Bolivia, Colombia, Venezuela) …………………………………………L. biconstricta Mann, 1823 L. nomada Donoso et al., 2006 Key to workers of Leptanilloides 3D) (Ecuador) …………………………………… …………………………………………………………L. copalinga Delsinne & Donoso sp. nov. Leptanilloides copalinga Delsinne & Donoso sp. nov. urn:lsid:zoobank.org:act:4324B13A-500B-464C-AE3F-62BCAF95A2CC Figs 2A–F, 3A–F Leptanilloides copalinga Delsinne & Donoso sp. nov. urn:lsid:zoobank.org:act:4324B13A-500B-464C-AE3F-62BCAF95A2CC Figs 2A–F, 3A–F Key to workers of Leptanilloides 7 European Journal of Taxonomy 143: 1–35 (2015) European Journal of Taxonomy 143: 1–35 (2015) 8. In lateral view, petiolar sternite distinctly bulging medially ……………………………………9 – In lateral view, petiolar sternite straight (not bulging) or bulging anteriorly …………………10 9. Hindtibia with two very small, simple spurs, without pectinate spur clearly visible under 50× magnification. Petiolar spiracle opening in an excavation distinctly larger than propodeal spiracle (Borowiec & Longino 2011: fig. 5g–h). Flange over metapleural gland opening sharply pointed posteriorly (Mexico, Guatemala) …………………………L. gracilis Borowiec & Longino, 2011 – Hindtibia with large pectinate spur, clearly discernable under 50× magnification. Petiolar spiracle not in excavation, similar and subequal to or smaller in diameter than propodeal spiracle (Borowiec & Longino 2011: fig. 4g–h). Flange over metapleural gland opening rounded posteriorly (Venezuela) ………………………………………………L. femoralis Borowiec & Longino, 2011 10. Head narrow, HL < 0.30, HW < 0.20, CI < 60. Head dorsum densely foveolate with foveolae separated by less than their diameter, often contiguous (Colombia) ……………………………… …………………………………………………………………L. sculpturata Brandão et al., 1999 – Head broader, HL > 0.30, HW > 0.20, CI > 65. Head dorsum less densely foveolate, with foveolae separated by about their diameter or more ……………………………………………11 11. Size large, HL ≥ 0.50, HW ≥ 0.38. Flange over metapleural gland opening short, not surpassing propodeum declivity margin in lateral view, and rounded posteriorly ………………………12 – Smaller, HL < 0.45, HW < 0.35. Flange over metapleural gland opening long, surpassing propodeum declivity margin in lateral view, and sharply pointed posteriorly ………………13 12. HL = 0.50 on single known specimen. Masticatory margin of mandible distinctly dentate, with regularly spaced and well-developed teeth (Brandão et al. 1999: fig. 16) (Ecuador) …………… ……………………………………………………………………L. improvisa Brandão et al., 1999 – HL ≥ 0.58. Masticatory margin of mandible dentate but teeth extremely minute, blunt and irregularly shaped, difficult to discern even under magnifications of about 100× (Figs 4B, 6C) (Ecuador) ………………………………………………L. prometea Delsinne & Donoso sp. nov. 13. Ventral margin of petiolar sternite forming an even convexity in lateral view. Reticulation on mesopleuron, metapleuron and lateral side of petiole uninterrupted. In dorsal view, petiole subquadrate, 67 ≤ PI ≤ 80 (Ecuador) ………………………L. erinys Borowiec & Longino, 2011 – Ventral margin of petiolar sternite relatively straight in lateral view (Fig. 3C). Reticulation on mesopleuron, metapleuron and lateral side of petiole superficial and interrupted. In dorsal view, petiole slender, rectangular, 52 ≤ PI ≤ 63 (Fig. 3D) (Ecuador) …………………………………… …………………………………………………………L. Key to workers of Leptanilloides copalinga Delsinne & Donoso sp. nov. Leptanilloides copalinga Delsinne & Donoso sp. nov. urn:lsid:zoobank.org:act:4324B13A-500B-464C-AE3F-62BCAF95A2CC Figs 2A–F, 3A–F 8. In lateral view, petiolar sternite distinctly bulging medially ……………………………………9 – In lateral view, petiolar sternite straight (not bulging) or bulging anteriorly …………………10 9. Hindtibia with two very small, simple spurs, without pectinate spur clearly visible under 50× magnification. Petiolar spiracle opening in an excavation distinctly larger than propodeal spiracle (Borowiec & Longino 2011: fig. 5g–h). Flange over metapleural gland opening sharply pointed posteriorly (Mexico, Guatemala) …………………………L. gracilis Borowiec & Longino, 2011 – Hindtibia with large pectinate spur, clearly discernable under 50× magnification. Petiolar spiracle not in excavation, similar and subequal to or smaller in diameter than propodeal spiracle (Borowiec & Longino 2011: fig. 4g–h). Flange over metapleural gland opening rounded posteriorly (Venezuela) ………………………………………………L. femoralis Borowiec & Longino, 2011 10. Head narrow, HL < 0.30, HW < 0.20, CI < 60. Head dorsum densely foveolate with foveolae separated by less than their diameter, often contiguous (Colombia) ……………………………… …………………………………………………………………L. sculpturata Brandão et al., 1999 – Head broader, HL > 0.30, HW > 0.20, CI > 65. Head dorsum less densely foveolate, with foveolae separated by about their diameter or more ……………………………………………11 11. Size large, HL ≥ 0.50, HW ≥ 0.38. Flange over metapleural gland opening short, not surpassing propodeum declivity margin in lateral view, and rounded posteriorly ………………………12 – Smaller, HL < 0.45, HW < 0.35. Flange over metapleural gland opening long, surpassing propodeum declivity margin in lateral view, and sharply pointed posteriorly ………………13 12. HL = 0.50 on single known specimen. Masticatory margin of mandible distinctly dentate, with regularly spaced and well-developed teeth (Brandão et al. 1999: fig. 16) (Ecuador) …………… ……………………………………………………………………L. improvisa Brandão et al., 1999 – HL ≥ 0.58. Masticatory margin of mandible dentate but teeth extremely minute, blunt and irregularly shaped, difficult to discern even under magnifications of about 100× (Figs 4B, 6C) (Ecuador) ………………………………………………L. prometea Delsinne & Donoso sp. nov. 13. Ventral margin of petiolar sternite forming an even convexity in lateral view. Reticulation on mesopleuron, metapleuron and lateral side of petiole uninterrupted. In dorsal view, petiole subquadrate, 67 ≤ PI ≤ 80 (Ecuador) ………………………L. erinys Borowiec & Longino, 2011 – Ventral margin of petiolar sternite relatively straight in lateral view (Fig. 3C). Reticulation on mesopleuron, metapleuron and lateral side of petiole superficial and interrupted. In dorsal view, petiole slender, rectangular, 52 ≤ PI ≤ 63 (Fig. Paratypes ECUADOR: Same data as holotype: 1 worker, gold-coated for SEM (RBINS, sc 4006301); 1 pinned (MUTPL, sc 4006303); 1 worker, 96% ethanol (RBINS, sc 4006302, specimen with DNA data). GenBank accession number: KT601697. Measurements (in mm) and indices Holotype (paratype 4006303): HW 0.33 (0.34), HL 0.44 (0.44), SL 0.23 (0.21), PrW 0.2 (0.22), WL 0.56 (0.57), MH 0.17 (0.17), PL 0.17 (0.18), PW 0.09 (0.11), PPL 0.16 (0.18), PPW 0.16 (0.18), AivL 0.26 (0.26), AivW 0.3 (0.33), FFeL 0.23 (0.27), FFeW 0.09 (0.11), HFeL 0.29 (0.29), HTL 0.3 (0.29), CI 75 (76.25), SI 70 (60.66), PI 51.61 (62.5), MI 30 (30.39). Holotype (paratype 4006303): HW 0.33 (0.34), HL 0.44 (0.44), SL 0.23 (0.21), PrW 0.2 (0.22), WL 0.56 (0.57), MH 0.17 (0.17), PL 0.17 (0.18), PW 0.09 (0.11), PPL 0.16 (0.18), PPW 0.16 (0.18), AivL 0.26 (0.26), AivW 0.3 (0.33), FFeL 0.23 (0.27), FFeW 0.09 (0.11), HFeL 0.29 (0.29), HTL 0.3 (0.29), CI 75 (76.25), SI 70 (60.66), PI 51.61 (62.5), MI 30 (30.39). Etymology Name in apposition which refers to the Copalinga Private Reserve, the type locality, in honour of its owners Catherine Vits and Boudewijn de Roover to acknowledge their efforts for the conservation of Ecuadorian biodiversity. Diagnosis Leptanilloides copalinga sp. nov. belongs to the L. biconstricta species-group. It can be distinguished from other species of this group by the combination of the following characters: masticatory margin of mandible feebly dentate, with teeth extremely minute, blunt and irregularly shaped (Fig. 3A); lateroclypeal tooth present (difficult to observe in stereo microscopy even at high magnification due to the small size of the species but obvious in SEM; Fig 3A); head with piligerous foveolae separated by smooth interspaces equaling 2–3 diameters; reticulation on mesopleuron, metapleuron and lateral side of petiole superficial and interrupted; flange over metapleural gland opening conspicuous and sharply 8 DELSINNE T., SONET G. & DONOSO D.A., Two new Leptanilloides from Ecuador pointed posteriorly, resulting in a projection which surpasses propodeum declivity margin in lateral view (Fig. 3B); petiole slender in dorsal view, 52 ≤ PI ≤ 63 (Fig. 3D); and subpetiolar process straight (not distinctly bulging), with a rounded projection anteriorly (Fig. 3C). pointed posteriorly, resulting in a projection which surpasses propodeum declivity margin in lateral view (Fig. 3B); petiole slender in dorsal view, 52 ≤ PI ≤ 63 (Fig. 3D); and subpetiolar process straight (not distinctly bulging), with a rounded projection anteriorly (Fig. 3C). Holotype Holotype ECUADOR: Worker, Zamora-Chinchipe Prov., Copalinga Private Reserve, 1510 m, 4°4’56.6” S, 78°58’5.71” W, 2 Apr. 2010, soil sample, coll. Thibaut Delsinne and Tania Milena Arias-Penna (QCAZ, sc 4006304). Worker With the characters typical of the genus (see diagnosis and description in Brandão et al. 1999 and Borowiec & Longino 2011) and of the L. biconstricta species-group (see diagnosis of the group above). Other characters or differences are as follows: Head. Elongate and rectangular with lateral margin nearly straight and parallel. Posterior corner rounded. Posterior margin modestly convex, almost straight. Parafrontal ridge absent. Clypeal lamella strongly convex (Fig. 3A). Basal and masticatory margins of mandible dentate but teeth extremely minute, blunt and irregularly shaped, difficult to discern even under magnifications of about 100× (Fig. 3A). Basal and masticatory margins united by a broad convexity. Basal margin faintly crenulate. Labial and maxillary palps difficult to discern in situ but formula apparently 2,2. Hypostomal anterior border without distinct tooth. Scape when laid back failing to reach medial distance to posterior margin of head by nearly one maximum diameter. Mesosoma. Flange over metapleural gland opening conspicuous and sharply pointed posteriorly, forming a projection which surpasses propodeum declivity margin (Fig. 3B). Femur not conspicuously enlarged, relatively slender. Midtibia with one short (i.e., half size of foretibia strigil), pectinate spur (Fig. 3F), although pectination may be difficult to observe even at high magnification. Hindtibia with one broadly pectinate spur roughly as long as strigil. Metatibial gland absent or very reduced and not visible even in SEM. Metasoma. Long and relatively slender. In dorsal view, petiole rectangular with lateral margin slightly convex, twice as long as wide (Fig. 3D), and as long as abdominal segment III (postpetiole). Anterior 9 European Journal of Taxonomy 143: 1–35 (2015) face concave, posterior face straight. In lateral view, petiole height approximately ¼ smaller than height of abdominal segment III. Petiolar tergite dome-shaped, with short anterior and posterior faces, maximum height situated in posterior half. Short tubulated portion present posteriorly. Petiolar spiracle inconspicuous, not in excavation, set near anterior rim of tergite, similar in form and slightly smaller in diameter than propodeal spiracle. Subpetiolar process with ventral margin straight to slightly concave, not distinctly bulging, anteriorly forming rounded projection (Fig. 3C). Maximum height of petiolar sternite situated medially. In dorsal view, abdominal segment III (postpetiole) trapezoid, with straight, parallel anterior and posterior faces. Posterior face twice as long as anterior face. In lateral view, tergite evenly convex, without well-differentiated posterior face. Sternite evenly rounded. In dorsal view, abdominal segments IV–VI subequal in length. face concave, posterior face straight. Worker In lateral view, petiole height approximately ¼ smaller than height of abdominal segment III. Petiolar tergite dome-shaped, with short anterior and posterior faces, maximum height situated in posterior half. Short tubulated portion present posteriorly. Petiolar spiracle inconspicuous, not in excavation, set near anterior rim of tergite, similar in form and slightly smaller in diameter than propodeal spiracle. Subpetiolar process with ventral margin straight to slightly concave, not distinctly bulging, anteriorly forming rounded projection (Fig. 3C). Maximum height of petiolar sternite situated medially. In dorsal view, abdominal segment III (postpetiole) trapezoid, with straight, parallel anterior and posterior faces. Posterior face twice as long as anterior face. In lateral view, tergite evenly convex, without well-differentiated posterior face. Sternite evenly rounded. In dorsal view, abdominal segments IV–VI subequal in length. Pilosity and sculpture. Mandible smooth and shiny, with few, scattered piligerous punctures. Head with abundant deep piligerous foveolae and smooth interspaces on average equaling two or three puncture diameters. Mesosoma and abdomen more finely and sparsely punctate. Fine reticulate sculpture present laterally on lower pronotum, mesopleuron, propodeum and petiole. Dorsal part of pronotum, mesopleuron, propodeum and petiole smooth and shiny. Body and appendages with abundant, short and subdecumbent to suberect hairs. Body color yellow to reddish (Fig. 2), with head and mesosoma darker than petiole and gaster. Legs and antennae yellowish. Fig. 2. Leptanilloides copalinga Delsinne & Donoso sp. nov. A–C. Paratype worker (specimen code 4006302). D–F. Holotype worker (specimen code 4006304). A, D. Habitus, dorsal view. B, E. Head in full-face view. C, F. Habitus, lateral view. Fig. 2. Leptanilloides copalinga Delsinne & Donoso sp. nov. A–C. Paratype worker (specimen code 4006302). D–F. Holotype worker (specimen code 4006304). A, D. Habitus, dorsal view. B, E. Head in full-face view. C, F. Habitus, lateral view. 10 DELSINNE T., SONET G. & DONOSO D.A., Two new Leptanilloides from Ecuador Fig. 3. Leptanilloides copalinga Delsinne & Donoso sp. nov. Paratype worker (specimen code 4006301) A. Anterior part of the head in full-face view, showing vertical and fused frontal carinae, expose antennal socket, lateroclypeal tooth, strongly convex clypeal lamella and subtriangular mandible wit minute denticles along its inner margin. B. Lower part of propodeum in lateral view, showing the lon and sharp flange over the metapleural gland opening. C. Petiole in lateral view. D. Propodeum an petiole in dorsal view. E. Abdominal segment III (postpetiole) in lateral view. F. Mesotibial spur, shor and pectinate Fig. 3. Biology Unknown. The type series was found by visual search in a 15×15×15 cm soil core inspected for ants during 20 person-minutes (the soil content was examined over a white plastic board using a headlamp to facilitate ant detection), which suggests subterranean habits as observed in other Leptanilloides species. The habitat is a relatively well-preserved evergreen lower montane forest. Soil texture is clay loam; proportion of sand, silt and clay is 44%, 22%, and 35%, respectively; mean pH = 4 ± 0.2 SD (n = 24 soil samples). Diagnosis Leptanilloides prometea belongs to the L. biconstricta species-group and can be distinguished by the combination of the following characters: masticatory margin of mandible dentate but teeth extremely minute, blunt and irregularly shaped (Fig. 6C); lateroclypeal tooth present (Fig. 6C); head with piligerous foveolae separated by smooth interspaces equaling, on average, puncture diameters; fine, uninterrupted reticulation on mesopleuron, metapleuron and lateral side of petiole; flange over metapleural gland opening forming a short, blunt projection (Fig. 6D); and subpetiolar process relatively straight (not distinctly bulging), without posterior angle (Fig. 6E). Remarks This species belongs to the L. biconstricta species-group. The most similar species are L. biconstricta and L. atlantica, which also possess a long and sharply pointed flange over the metapleural gland opening and a straight (not bulging) petiolar sternite with a rounded anterior projection. However, these two species have a deeper constriction between the postpetiole and abdominal segment IV. Besides, L. copalinga sp. nov. has a petiole roughly as long as the postpetiole (longer in L. biconstricta) and its postpetiolar sternite is evenly rounded (distinctly bulging anteriorly in L. biconstricta). Finally, L. copalinga sp. nov. is larger than L. atlantica (Fig. 1). The other species of the L. biconstricta species- group are easily distinguished from L. copalinga sp. nov. by, among other characters, the shape of their petiolar sternite (bulging medially in L. gracilis and L. femoralis; bulging anteriorly in L. erinys), the shape of the flange over the metapleural gland opening (short and rounded posteriorly in L. improvisa and L. prometea), the head sculpture (more densely foveolate in L. sculpturata) and the lateroclypeal tooth (absent in L. caracola). Leptanilloides prometea Delsinne & Donoso sp. nov. urn:lsid:zoobank.org:act:CC756523-8025-4C13-8987-5F91A1E04B7E Figs 4A–D, 5, 6A–F, 7A–C Leptanilloides prometea Delsinne & Donoso sp. nov. urn:lsid:zoobank.org:act:CC756523-8025-4C13-8987-5F91A1E04B7E Figs 4A–D, 5, 6A–F, 7A–C Distribution Only known from the type locality. Male Unknown. Unknown. Worker Leptanilloides copalinga Delsinne & Donoso sp. nov. Paratype worker (specimen code 4006301). A. Anterior part of the head in full-face view, showing vertical and fused frontal carinae, exposed antennal socket, lateroclypeal tooth, strongly convex clypeal lamella and subtriangular mandible with minute denticles along its inner margin. B. Lower part of propodeum in lateral view, showing the long and sharp flange over the metapleural gland opening. C. Petiole in lateral view. D. Propodeum and petiole in dorsal view. E. Abdominal segment III (postpetiole) in lateral view. F. Mesotibial spur, short and pectinate. Fig. 3. Leptanilloides copalinga Delsinne & Donoso sp. nov. Paratype worker (specimen code 4006301). A. Anterior part of the head in full-face view, showing vertical and fused frontal carinae, exposed antennal socket, lateroclypeal tooth, strongly convex clypeal lamella and subtriangular mandible with minute denticles along its inner margin. B. Lower part of propodeum in lateral view, showing the long and sharp flange over the metapleural gland opening. C. Petiole in lateral view. D. Propodeum and petiole in dorsal view. E. Abdominal segment III (postpetiole) in lateral view. F. Mesotibial spur, short and pectinate. 11 European Journal of Taxonomy 143: 1–35 (2015) Gyne Unknown. Etymology Name in apposition, in honour of the Prometeo initiative of the Ecuadorian government that seeks to strengthen research and knowledge transference by getting national and international experts to work together. This project is aligned to the Ecuadorian “National Development Plan for Good Living”, which has the objective, among others, of guaranteeing the rights of Nature and promoting a healthy and 12 DELSINNE T., SONET G. & DONOSO D.A., Two new Leptanilloides from Ecuador sustainable environment. Furthermore, the species is the largest of the L. biconstricta species-group and its name nicely reminds us of ‘Prometheus’, the Titan in Greek mythology who brought fire, a symbol of enlightenment, to mankind. We hope that this new ant species will symbolize the promise of a bright future for Ecuadorian biodiversity. sustainable environment. Furthermore, the species is the largest of the L. biconstricta species-group and its name nicely reminds us of ‘Prometheus’, the Titan in Greek mythology who brought fire, a symbol of enlightenment, to mankind. We hope that this new ant species will symbolize the promise of a bright future for Ecuadorian biodiversity. Holotype ECUADOR: Worker, Zamora-Chinchipe Prov., Reserva Biológica San Francisco (RBSF), 2070 m, 3°58’ S, 79°05’ W, 13 May 2010, within 0.5 m2 of leaf litter extracted with a mini-Winkler apparatus for 96 h, coll. Thibaut Delsinne and Tania Milena Arias-Penna (QCAZ, sc 4267803). Paratypes (n = 67) ECUADOR: Same data as holotype except that specimens were captured after 48 h of Winkler extraction: 30 workers, 96% ethanol (RBINS, General Inventory Number 33044: sc 4052302 [1 specimen with DNA data], sc 4052314 [1 specimen with DNA data], sc 4052316 [21 specimens without DNA data]; BMNH [1 specimen, sc 4052317]; CASC [1 specimen, sc 4052318]; ICN [1 specimen, sc 4052319]; JTLC [1 specimen, sc 4052320]; MCZC [1 specimen, sc 4052321]; MZSP [1 specimen, sc 4052322]; QCAZ [1 specimen, sc 4052323]); 3 workers, pinned (MUTPL, sc 4052310, 4052315; RBINS, sc 4052313); Fig. 4. Leptanilloides prometea Delsinne & Donoso sp. nov. – A–C. Paratype worker (specimen code 4052301). A. Habitus, dorsal view. B. Head in full-face view. C. Habitus, lateral view. – D. Apex of hindtibia; reservoir of metatibial gland is visible under translucent cuticle at the base of the tibial spur (paratype, specimen code 4052313). Fig. 4. Leptanilloides prometea Delsinne & Donoso sp. nov. – A–C. Paratype worker (specimen code 4052301). A. Habitus, dorsal view. B. Head in full-face view. C. Habitus, lateral view. – D. Apex of hindtibia; reservoir of metatibial gland is visible under translucent cuticle at the base of the tibial spur (paratype, specimen code 4052313). 13 European Journal of Taxonomy 143: 1–35 (2015) 2 workers, 96% ethanol after non-destructive DNA extraction (RBINS, sc 4052311, 4052312); 1 worker, gold-coated for SEM (RBINS, sc 4052301). – Same data as holotype except 21 Mar. 2010, within upper 5 cm of organic layer of a core (Ø 5 cm) extracted by heat using a modified high gradient extractor for 4 days: 28 workers, 96% ethanol (RBINS, sc 4060601); 2 workers, 96% ethanol after non-destructive DNA extraction (RBINS, sc 4060602, 4060603); 1 worker, pinned (MUTPL, sc 4060604). GenBank accession numbers: KT601698–KT601703 and KT750331. Measurements (in mm) and indices Holotype (3 paratypes): HW 0.4 (0.4–0.44), HL 0.58 (0.61–0.62), SL 0.27 (0.29), PrW 0.27 (0.29), WL 0.71 (0.76–0.78), MH 0.23 (0.24), PL 0.22 (0.22–0.23), PW 0.11 (0.12), PPL 0.22 (0.22–0.23), PPW 0.2 (0.22–0.23), AivL 0.31 (0.31–0.33), AivW 0.37 (0.38–0.4), FFeL 0.38 (0.38–0.4), FFeW 0.11 (0.11), HFeL 0.44 (0.44–0.45), HTL 0.42 (0.44–0.46), CI 69.23 (65.45–71.43), SI 66.67 (66.25–72.22), PI 50.00 (52.5–53.66), MI 32.81 (31.43–32.36). Holotype (3 paratypes): HW 0.4 (0.4–0.44), HL 0.58 (0.61–0.62), SL 0.27 (0.29), PrW 0.27 (0.29), WL 0.71 (0.76–0.78), MH 0.23 (0.24), PL 0.22 (0.22–0.23), PW 0.11 (0.12), PPL 0.22 (0.22–0.23), PPW 0.2 (0.22–0.23), AivL 0.31 (0.31–0.33), AivW 0.37 (0.38–0.4), FFeL 0.38 (0.38–0.4), FFeW 0.11 (0.11), HFeL 0.44 (0.44–0.45), HTL 0.42 (0.44–0.46), CI 69.23 (65.45–71.43), SI 66.67 (66.25–72.22), PI 50.00 (52.5–53.66), MI 32.81 (31.43–32.36). Worker With the characters typical of the genus (see diagnosis and description in Brandão et al. 1999 and Borowiec & Longino 2011) and of the L. biconstricta species-group (see diagnosis of the group above). Other characters or differences are as follows: Head. Elongate and rectangular with lateral margin nearly straight and parallel. Posterior corner rounded. Posterior margin modestly convex, almost straight. Parafrontal ridge absent. Clypeal lamella Fig. 5. Habitus in lateral view of paratype (specimen code 4060601) of Leptanilloides prometea Del- sinne & Donoso sp. nov. Note the rounded anterior projection of the subpetiolar process, which differs from the acute shape observed in the specimen on Figs 4C and 6E. Fig. 5. Habitus in lateral view of paratype (specimen code 4060601) of Leptanilloides prometea Del- sinne & Donoso sp. nov. Note the rounded anterior projection of the subpetiolar process, which differs from the acute shape observed in the specimen on Figs 4C and 6E. 14 DELSINNE T., SONET G. & DONOSO D.A., Two new Leptanilloides from Ecuador strongly convex (Fig. 6C). Masticatory margin of mandible dentate but teeth extremely minute, blunt and irregularly shaped, difficult to discern even under magnifications of about 100× (Fig. 6C). Basal and masticatory margins united by a broad convexity. Basal margin faintly crenulate. Labial palp not readily visible but, at least, one-segmented, maxillary palp two-segmented. Hypostomal anterior border rounded and slightly projected outward but without forming a distinct tooth (Fig. 6A). Scape when laid back reaching about medial distance to posterior margin of head. Fig. 6. Leptanilloides prometea Delsinne & Donoso sp. nov. Paratype worker (specimen code 4052301). A. Head, ventral view. B. Mesotibial pectinate spur. C. Anterior part of the head in full-face view, showing vertical and fused frontal carinae, exposed antennal socket, well-developed lateroclypeal tooth, strongly convex clypeal lamella and subtriangular mandible with minute denticles along its inner margin. D. Lower part of propodeum in lateral view, showing the flange over the metapleural gland opening. E. Petiole in lateral view. F. Simple claw (hindleg). Fig. 6. Leptanilloides prometea Delsinne & Donoso sp. nov. Paratype worker (specimen code 4052301). A. Head, ventral view. B. Mesotibial pectinate spur. C. Anterior part of the head in full-face view, showing vertical and fused frontal carinae, exposed antennal socket, well-developed lateroclypeal tooth, strongly convex clypeal lamella and subtriangular mandible with minute denticles along its inner margin. D. Gyne Unknown. Male Unknown. Distribution Only known from the type locality. Worker Lower part of propodeum in lateral view, showing the flange over the metapleural gland opening. E. Petiole in lateral view. F. Simple claw (hindleg). 15 European Journal of Taxonomy 143: 1–35 (2015) Mesosoma. Flange over metapleural gland opening conspicuous and posteriorly forming short, blunt projection, not surpassing propodeum declivity margin (Fig. 6D). Femur not conspicuously enlarged, relatively slender. Midtibia with one short (i.e., half size of foretibia strigil), pectinate spur (Fig. 6B), although pectination may be difficult to observe even at high magnification. Hindtibia with one broadly pectinate spur roughly as long as strigil. Metatibial gland present, visible at high magnification and good lighting as translucent oval area at apex of tibia, behind spur insertion (Fig. 4D). Metatibial gland pore plate observable in SEM (Fig. 7A–C). Metasoma. Long and relatively slender. In dorsal view, petiole uniformly rectangular, twice as long as wide, as long as abdominal segment III (postpetiole). Anterior face concave, posterior face straight. In lateral view, petiole height approximately ¼ smaller than height of abdominal segment III. Petiolar tergite dome-shaped, with short and poorly differentiated anterior and posterior faces, maximum height situated in posterior half. Short tubulated portion present posteriorly. Petiolar spiracle inconspicuous, not in excavation, set near anterior rim of tergite, similar in form and slightly smaller in diameter than propodeal spiracle. Subpetiolar process with ventral margin relatively straight (not distinctly bulging), and without posterior angle (Fig. 6E). Anterior projection of subpetiolar process variable in shape, acute to rounded (Figs 5, 6E). Maximum height of petiolar sternite situated in its anterior half. In dorsal view, abdominal segment III (postpetiole) trapezoid, with straight, parallel anterior and posterior faces. Posterior face almost twice as long as anterior face. In lateral view, tergite evenly convex, without well-differentiated posterior face. Sternite evenly rounded, slightly bulging anteriorly. In dorsal view, abdominal segments IV–VI subequal in length. Pilosity and sculpture. Mandible smooth and shiny, with few scattered piligerous punctures. Head with abundant deep piligerous punctures and smooth interspaces on average equaling puncture diameter, except on ventral side and front where punctures are sparser, separated by more than their diameter. Mesosoma and abdomen more finely and sparsely punctate. Fine reticulate sculpture present laterally on lower pronotum, entire mesopleuron, propodeum, and petiole. Body and appendages with abundant, short and subdecumbent to suberect hairs. Body color brownish to reddish, with head and mesosoma tending to be darker than petiole and gaster. Legs and antennae yellowish. Worker Gyne Unknown. Biology Unknown. All the specimens were collected from a single reserve bordering the Podocarpus National Park on the eastern Andean slope of southern Ecuador. The habitat is an evergreen lower montane forest (Homeier et al. 2008) which is in nearly pristine condition. The forest harbours more than 300 tree species, with Lauraceae, Melastomataceae and Rubiaceae being the most species-rich families (Homeier et al. 2012). The mean annual temperature is c. 15 °C and mean annual precipitation is c. 2200 mm, with low seasonality (Bendix et al. 2008). Soils of the sampling area are cambisols, with a very thick (often > 50 cm) leaf litter layer (Homeier et al. 2012). Soil texture is sandy silt loam; proportion of sand, silt and clay is 41%, 52%, and 6%, respectively; mean pH = 3.2 ± 0.1 SD (n = 24 soil samples). 16 DELSINNE T., SONET G. & DONOSO D.A., Two new Leptanilloides from Ecuador Material examined Material examined ECUADOR: 1 ♂, Zamora-Chinchipe Prov., Reserva Biológica San Francisco, entrance of the T1 trail, 1920 m, 3°58’44.09” S, 79°05’8.26” W, 16–29 Mar. 2011, Malaise trap, coll. Thibaut Delsinne and Tania Milena Arias-Penna (QCAZ, sc 4870101). GenBank accession number: KT601704. Material examined ECUADOR: 1 ♂, Zamora-Chinchipe Prov., Reserva Biológica San Francisco, entrance of the T1 trail, 1920 m, 3°58’44.09” S, 79°05’8.26” W, 16–29 Mar. 2011, Malaise trap, coll. Thibaut Delsinne and Tania Milena Arias-Penna (QCAZ, sc 4870101). GenBank accession number: KT601704. Measurements (in mm) and indices HW 0.34, HL 0.31, SL 0.24, LAII 0.07, LAIII 0.09, LAIV 0.09, LAXIII 0.17, EL 0.13, MaL 0.16, PrW 0.33, WL 0.66, MH 0.36, PL 0.12, PW 0.11, PPL 0.33, PPW 0.33, AivL 0.13, AivW 0.33, FFeL 0.36, FFeW 0.06, HFeL 0.49, HTL 0.4, CI 110.71, SI 70.97, PI 90.91, MI 54.24. Remarks Leptanilloides prometea sp. nov. is the largest species of the L. biconstricta species-group (Fig. 1). The most similar species in habitus and size is L. improvisa, but head of L. prometea sp. nov. is longer (Fig. 1) Leptanilloides prometea sp. nov. is the largest species of the L. biconstricta species-group (Fig. 1). The most similar species in habitus and size is L. improvisa, but head of L. prometea sp. nov. is longer (Fig. 1) Fig. 7. Leptanilloides prometea Delsinne & Denoso sp. nov. Scanning electron micrographs of the metatibial apex of paratype worker (specimen code 4052301), showing the meta- tibial gland pore plate (arrow) at different magnifications. Fig. 8. Leptanilloides ‘Male 4’ (specimen code 4870101) from southern Ecuador. A. Head in full face view. B. Habitus in dorsal view. C. Habitus in lateral view. Fig. 7. Leptanilloides prometea Delsinne & Denoso sp. nov. Scanning electron micrographs of the metatibial apex of paratype worker (specimen code 4052301), showing the meta- tibial gland pore plate (arrow) at different magnifications. Fig. 8. Leptanilloides ‘Male 4’ (specimen code 4870101) from southern Ecuador. A. Head in full face view. B. Habitus in dorsal view. C. Habitus in lateral view. 17 European Journal of Taxonomy 143: 1–35 (2015) and its mandible has only minute and irregular teeth along the masticatory margin (teeth are conspicuous and regularly spaced in L. improvisa). Moreover, L. prometea sp. nov. can easily be distinguished from other species in the group by: the presence of a conspicuous lateroclypeal tooth (absent in L. caracola), the evenly rounded sternite of abdominal segment III (bulging anteriorly in L. biconstricta), the petiole being as long as the postpetiole (petiole longer than postpetiole in L. biconstricta), the petiolar sternite being higher in its anterior half (bulging medially in L. gracilis and L. femoralis), the head dorsum with foveolae separated by about their diameter (more densely foveolate in L. sculpturata, with foveolae separated by less than their diameter), and the short, blunt flange over the metapleural gland opening (long and sharply pointed in L. atlantica, L. copalinga and L. erinys). Leptanilloides sp. ‘Male 4’ (unassociated to workers) Figs 8A–C, 9A–B, 10A–B Leptanilloides sp. ‘Male 4’ (unassociated to workers) Figs 8A–C, 9A–B, 10A–B Description Head. Broader than long, with large convex eye that occupies anterior half of side of head. Mandible slender, falcate, twisted at apex, overlapping at closure (Fig. 8A). Basal and masticatory margins strongly concave, separated by well-developed tooth. External margin straight along basal and medial lengths but strongly bent at apex. Mandible longer than eye length. Lateroclypeal tooth and hypostomal tooth lacking, clypeus short and transverse, with narrow clypeal lamella (apron). Antennal socket horizontal and exposed, located at anterior clypeal margin, margin not projecting anteriorly beyond ventral articulation with labrum. Antenna 13-segmented, each segment longer than wide, with second segment shortest. Scape of moderate length, 1.5 times longer than length of ultimate antennal segment. Scape length nearly four times length of second antennal segment. Lateral ocellus separated from median ocellus by its diameter. Distance between lateral ocelli similar to distance between median and lateral ocellus; as a result, ocelli forming equilateral triangle. Mesosoma. Pronotum U-shaped in dorsal view and reduced anteromedially to thin horizontal strip, set below level of dorsally protruding mesonotum and triangular in lateral view, with pointed posterior apex directed towards wing base. Mesoscutum lacking notaulus. Parapsidal line not discerned, apparently absent. Axillae depressed, not meeting medially, connected by narrow furrow; tegula very small and inconspicuous. Mesopleuron lacking oblique transverse sulcus and hence not divided into anepisternum and katepisternum. Transcutal sulcus deeply impressed. Mesoscutum and mesoscutellum prominently bulging, in lateral view. Metapleural gland not discernable. Propodeum rounded in profile, with dorsal and declivous faces only poorly differentiated; dorsal surface somewhat shorter than declivous. Propodeal spiracle small, circular, positioned slightly above midheight of propodeum and slightly posterior to metanotum. Leg slender, mid tibia with one simple and hind tibia with one simple spur (observed under 50× magnification), pretarsal claw lacking preapical tooth. Wings (Fig. 9A–B). With relatively well-developed venation (for Leptanilloides). Forewing with C present, tubular and pigmented. Sc+R approximated to wing margin, very narrow. Sc+R1 in line with 18 DELSINNE T., SONET G. & DONOSO D.A., Two new Leptanilloides from Ecuador DELSINNE T., SONET G. & DONOSO D.A., Two new Leptanilloides from Ec 19 9. Wings of Leptanilloides ‘Male 4’ (specimen code 4870101) and terminology used iption. A. Forewing. B. Hindwing. Description Abbreviations: 2r-rs = second radial-radial sector cros nal vein, C = costal vein, Cu = cubital vein, cu-a = cubital-anal crossvein, M = medial vein, M ree abscissa of medial vein, M+Cu = fused medial-cubital veins, Pst = pterostigma, Rs+M = al and radial sector veins, Rs = radial sector, Rs·f1 (to Rs·f5) = first (to fifth) free abscissa of r vein, Sc+R = fused subcostal and radial veins, Sc+R1: fused subcostal and first radial vein Fig. 9. Wings of Leptanilloides ‘Male 4’ (specimen code 4870101) and terminology used in the description. A. Forewing. B. Hindwing. Abbreviations: 2r-rs = second radial-radial sector crossvein, A = anal vein, C = costal vein, Cu = cubital vein, cu-a = cubital-anal crossvein, M = medial vein, M·f1 = first free abscissa of medial vein, M+Cu = fused medial-cubital veins, Pst = pterostigma, Rs+M = fused medial and radial sector veins, Rs = radial sector, Rs·f1 (to Rs·f5) = first (to fifth) free abscissa of radial sector vein, Sc+R = fused subcostal and radial veins, Sc+R1: fused subcostal and first radial veins. 19 European Journal of Taxonomy 143: 1–35 (2015) Sc+R, tubular. Pterostigma well-marked. M+Cu tubular and pigmented, curved towards posterior wing margin before division. Rs·f1 nebulous. M·f1 pigmented, tubular. Rs+M, Rs·f2, and Rs·f3 all joined, tubular and pigmented. 1r-rs absent. 2r-rs present, tubular and pigmented. Rs·f4 and Rs·f5 joined and not differentiated in absence of 2rs-m. Rs·f4&Rs·f5 partly tubular, then nebulous, terminating before wing margin. M and Cu diverging at cu-a. Free abscissa of M partly nebulous, then spectral, very weakly visible, joining to Rs+M&Rs·f2&Rs·f3. Abscissa of Cu joined, nebulous throughout most of length and continuing as spectral. Vein A tubular, joining cu-a at obtuse angle and confluent with Rs+M, apparently absent beyond cu-a. Posterior margin of fore wing with fold where hamuli attach, narrow, conspicuous. Hindwing with C present, narrow and faint. Anterior hindwing margin with small differentiated pigmentation area located in distal half of wing. Three hamuli originate in pigmented region. Sc+R present, nebulous, almost reaching third of wing length. Sc+R1 absent. Rs·f1&Rs·f2 partly nebulous, then spectral. Jugal lobe absent. Metasoma. Slender in lateral view, obovate in dorsal view, widest at abdominal segment V. Petiole (abdominal segment II) longer than high or wide, rectangular in dorsal view, with convex lateral margin, ovate in lateral view, and weakly constricted posteriorly, helcium thus apparently quite broad. Distribution Only known from the sampling locality. Description Petiolar spiracle located on anterior third of segment, near anterodorsal extremity. Abdominal segment III three times larger than petiole at its maximum width, not developed as postpetiole nor separated from abdominal segment IV by marked constriction. Abdominal spiracle III located on anterior third of tergite. Abdominal segments II and III with tergosternal fusion. Abdominal segment IV and succeeding segments lacking tergosternal fusion. Spiracle present on anterior third of tergite IV. Abdominal segments V and VI not separated from succeeding segments by constrictions. Abdominal spiracles V and VI indiscernible. Abdominal tergite VIII (pygidium) small and simple but visible dorsally, not wholly covered by abdominal tergite VII. Genitalia (Fig. 10A–B). Pygostyle absent. Abdominal sternite IX (subgenital plate) with posterior margin broadly and deeply concave but not bifurcate. Basal ring present, not hypertrophied. Paramere relatively small, harpago rounded at apex; paramere shorter than petiole length. Volsella a simple, broad lobe, lacking differentiated cuspis. Aedeagus subequal in length to paramere and volsella, simple, narrow, distally spatulate. Pilosity and sculpture. Integument mostly smooth and shiny, with scattered piligerous punctures. Pilosity common on most of body, suberect to decumbent. Color light brown, head and metasoma past abdominal segment III slightly darker. Appendages (antennae, mandibles, legs) lighter than body. DNA results The COI sequences of the two new species and ‘Male 4’ differ significantly from each other (p-distances ranging from 18.8 to 21.7%) (Fig. 11). They also diverge (18–21.8%) from the other Leptanilloides DNA barcodes available in BOLD (6 specimens, 2 species: L. gracilis from the L. biconstricta species- group and L. nubecula from the L. legionaria species-group). Our phylogenetic analyses constructed on the basis of the nuclear gene Wg support one clade consisting of L. prometea sp. nov., L. femoralis, L. gracilis and Amyrmex sp. and another clade composed of L. nubecula, L. nomada and L. mckennae. Both clades are supported by bootstrap values of 98% and 99% in the neighbour-joining tree, 97% and 100% in the most parsimonious tree, 83% and 100% in the tree with maximum likelihood and by posterior probabilities of 1 in the tree built with Bayesian inference (Fig. 12). Remarks The male (specimen code 4870101) collected in southern Ecuador is easily distinguished from any other leptanilloid males (either of Leptanilloides or Amyrmex species, males of all species of Asphinctanilloides being unknown) by the unique shape of its mandible. We did not give it a specific name because it is not associated with workers. However, we called it ‘Male 4’ to indicate that it is different from the three males (‘Male 1’ to ‘Male 3’) described in Borowiec & Longino (2011), which are also without specific names. The wing venation of ‘Male 4’ is relatively similar to that of ‘Male 3’ in Borowiec & Longino (2011), except that the free abscissa of M on the forewing joins Rs+M&Rs·f2&Rs·f3 (not joining in ‘Male 3’) and Sc+R1 is not visible on the hindwing (present as a short nebulous stub in ‘Male 3’). ‘Males 1 and 2’ of Borowiec & Longino (2011) have more reduced venation, as do the males of Amyrmex (Ward & 20 DELSINNE T., SONET G. & DONOSO D.A., Two new Leptanilloides from Ecuador DELSINNE T., SONET G. & DONOSO D.A., Two new Leptanilloides from Ecua 21 Fig. 10. Genitalia of Leptanilloides ‘Male 4’ (specimen code 4870101). A. In posterior view. B entral view. g. 10. Genitalia of Leptanilloides ‘Male 4’ (specimen code 4870101). A. In posterior view. B ntral view. Fig. 10. Genitalia of Leptanilloides ‘Male 4’ (specimen code 4870101). A. In posterior view. B. In ventral view. 21 21 European Journal of Taxonomy 143: 1–35 (2015) Brady 2009). The forewing venation of L. mckennae and L. nubecula is also closely similar but, in these species, veins M and Cu diverge distal to crossvein cu-a (diverging at cu-a in ‘Male 4’). Table 1 lists informative characters to separate leptanilloid males. Brady 2009). The forewing venation of L. mckennae and L. nubecula is also closely similar but, in these species, veins M and Cu diverge distal to crossvein cu-a (diverging at cu-a in ‘Male 4’). Table 1 lists informative characters to separate leptanilloid males. Discussion The discovery of two new Leptanilloides species and a distinct male, which potentially belongs to another species, confirms assertions such as “there are many more species to be found and we have no idea how species-rich the [leptanilloid clade] may be” (Longino 2003), “new species [of Leptanilloides] will continue to be discovered” (Borowiec & Longino 2011), and “the ants may be much more common and widespread than presently known” (Silva et al. 2013). The rate of species discovery within the leptanilloid clade is indeed rapidly increasing (Fig. 13), and should continue to do so, thanks to growing interest in the subterranean ant assemblages, considered as the “final frontier” in the study of the biodiversity of Formicidae (Ryder Wilkie et al. 2007) and linked with the development of new Fig. 11. Neighbour-joining tree showing p-distances among DNA sequences of the mitochondrial COI barcode fragment obtained for all specimens of Leptanilloides Mann, 1823 sequenced here and available in GenBank and BOLD. The tree was rooted with a COI sequence of Cylindromyrmex striatus Mayr, 1870 (GenBank accession number AY233723). Labels provide species identifications and field IDs (in bold) or GenBank or BOLD numbers. Values at nodes indicate bootstrap support only if it was above 80%. Fig. 11. Neighbour-joining tree showing p-distances among DNA sequences of the mitochondrial COI barcode fragment obtained for all specimens of Leptanilloides Mann, 1823 sequenced here and available in GenBank and BOLD. The tree was rooted with a COI sequence of Cylindromyrmex striatus Mayr, 1870 (GenBank accession number AY233723). Labels provide species identifications and field IDs (in bold) or GenBank or BOLD numbers. Values at nodes indicate bootstrap support only if it was above 80%. Fig. 11. Neighbour-joining tree showing p-distances among DNA sequences of the mitochondrial COI barcode fragment obtained for all specimens of Leptanilloides Mann, 1823 sequenced here and available in GenBank and BOLD. The tree was rooted with a COI sequence of Cylindromyrmex striatus Mayr, 1870 (GenBank accession number AY233723). Labels provide species identifications and field IDs (in bold) or GenBank or BOLD numbers. Values at nodes indicate bootstrap support only if it was above 80%. 22 DELSINNE T., SONET G. Discussion & DONOSO D.A., Two new Leptanilloides from Ecuador Species or morphospecies Mandible shape External margin of mandible Clypeal apron HL, HW, CI Parapsidal line Forewing venation Forewing sub- marginal cell Veins M and Cu Paramere ‘Male 4’ falcate, twisted at apex, basal and masticatory strongly concave, separated by a well-developed tooth straight along its basal and medial lengths but strongly bent at apex narrow 0.31, 0.34, 110 not discernable, apparently absent relatively well-developed for Leptanilloides (e.g., Rs·f1 nebulous, M+Cu tubular, Rs·f4&f5 partly tubular and partly nebulous, free abscissa of M partly nebulous and partly spectral, joining Rs+M&Rs·f2&Rs·f3) relatively short, no more than three times longer than wide, not extending distad of stigma diverging at crossvein cu-a shorter than petiole length L. mckennae elongate-triangular, masticatory margin edentate and rounding into unarmed basal margin weakly concave prominent, subtriangular, bluntly pointed medially 0.59–0.64, 0.45–0.47, 131–135 present (long, according to observations made on pictures available on AntWeb 2015a) relatively well-developed for Leptanilloides (e.g., according to fig. 3 in Ward 2007: Rs·f1 tubular, M+Cu tubular, Rs·f4&f5 tubular, free abscissa of M nebulous, joining Rs+M&Rs·f2&Rs·f3) relatively short, no more than three times longer than wide, not extending distad of stigma diverging distal to cu-a by a distance greater than the length of the crossvein longer (about 1.5×) than petiole length L. nubecula falcate, without differentiated masticatory margin, edentate strongly and evenly curved not specified, apparently absent 0.32, 0.32, 100 (perhaps erroneous; see Ward & Brady 2009) not specified not described but, according to fig. 26 in Donoso et al. (2006), relatively well- developed (e.g., Rs·f1 tubular, M+Cu tubular, Rs·f4&f5 tubular, free abscissa of M nebulous, joining Rs+M&Rs·f2&Rs·f3) relatively short, not extending distad of stigma diverging distal to cu-a by a distance shorter than the length of the crossvein not specified ‘Male 1’ of Borowiec & Longino 2011 (good candidate for male caste of L. gracilis) falcate, without differentiated masticatory margin, edentate evenly curved absent 0.18–0.24, 0.25–0.31, 115–130 not discernable extremely reduced (e.g., Rs·f1 absent, M+Cu nebulous and inconspicuous, Rs·f4&f5 nebulous and poorly visible, free abscissa of M absent (or at least nebulous and not joining Rs+M&Rs·f2&Rs·f3, according to fig. Discussion 6f in Borowiec & Longino 2011) no closed submarginal cell diverging at cu-a a little longer than petiole length ‘Male 2’ of Borowiec & Longino 2011 falcate to linear, without differentiated masticatory margin, edentate straight, slightly bent at apex absent 0.3–0.31, 0.37–0.41, 125–127 weakly marked but long, running about two thirds of mesoscutum length relatively well developed (intermediate with, e.g., Rs·f1 reduced, M+Cu nebulous but conspicuous, Rs·f4&f5 partly tubular and partly nebulous, free abscissa of M nebulous and joining Rs+M&Rs·f2&Rs·f3) partially closed; relatively short, not extending distad of stigma diverging at cu-a a little longer than petiole length ‘Male 3’ of Borowiec & Longino 2011 elongate-triangular to sublinear, masticatory margin edentate and weakly differentiated from the unarmed basal margin straight, slightly bent at apex narrow 0.32–0.33, 0.42–0.43, 127–136 long, running about one third of mesoscutum length relatively well-developed (e.g., Rs·f1 nebulous, M+Cu tubular, Rs·f4&f5 tubular, free abscissa of M nebulous, not joining Rs+M&Rs·f2&Rs·f3) relatively short, less than four times longer than wide, not extending distad of stigma diverging at cu-a shorter than petiole length Amyrmex spp. elongate-triangular to sublinear, masticatory margin edentate and weakly differentiated from the unarmed basal margin curved basally, straight medially, and bent slightly mesad at apex narrow HL not specified, HW 0.32–0.41; CI 125–137 very weakly impressed, barely discernable reduced (e.g., based on Fig. 6 in Ward & Brady 2009, Rs·f1 tubular, M+Cu tubular, Rs·f4&f5 tubular, free abscissa of M absent) elongate, four times longer than wide, extending distad of stigma diverging at cu-a shorter (about 0.8×) than petiole length Table 1. List of informative characters to separate the Leptanilloides male collected in southern Ecuador (‘Male 4’) from males of L. mckennae Longino, 2003 (Ward 2007), L. nubecula (Donoso et al. 2006), three males without specific names (Borowiec & Longino 2011), and from males of the genus Amyrmex Kusnezov, 1953 (Ward & Brady 2009). 23 European Journal of Taxonomy 143: 1–35 (2015) techniques designed to specifically collect this subterranean ant fauna (Weissflog et al. 2000; Berghoff et al. 2003; Ryder Wilkie et al. 2007; Brandão et al. 2008; Schmidt & Solar 2010). techniques designed to specifically collect this subterranean ant fauna (Weissflog et al. 2000; Berghoff et al. 2003; Ryder Wilkie et al. 2007; Brandão et al. 2008; Schmidt & Solar 2010). Workers of L. copalinga sp. nov. and L. prometea sp. nov. presented the characteristic morphology of species from the L. biconstricta species-group. Discussion Interestingly, our phylogenetic analyses, albeit based on a single nuclear gene, supported the existence of the L. biconstricta and L. legionaria species-groups, and placed L. prometea sp. nov. within the clade composed of the L. biconstricta species-group, supporting the morphology-based hypothesis. Our results also suggest that COI can be used as a molecular tool to identify species of Leptanilloides, since large divergences were observed at COI among specimens that were assigned to different species on the basis of morphology. Unfortunately, these taxa are currently known from a limited number of workers and are not all represented in the library of DNA barcodes. Therefore, we could not compare the new sequences with those of all other species in the genus. It remains necessary to accumulate information concerning the leptanilloid clade to facilitate male-worker caste association and to establish generic limits with confidence. This case study highlights the interest in building a comprehensive reference library of COI barcodes and other DNA markers. The characters of the L. legionaria species-group are reminiscent of the three currently known species of Asphinctanilloides (Longino 2003; Donoso et al. 2006; Borowiec & Longino 2011) [two workers of a fourth species were recently discovered but have not yet been formally described (Silvestre et al. 2012; Silvestre pers. comm.)]. However, Asphinctanilloides does not exhibit constrictions (cincti) between the gastral segments, a condition considered as plesiomorphic within the leptanilloid clade (Brandão et al. 1999). In addition, four sting characters were identified as apomorphies for Asphinctanilloides, but because the sting apparatus of only one species of the L. legionaria species-group has been studied so far (L. legionaria in Brandão et al. 1999), it is not possible to assert that these traits are absent in all species of Leptanilloides. The monotypic Amyrmex is only known from its male (Ward & Brady 2009). The genus was initially placed in Dolichoderinae Forel, 1878, but genetic studies, based on fragments of 7–11 nuclear genes, confirmed that it is nested within Leptanilloides, apparently as sister of the L. biconstricta species-group Fig. 12. Neighbour-joining tree showing p-distances among DNA sequences of the wingless nuclear marker obtained for specimens of Leptanilloides Mann, 1823, Amyrmex Kusnezov, 1953 and Cylindro- myrmex Mayr, 1870 (as outgroup), available in GenBank and sequenced here (specimen code 4052311). Labels provide species identifications and field IDs (in bold) or GenBank or BOLD numbers. Discussion Values at nodes correspond to the bootstrap values (%) and posterior probabilities obtained in the Neighbour- joining/parsimony/maximum likelihood/Bayesian inference analyses. Bootstrap values < 80% and posterior probabilities < 0.95 are not indicated. Fig. 12. Neighbour-joining tree showing p-distances among DNA sequences of the wingless nuclear marker obtained for specimens of Leptanilloides Mann, 1823, Amyrmex Kusnezov, 1953 and Cylindro- myrmex Mayr, 1870 (as outgroup), available in GenBank and sequenced here (specimen code 4052311). Labels provide species identifications and field IDs (in bold) or GenBank or BOLD numbers. Values at nodes correspond to the bootstrap values (%) and posterior probabilities obtained in the Neighbour- joining/parsimony/maximum likelihood/Bayesian inference analyses. Bootstrap values < 80% and posterior probabilities < 0.95 are not indicated. 24 DELSINNE T., SONET G. & DONOSO D.A., Two new Leptanilloides from Ecuador (Ward & Brady 2009; Brady et al. 2014) or even nested within this species-group (this study). It is therefore clear that generic limits within the leptanilloid clade should be reassessed (Longino 2003; Ward & Brady 2009; Borowiec & Longino 2011). However, a broad set of uncertainties hampers that task, including (1) the unknown molecular-based phylogenetic position of Asphinctanilloides, (2) the unknown worker and male caste of Amyrmex and Asphinctanilloides, respectively, (3) the poor morphological distinction between the L. legionaria species-group and Asphinctanilloides, (4) the lack of collection series for nearly all species to correctly appreciate intra- and interspecific morphological variations, and (5) the availability of molecular data for only a limited number of species of Leptanilloides. The morphology of the two new species of Leptanilloides corresponds to the diagnosis of the genus based on the worker caste offered by Borowiec & Longino (2011), except that these species have one pectinate spur on both mid- and hindtibiae, giving the spur formula 1p,1p. The midtibial spur is small, making the pectination difficult to observe with a stereo microscope even at high magnification; however, SEM examinations were conclusive. Hitherto, the spur formula in species of Leptanilloides has been considered to be either 1s,1p or 2s,2s (Borowiec & Longino 2011). We had the opportunity to study paratypes of L. nomada (QCAZ I 119692) and L. nubecula (QCAZ I 119693, QCAZ I 119694). The three specimens possess a single short, but pectinate midtibial spur. This character also seems to be present in L. atlantica (Silva et al. 2013: fig. 2d). Discussion It could be interesting to reassess this criterion in previously described species of Leptanilloides and Asphinctanilloides, in order to evaluate its taxonomic value. A metatibial gland was detected in L. prometea sp. nov. (Fig. 7). In stereo microscopy, the gland reservoir is visible under the translucent cuticle at the base of the hindtibial spur, but observing it requires good Fig. 13. Cumulative number of described species of Leptanilloides Mann, 1823 over time, since the description of L. biconstricta Mann, 1823. Fig. 13. Cumulative number of described species of Leptanilloides Mann, 1823 over time, since the description of L. biconstricta Mann, 1823. 25 European Journal of Taxonomy 143: 1–35 (2015) lighting. In SEM, the gland area appears as an irregular patch of porous cuticle, contrasting with the smoother surrounding surface, as in L. erinys of the L. biconstricta species-group and L. nubecula of the L. legionaria species-group (Borowiec & Longino 2011). We were not able, however, to discern a metatibial gland in L. copalinga sp. nov. lighting. In SEM, the gland area appears as an irregular patch of porous cuticle, contrasting with the smoother surrounding surface, as in L. erinys of the L. biconstricta species-group and L. nubecula of the L. legionaria species-group (Borowiec & Longino 2011). We were not able, however, to discern a metatibial gland in L. copalinga sp. nov. As already noted by Donoso et al. (2006), the mandibles of Leptanilloides males vary greatly in shape, ranging from elongate-triangular to falcate (sickle-shaped), and with basal and masticatory margins ranging from undifferentiated to forming two distinct concavities. In Eciton Latreille, 1804 and Dorylus Fabricius, 1793, two other genera of Dorylinae, males use their sickle-shaped mandibles to grab the dichthadiigyne at her petiolar horns during copulation (Kronauer & Boomsma 2007). In Leptanilloides, the gyne caste is known for only two species, L. nubecula (Donoso et al. 2006) and L. erinys (Borowiec & Longino 2011). In both species, the gyne is subdichthadiiform with an enlarged petiole but without the dorsal or lateral horns typical of Eciton and Dorylus gynes. It is reasonable to expect that most males of Leptanilloides also use their elongate mandibles to grab the female behind the petiole during copulation. However, the variety of mandibular shapes also suggests that males may display different reproductive behaviors (Shik et al. 2013). The recent discovery of L. Note added in press During the editing process, a 15th species of Leptanilloides, namely L. chihuahuaensis MacGown, Schiefer & Branstetter, 2015, was described based on six males from western Texas, USA. This species extends the range of the genus 2500 km to the north and indicates that it possesses a larger ecological niche than suspected, since it can also be found in xeric ecosystems. The COI sequence of L. chihuahuaensis is distant by more than 10 % from L. copalinga sp. nov., L. prometea sp. nov. and ‘Male 4’, providing convincing support that L. chihuahuaensis is neither the male of L. copalinga or L. prometea, nor the same species as ‘Male 4’. Moreover, the mandible shape of ‘Male 4’ remains unique within the genus. MacGown J.A., Schiefer T.L. & Branstetter M.G. 2015. First record of the genus Leptanilloides (Hymeno- ptera: Formicidae: Dorylinae) from the United States. Zootaxa 4006: 392-400. http://dx.doi.org/10.11646/ zootaxa.4006.2.10 Discussion atlantica in the Brazilian Atlantic Forest considerably expanded the geographic range of the genus (Silva et al. 2013). Nevertheless, it seems that the highest diversity of Leptanilloides occurs in the Andes, especially at high elevations. In this respect, the montane forests and páramos of Ecuador are noteworthy, since half of the currently known species of Leptanilloides (i.e., 7 out of 14) were collected in these habitats. Unfortunately, Andean ecosystems are currently being destroyed at an alarming rate (Goerner et al. 2007; Tejedor Garavito et al. 2012; Thies et al. 2014; Curatola Fernández et al. 2015). To determine whether the observed distribution of species of Leptanilloides is a mere sampling artifact or an actually existing pattern (Silva et al. 2013), it would be necessary to (1) carry out more inventories of the subterranean ant fauna in the Andes and elsewhere, and (2) secure the conservation of Andean habitats. Acknowledgements We warmly thank Catherine Vits and Boudewijn de Roover from the Copalinga Private Reserve for access to their estate; Jörg Bendix, Felix Matt, Jörg Zeilinger, the DFG-Deutsche Forschungsgemeinschaft Research Unit 816, the team of the Estación Científica San Francisco (ECSF) and Naturaleza y Cultura Internacional for allowing and extensively facilitating our work at ECSF and RBSF; the Ministerio del Ambiente of the Republic of Ecuador for collection permits; Isabelle Bachy, Julien Cillis, Yves Laurent, and Maurice Leponce from RBINS for their help with ant digitization; and Tania Milena Arias-Penna, Delia Magaly Montalván Carrión, and Jaime Peña for assistance during fieldwork. The 26 DELSINNE T., SONET G. & DONOSO D.A., Two new Leptanilloides from Ecuador authors thank Marek Borowiec and one anonymous reviewer for insightful comments and suggestions. 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Genetic Algorithm Approaches for the Phylogenetic Analysis of Large Biological Sequence Datasets under the Maximum Likelihood Criterion. PhD Thesis. University of Texas at Austin, Texas. Manuscript received: 22 June 2015 Manuscript accepted: 12 August 2015 Published on: 8 October 2015 Topic editor: Koen Martens Desk editor: Danny Eibye-Jacobsen Printed versions of all papers are also deposited in the libraries of the institutes that are members of the EJT consortium: Muséum National d’Histoire Naturelle, Paris, France; Botanic Garden Meise, Belgium; Royal Museum for Central Africa, Tervuren, Belgium; Natural History Museum, London, United Kingdom; Royal Belgian Institute of Natural Sciences, Brussels, Belgium; Natural History Museum of Denmark, Copenhagen, Denmark. 30 DELSINNE T., SONET G. & DONOSO D.A., Two new Leptanilloides from Ecuador Appendix 1 Nexus file with all aligned sequences of the wingless gene (Wg) used in this paper and Mr Bayes command block. #NEXUS #NEXUS begin data; dimensions ntax=10 nchar=412; format missing=? gap=- datatype=dna; matrix 4052311_L_prometea ACTACCGAACTTCCGCGTTGTCGGCGACAACCTGAAGGATCGTTTCGACGGCGCGTCC CGGGTGATGGTGACCAATTCGGACCGCGCCCGCATCATCGCAGCTAACGCGATTACCA GCAACTCGGCCAGCAACTCCGTGCACCAGCACCGCGGCGGTCTCGCACGCCGGCAGCGC TACAATTTCCAATTAAAACCGTACAATCCGGAGCACAAGCCGCCCGGGCGCAAGGACCTC GTCTACGTGGAAACGTCGCCGGGCTTCTGCGAGAAGAACCCGAAATTCGGCATCCTCGG CACGCAGGGCCGTCAGTGCAACGACACCAGCATCGGCGTCGACGGGTGCGACTTGATGT GCTGCGGCAGAGGCTACAAGACCCAGGAGGTGACGGTGA?CGAGAGGT?C??CTGCTCC FJ588490_Amyrmex_BR01 GCTGCCGAACTTCCGCGTTGTCGGCGACAACCTGAAGGACCGTTTCGACGGCGCGTCG CGGGTAATGGTGACCAACTCGGACCGCGCCCGCATCATCGCGGCTAACGCGATTACCA GCAACTCGGCCAGCAACTCCGTGCACCAGCACCGCGGCGGTCTCGCACGCCGGCAGCGC TACAATTTCCAACTGAAACCGTACAATCCGGAGCACAAGCCGCCCGGGCGCAAGGACCT CGTCTACGTGGAGGCGTCGCCGGGCTTCTGCGAGAAGAACCCGAAATTCGGCATCCTCG GCACCCACGGCCGCCAGTGCAACGATACCAGCATCGGCGTCGACGGATGCGACCTGATGT GCTGCGGCAGGGGCTACAAGACCCAGGAGGTAACAGTGATCGAGAGGTGCGCCTGCACC J _ _ GCTGCCGAACTTCCGCTCTGTCGGCGACAACCTGAAGGACCGTTTCGACGGCGCGTCCCG GGTGATGATGACCCTCTCGGACCGCCCCCGCAGC---GCGGCGAACGCGATTATCAGCAACT CGGCCAGCAACTCCGTGCACCAGCACCGGAGCGGTCTCGCGCGCCGCCAGCGCTACAAC TTCCAGCTGAAACCGTACAATCCGGAGCACAAGCCGCCCGGGCGCAAGGACCTCGTTTA CGTGGAAGCGTCGCCGGGTTTCTGCGAGAGGAACCCGAAATTCGGCATCATGGGCACC CACGGCCGCCAGTGCAACGACACCAGCATCGGCGTCGACGGCTGCGACCTCATGTGCTG CGGCAGGGGCTACAAGACCCAGGAGGTGACGGTGATCGAGAGGTGCGCCTGCACG KJ523540_L_gracilis GCTGCCCAACTTCCGCGTCGTCGGCGACAACCTGAAGGACCGTTTCGACGGCGCGTCC CGGGTAATGGTGACCAATTCCGACCGCGCCCGCATCATCGCGGCTAACGCGATTACCA GCAACTCGGCCAGCAATTCCGTGCACCAGCACCGCGGCGGCCTCGTGCGCCGGCAGCGC TACCATTTCCAGCTGAAACCGTACAATCCGGAGCACAAGCCGCCCGGGCGCAAGGACCT CGTCTACGTGGAGGCGTCGCCGGGCTTCTGCGAGAAGAACCCGCGATTCGGCATCCTCGG CACCCACGGACGCCAGTGCAACGACACCAGCATCGGCGTCGACGGCTGCGACCTCATGT GCTGCGGCAGGGGCTACAAGACCCAGGAGGTCACGGTGATCGAGAGGTGCGCCTGCACC KJ523539_L_femoralis _ _ GCTACCGAACTTCCGCGTTGTCGGCGACAACTTGAAGGACCGTTTCGACGGGGCGTCC CGGGTGATGGTGACCAACTCGGACCGCGCCCGCATCATCGCGGCTAACGCGATTACCA GCAACTCGGCCAGCAACTCCGTGCACCAGCACCGCGGCGGTCTTGCACGCCGGCAGCGC 31 European Journal of Taxonomy 143: 1–35 (2015) TACAATTTCCAACTGAAACCGTACAACCCGGAGCACAAGCCGCCAGGGCGCAAGGACCT CGTGTACGTGGAGACGTCGCCGGGCTTCTGCGAGAAGAACCCGAAATTCGGCATTATGGG CACCCACGGCCGGCAATGCAACGACACCAGCATTGGCGTCGACGGGTGCGACCTCATGT GCTGCGGCAGGGGTTACAAGACCCAGGAGGTGACGGTGATCGAGAGGTGCGCCTGCACC DQ353106_L_nomada DQ353106_L_nomada GCTGCCCAACTTCCGCTCAGTCGGCGACAACCTGAAGGACCGTTTCGACGGCGCCTC GCGGGTGATGATTACCCTCTCGGACCGTCCCCGCAGC---GCGGCGAACGCGATTATCA GCAACTCGGCCAGCAACTCCGTGCACCAGCACCGGAGCGGTCTCGCGCGCCGCCAGCGC TACAACTTCCAGCTGAAACCGTACAATCCGGAGCACAAGCCGCCCGGGCGCAAGGACCT CGTGTACGTGGAAGCGTCGCCGGGTTTCTGCGAGAGGAACCCGAAATTCGGCATCATGGG CACCCACGGCCGGCAGTGCAACGACACCAGCATCGGCGTCGACGGCTGCGACCTGATGT GCTGCGGCAGGGGCTACAAGACCCAGGAGGTGACGGTGATCGAGAGGTGCGCCTGCACG AY867429_L_mckennae AY867429_L_mckennae GCTGCCGAACTTCCGCTCTGTCGGCGACAACCTGAAGGACCGTTTCGACGGCGCGTCC CGGGTGATGATGACCCTGTCGGACCGCCCCCGCAGC---GCGGCGAACGCGATTATCA GCAACTCGGCCAGCAACTCCGTGCACCAGCACCGTAGCGGTCTCGCGCGCCGCCAGCGC TACAACTTCCAGCTGAAGCCGTACAATCCGGAGCACAAGCCGCCCGGGCGCAAGGACCT CGTGTACGTGGAGGCGTCGCCGGGTTTCTGCGAGAAGAACCCGAAATTCGGCATCATGGG CACCCACGGCCGGCAGTGCAACGACACCAGCATCGGCGTCGACGGCTGCGACCTAATGT GCTGCGGCAGGGGCTACAAGACCCAGGAGGTGACGGTGATCGAGAGGTGCGCCTGCACG _ _ GCTGCCCAACTTCCGCGTGGTCGGCGACAACCTGAAGGACCGCTTCGACGGCGCGTCA CGAGTGATGGTGACCAACTCGGATCGCGCCCGCAGC---AACGCCAACGCCATCATCA GCAACTCGGCCAGCAACTCCGTGCACAATCACCGCGGTGGTCTGGGACGTCGGCAGCGC TACAACATCCAGCTGAAGCCGTACAATCCGGAGCACAAGCCGCCCGGGAGCAAGGACCT CGTGTACGTGGAGCCGTCGCCGCCGTTCTGCGAGAAGAACCCGAAACTCGGGATCCTGG GCACCCACGGCCGGCAGTGCAACGACACGAGCATCGGCGTCGATGGCTGCGACCTGATG TGCTGCGGCAGAGGCTACAAGACCGAGGAGGTGACGGTGATCGAGAGGTGCGCCTGCA CG AY233640_C_striatus ACTGCCGAACTTCCGCGTGGTCGGCGACAACCTGAAGGACCGCTTCGACGGCGCGTC TCGAGTGATGGTGACGAACTCGGATCGCGCTCGCGGC---AACGCGAACGCCATCATCA GCAACTCGGCCAGCAACTCCGTGCACCATCACCGCGCTGGGCTGGGACGTCGGCAGCGC TACAACATCCAGCTGAAGCCGTACAATCCGGAGCACAAGCCGCCCGGCAGCAAGGACCT CGTCTACGTGGAGCCGTCGCCGCCGTTCTGCGAGAAGAACCCGAAACTCGGCATCCTGG GCACCCACGGCCGGCAGTGCAACGACACCAGCATCGGCGTCGACGGCTGCGACCTGATG TGCTGCGGCAGGGGCTACAAGACCGAGGAGGTGACGGTGATCGAGAGGTGCGCC?????? AY867426_C_striatus ACTGCCGAACTTCCGCGTGGTCGGCGACAATCTGAAGGACCGCTTCGACGGCGCGTC TCGAGTGATGGTGACGAACTCGGATCGCGCTCGCGGC---AACGCGAACGCCATCATCA GCAACTCGGCCAGCAACTCCGTGCACCATCACCGCGCTGGGCTGGGACGTCGGCAGCGC TACAACATCCAGCTGAAGCCGTACAATCCGGAGCACAAGCCGCCCGGCAGCAAGGACCT CGTCTACGTGGAGCCGTCGCCGCCGTTCTGCGAGAAGAACCCGAAACTCGGCATCCTGGG CACCCACGGCCGGCAGTGCAACGACACCAGCATCGGCGTCGACGGCTGCGACCTGATGT GCTGCGGCAGGGGCTACAAGACCGAGGAGGTGACGGTGATCGAGAGGTGCGCCTGCACG 32 DELSINNE T., SONET G. & DONOSO D.A., Two new Leptanilloides from Ecuador charset 1st = 2-412\3; charset 2nd = 3-412\3; charset 3rd = 1-412\3; charpartition bypart = wg1:1st, wg2:2nd, wg3:3rd; end; begin codons; codonposset * coding= 1:2-412\3, 2:3-412\3, 3:1-412\3; end; partition genes = 3: wg1, wg2, wg3 set partition = genes; lset applyto=(2,3) nst=1; lset applyto=(1) nst=6; lset applyto=(1) rates=Gamma; lset applyto=(2,3) rates=Equal; partition genes = 3: wg1, wg2, wg3; partition genes = 3: wg1, wg2, wg3; set partition = genes; lset applyto=(2,3) nst=1; lset applyto=(1) nst=6; lset applyto=(1) rates=Gamma; lset applyto=(2,3) rates=Equal; unlink statefreq=(all) revmat=(all) shape=(all) pinvar=(all); prset applyto=(all) ratepr=variable; mcmcp ngen=10000000 printfreq=1000 samplefreq=1000 nchains=4 savebrlens=yes filename=w ghok_BI; mcmc; end; unlink statefreq=(all) revmat=(all) shape=(all) pinvar=(all); unlink statefreq=(all) revmat=(all) shape=(all) pinvar=(all); prset applyto=(all) ratepr=variable; mcmcp ngen=10000000 printfreq=1000 samplefreq=1000 nchains=4 savebrlens=yes filename=w ghok_BI; mcmc; end; 33 European Journal of Taxonomy 143: 1–35 (2015) R script used for the parsimony analysis. library(ape) library(phangorn) dna<-read.dna(“input.fasta”,”fasta”) dnaphy<-as.phyDat(dna) tre.ini<-nj(dist.dna(dna,model=”raw”)) parsimony(tre.ini, dnaphy) tre.pars<-optim.parsimony(tre.ini, dnaphy) root(tre.pars,out=10) tre.Ratchet<-pratchet(dnaphy, trace = 0) tres.pars<-bootstrap.phyDat(dnaphy, pratchet, trace=0) tre.Ratchet<-acctran(tre.Ratchet, dnaphy) plotBS(tre.Ratchet, tres.pars)->treB.pars write.tree(treB.pars, “RParsimony.phy”) library(ape) library(phangorn) dna<-read.dna(“input.fasta”,”fasta”) dnaphy<-as.phyDat(dna) tre.ini<-nj(dist.dna(dna,model=”raw”)) parsimony(tre.ini, dnaphy) tre.pars<-optim.parsimony(tre.ini, dnaphy) root(tre.pars,out=10) tre.Ratchet<-pratchet(dnaphy, trace = 0) tres.pars<-bootstrap.phyDat(dnaphy, pratchet, trace=0) tre.Ratchet<-acctran(tre.Ratchet, dnaphy) plotBS(tre.Ratchet, tres.pars)->treB.pars write.tree(treB.pars, “RParsimony.phy”) 34 34 DELSINNE T., SONET G. & DONOSO D.A., Two new Leptanilloides from Ecuador Appendix 3 Configuration file used for the maximum likelihood analysis using GARLI ver. 2.0 (Zwickl 2006).. [general] datafname = wghok.nex constraintfile = none streefname = stepwise attachmentspertaxon = 50 ofprefix = MLgarlibest randseed = -1 availablememory = 512 logevery = 10 saveevery = 100 refinestart = 1 outputeachbettertopology = 0 outputcurrentbesttopology = 0 enforcetermconditions = 1 genthreshfortopoterm = 5000 scorethreshforterm = 0.001 significanttopochange = 0.01 outputphyliptree = 0 outputmostlyuselessfiles = 0 writecheckpoints = 0 restart = 0 outgroup = 10 resampleproportion = 1.0 inferinternalstateprobs = 0 outputsitelikelihoods = 0 optimizeinputonly = 0 collapsebranches = 1 numratecats = 1 invariantsites = non numratecats = 1 invariantsites = none [model3] datatype = nucleotide ratematrix = ( 0 1 2 2 3 0 ) statefrequencies = estimate ratehetmodel = gamma numratecats = 4 invariantsites = none [master] nindivs = 2 holdover = 1 selectionintensity = 0.5 holdoverpenalty = 0 stopgen = 5000000 stoptime = 5000000 startoptprec = 0.5 minoptprec = 0.01 numberofprecreductions = 10 treerejectionthreshold = 50.0 topoweight = 1 modweight = 0.05 brlenweight = 0.2 randnniweight = 0.1 randsprweight = 0.3 limsprweight = 0.6 intervallength = 100 intervalstostore = 5 limsprrange = 6 meanbrlenmuts = 5 gammashapebrlen = 1000 gammashapemodel = 1000 uniqueswapbias = 0.1 distanceswapbias = 1.0 [model1] datatype = nucleotide ratematrix = ( 0 0 0 0 0 0 ) statefrequencies = estimate ratehetmodel = none numratecats = 1 invariantsites = none [model2] datatype = nucleotide ratematrix = ( 0 0 0 0 0 0 ) statefrequencies = estimate ratehetmodel = none 35
https://openalex.org/W1976613160	https://docusalut.com/bitstream/20.500.13003/11275/1/000335503000001.pdf	English	null	Effectiveness of a complex intervention in reducing the prevalence of smoking among adolescents: study design of a cluster-randomized controlled trial	BMC public health	2,014	cc-by	6,367	STUDY PROTOCOL Open Access Open Access © 2014 Leiva et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Effectiveness of a complex intervention in reducing the prevalence of smoking among adolescents: study design of a cluster-randomized controlled trial so Leiva1, Andreu Estela2, Maties Torrent3, Amador Calafat4, Miquel Bennasar5 and Aina Yáñez6 Leiva et al. BMC Public Health 2014, 14:373 http://www.biomedcentral.com/1471-2458/14/373 Leiva et al. BMC Public Health 2014, 14:373 http://www.biomedcentral.com/1471-2458/14/373 Correspondence: aleiva@ibsalut.caib.es 1Primary Care Research Unit of Mallorca, Baleares Health services-IbSalut, Mallorca, Spain Full list of author information is available at the end of the article Abstract Trial registration: Current Controlled Trials: NCT01602796. Keywords: Adolescent, Intervention study, Randomized controlled trial, Risk-reducing behavior, Schools Keywords: Adolescent, Intervention study, Randomized controlled trial, Risk-reducing behavior, Schools Abstract Background: The likelihood of an adolescent taking up smoking may be influenced by his or her society, school and family. Thus, changes in the immediate environment may alter a young person’s perception of smoking. Methods/Design: The proposed multi-center, cluster-randomized controlled trial will be stratified by the baseline prevalence of smoking in schools. Municipalities with fewer than 100,000 inhabitants will be randomly assigned to a control or intervention group. One secondary school will be randomly selected from each municipality. These schools will be randomized to two groups: the students of one will receive any existing educational course regarding smoking, while those of the other school will receive a four-year, class-based curriculum intervention (22 classroom lessons) aimed at reinforcing a smoke-free school policy and encouraging smoking cessation in parents, pupils, and teachers. The intervention will also include annual meetings with parents and efforts to empower adolescents to change the smoking related attitudes and behaviors in their homes classrooms and communities g p p smoking-related attitudes and behaviors in their homes, classrooms and communities. g We will enroll children aged 12-13 years as they enter secondary school during two consecutive school years (to obtain sufficient enrolled subjects). We will follow them for five years, until two years after they leave secondary school. All external evaluators and analysts will be blinded to school allocation. y The aim of this study is to analyze the effectiveness of a complex intervention in reducing the prevalence of smoking in the third year of compulsory secondary education (ESO) and two years after secondary school, when the participants are 14-15 and 17-18 years old, respectively. The aim of this study is to analyze the effectiveness of a complex intervention in reducing the prevalence of smoking in the third year of compulsory secondary education (ESO) and two years after secondary school, when the participants are 14-15 and 17-18 years old, respectively. Discussion: Most interventions aimed at preventing smoking among adolescents yield little to no positive long-term effects. This clinical trial will analyze the effectiveness of a complex intervention aimed at reducing the incidence and prevalence of smoking in this vulnerable age group. Discussion: Most interventions aimed at preventing smoking among adolescents yield little to no positive long-term effects. This clinical trial will analyze the effectiveness of a complex intervention aimed at reducing the incidence and prevalence of smoking in this vulnerable age group. Trial registration: Current Controlled Trials: NCT01602796. Background approximately 15% of 14- and 15-year-olds smoking, al- though this percentage varies from 7% to 23% among countries [3]. Some countries have reported a change in the pattern of initial consumption in recent years, with more girls beginning to smoke, especially among younger age groups [4]. Within any given country, a high variability in smoking behavior is seen across schools [5-7]. Tobacco use is a known risk factor for many chronic illnesses; it is the main cause of avoidable mortality in Europe, and it is responsible for 4.2 million deaths per year worldwide [1,2]. The prevalence of tobacco use among adolescents in the European Union is high, with The various factors associated with taking up smoking can be grouped into three categories, individual, social and cultural factors [8]. Individual factors are intrinsically * Correspondence: aleiva@ibsalut.caib.es 1Primary Care Research Unit of Mallorca, Baleares Health services-IbSalut, Mallorca, Spain Full list of author information is available at the end of the article Page 2 of 8 Leiva et al. BMC Public Health 2014, 14:373 http://www.biomedcentral.com/1471-2458/14/373 Leiva et al. BMC Public Health 2014, 14:373 http://www.biomedcentral.com/1471-2458/14/373 Leiva et al. BMC Public Health 2014, 14:373 http://www.biomedcentral.com/1471-2458/14/373 in municipalities. There is 53 municipalities in Majorca and 8 in Minorca, between 300 to 350,000 inhabitants. All Balearic Island municipalities in Majorca and Minorca having fewer than 100,000 inhabitants and at least one public compulsory secondary-educational institution will be included. In municipalities containing more than one secondary school, the secondary school to be included will be determined through a randomization process. If the se- lected school declines to participate, another school from the same municipality will be invited to take part. linked to a given person and include the individual’s age and certain cognitive, emotional and biological aspects. The factors associated with the immediate social con- text (i.e., the environment closest to the adolescent) include the smoking-related attitudes and behaviors of family members and friends. The factors associated with the socio-cultural environment (i.e., the wider so- cial context) include the smoking policy of the adoles- cent’s school. Around 70% of current smokers began smoking before the age of 18 years, and those who do not begin to smoke by this age are highly unlikely to become smokers as adults [9]. Study population Th l i The populations of the included municipalities vary from 11,000 and 30,000 inhabitants. The main economic ac- tivities of these communities are tourism and farming. The study participants will consist of all 12-13 year- old students, who enrolled in the first grade of compul- sory secondary education (ESO) over two consecutive academic periods (i.e., 2011-12 and 2012-13). The inter- vention will take place over the four years during which these adolescents are enrolled in ESO. Written informed consent will be obtained from all students and from at least one parent/guardian per student, according to the declaration of Helsinki. A review of the effectiveness of distinct school- and/or community-based interventions to prevent smoking [10] indicates that multi-sector interventions (i.e. those involv- ing the classroom, school, extracurricular activities, family, and community) yield more promising results than single- sector interventions. Furthermore, the evidence suggests that school-based preventive programs are not effective on their own; instead, interventions should be community- based, and should have impacts in the classroom, the school environment, and the community [11]. Random assignment f d Cluster randomization is required because the inter- ventions will be implemented at the school level. Once informed consent and the commitment to partici- pate have been obtained from the students/parents and the school, respectively, a baseline survey will be per- formed to determine the prevalence of smoking. There- after, the participating schools will be subjected to stratified randomization according to the baseline preva- lence of tobacco use at each school by the “random” module, and the option separate strata of PEPI (version 4.0, Sagebrush Press). Measurements Table 1 summarizes the study measurements, variables and schedule. The baseline questionnaire will gather data on a given student’s sociodemographic characteristics, en- vironment and tobacco use, and the attitudes of his/her Background Preventing young people from starting to smoke and helping young smokers break the habit are currently the most effective means to reduce the number of deaths attributable to smoking in the medium and long terms. To this end, various European countries have introduced programs designed to prevent tobacco use. Such programs should be based on scientific evi- dence and seek to reach children before they commence smoking. Recruitment Headmasters at the selected schools will be contacted by a research team member, who will provide details about the study and invite the school to participate. Pilot study A pilot program is currently underway in a single public secondary education center to test the proposed inter- vention, the materials, and the data-collection networks. Following the four-year intervention and follow-up, data will be gathered and evaluated. We plan to implement the full study at the two-year mark of the pilot program. We propose to carry out a cluster-randomised con- trolled clinical trial to analyse the effectiveness of a multifactorial intervention addressed to adolescents and, in particular, their environment (educational centre, teachers and parents), as we believe that altering the adolescents’ immediate environment could have a considerable effect on reducing tobacco use among the young. Design and environment This prospective multi-center cluster-randomized con- trolled trial will involve schools and communities strati- fied by school-level tobacco consumption at baseline. The clusters will consist of municipalities in the Balearic Islands. Control schools will continue to implement any smoking-prevention activities that are already in place when the study begins. Inclusion criteria The Balearic island is organized into four insular coun- cils (Majorca, Minorca, Ibiza and Formentera) and those Leiva et al. BMC Public Health 2014, 14:373 http://www.biomedcentral.com/1471-2458/14/373 Leiva et al. BMC Public Health 2014, 14:373 http://www.biomedcentral.com/1471-2458/14/373 Page 3 of 8 Table 1 Measures, questionnaires and timeline Instrument Assessment area Timing of assessment Pre-test questionnaire Baseline prevalence of smoking Before randomization Baseline data form Student smoking behaviors At baseline Teacher baseline data form Teacher smoking behaviors and attitudes towards smoking and prevention At baseline Parent baseline data form Parent smoking behaviors and attitudes towards smoking At baseline Smoking prevalence form Prevalence of smoking among adolescents After the fourth year of ESO and two years after graduation Table 1 Measures, questionnaires and timeline Instrument Assessment area Before randomization workshops that are designed to sensitize participants to the power of advertising. The intervention will also seek to: debunk false beliefs about tobacco; encourage posi- tive means for coping with emotion, stress and peer pressure; and help students develop interpersonal rela- tionship skills, self-esteem, strategies for accepting rules and limits, critical thinking skills, problem-solving skills, and the ability to recognize risky situations. parents and teachers towards smoking. The smoking ques- tion was adapted from a previously validated questionnaire for adolescents [12]. Information on tobacco use will be collected through the following question: Which of the following statements best describes you? (A) I have never tried to smoke; (B) I have tried cigarettes a few times, but I do not smoke now; (C) I currently smoke less than one cigarette per month; (D) I currently smoke at least one cigarette per month, but less than one cigarette per week; (E) I currently smoke at least one cigarette per week; (F) I smoke every day; (G) I used to smoke regularly in the past, but I do not smoke now. The classroom sessions will be given by trained teachers in the intervention components, smoking prevention and Health. It will consist of seven lessons in the first year (ages 12-13), six lessons in the second year (ages 13-14), five les- sons in the third year (ages 14-15) and four lessons in the fourth year (ages 15-16) of ESO. All lessons will be age- appropriate and relevant to the students’ curricula. All lessons will include material that students will work on together with their parents. Intervention ITACA (“multifactorial intervention to reduce the preva- lence of smoking in the adolescent population: a cluster- randomized trial”) is a cognitive-behavioral intervention based on the social-influences model. It is designed to prevent adolescents from starting to smoke and to em- power them to change the smoking-related attitudes and behaviors within their immediate environments, including their homes, classrooms and communities (e.g., neighbor- hoods, town squares and/or villages). This intervention will be integrated into the schools’ curricular activities. The main components of this intervention and the activities to be performed are summarized in Table 2. Families Th The parents of our participants will meet with the personnel of the study, at least once per year at the be- ginning of each academic to receive information about the intervention and learn how they can help keep their children from smoking. These meetings will focus on en- couraging parents to: have an appropriate attitude to- wards drug use; recognize situations that constitute a risk for adolescents; establish rules and limits; be sensi- tive to the role that family plays in tobacco use; and par- ticipate in the smoke-free home initiative. At least once per year, a workshop on smoking cessation will be held for the participating families of each municipality. Fourth year (age 15-16 years) Second year (age 13-14 years) Third year (age 14-15 years) Second year (age 13-14 years) Third year (age 14-15 years) Four lessons: one on the harmful effects of smoking; one on refusal skills (critical thinking and problem-solving skills); two on identifying social influences that encourage people to start smoking (tobacco company advertising campaigns, debunking false beliefs about tobacco), and smoking cessation Seven lessons: two on information about the harmful effects of smoking; four on refusal skills (self-esteem, interpersonal relationship skills, problem-solving skills, ability to recognize risky situations, and strategies for accepting rules and limits); and one on identifying social influences that encourage people to start smoking, and smoking cessation Five lessons: three on refusal skills (strategies for accepting rules and limits, problem-solving skills, group pressure, self-esteem and coping with emotion and stress); two on identifying social influences (debunking false beliefs about tobacco, and anti-advertising workshops designed to sensitize people to the power of advertising), and smoking cessation Six lessons: three about the harmful effects of smoking; one on refusal skills (interpersonal relationship skills and critical thinking); two on identifying social influences that encourage people to start smoking (tobacco company advertising campaigns, debunking false beliefs about tobacco), and smoking cessation Five lessons: three on refusal skills (strategies for accepting rules and limits, problem-solving skills, group pressure, self-esteem and coping with emotion and stress); two on identifying social influences (debunking false beliefs about tobacco, and anti-advertising workshops designed to sensitize people to the power of advertising), and smoking cessation Two work meetings focused on parental attitudes towards drug consumption and helping parents recognize risky situations, establish rules and limits, understand the family’s role in tobacco use and the smoke-free home initiative, and implement smoking cessation Parental interventions Two work meetings focused on parental attitudes towards drug consumption and helping parents recognize risky situations, establish rules and limits, understand the family’s role in tobacco use and the smoke-free home initiative, and implement smoking cessation Two work meetings focused on parental attitudes towards drug consumption and helping parents recognize risky situations, establish rules and limits, understand the family’s role in tobacco use and the smoke-free home initiative, and implement smoking cessation Two work meetings focused on parental attitudes towards drug consumption and helping parents recognize risky situations, establish rules and limits, understand the family’s role in tobacco use and the smoke-free home initiative, and implement smoking cessation Collaborate with the student on homework regarding parental tobacco habits and attitudes, social norms and passive smoking Collaborate with the student on homework regarding parental tobacco habits and attitudes, social norms and passive smoking Collaborate with the student on homework regarding parental tobacco habits and attitudes, social norms and passive smoking Collaborate with the student on homework regarding parental tobacco habits and attitudes, social norms and passive smoking Classroom sessions h f The four-year curricular component will consist of 22 lessons of approximately 50 minutes each, including eight lessons that will be presented as part of the envir- onmental sciences curriculum, six as part of the social sciences curriculum, two as part of the physical educa- tion curriculum, one in the mathematics curriculum, and five that will be presented as student tutorials. One novel aspect of the intervention is that 20% of the curricular sessions will ask the student to involve their families in home-based tasks related to smoking preven- tion. Furthermore, a minimum of three leaflets will be provided each year regarding smoking in adolescents. The leaflets will contain answers to questions frequently asked by families, along with support material from the curricular sessions that parents and students can work on together. Parents will also receive information on make your home a smoke-free. Finally, parents will have access to a webpage that offers information on the school The components of the social-influences model that will integrated into this curriculum include teaching stu- dents to identify the social influences that encourage people to start smoking (e.g., tobacco company advertis- ing campaigns, peer pressure), providing information about the harmful effects of smoking, and nurturing re- fusal skills. The curriculum will include anti-advertising Table 2 Intervention components Component of the intervention First year (age 12-13 years) School-based interventions Seven lessons: two on inform Fourth year (age 15-16 years) Statistical analysis S l i A y Sample size A recent study on smoking in adolescents aged 14-15 years (third year of ESO) found that the prevalence of regular tobacco use in the Balearic Islands was 14% [13]. Using this as the prevalence for our control group and presuming that our intervention will reduce the prevalence of tobacco consumption in the intervention group by at least 40%, the use of 1,002 students per group will yield a statistical power > 80% for our between-group comparisons, assuming a bilateral α error of 5% and a 10% loss of participants to follow-up. The cluster size will be approximately 100 adolescents per municipality; we expect a 0.02 intra-class correlation coefficient [14], yielding a 2.98 cluster design effect. The design effect was calculated using the formula: Deff = 1 + (m – 1) * ICC; where “Deff” corresponds to the design effect, “m” is the cluster size and “ICC” is the intraclass correlation coefficient. The tar- get sample size for each group was calculated to be 1,113 students, or 2,226 students in total. To achieve this sample The protocol for implementing the basic curriculum and the supplementary components of the intervention have been standardized in an effort to homogenize the intervention. The supplementary components include: the timing and contents of the lessons; agendas for the teachers’ meetings; the contents of the initial teacher- training workshop; the contents of the parental meetings; and the establishment of a professional advice network for parents and teachers who decide to quit smoking during the study. Teacher interventions Teacher training on: competences related to smoking prevention in adolescents; managing conflict situations; attitudes towards preventive activities; information on the prevalence of smokers in the educational center; and smoking cessation Smoking cessation Smoking cessation. Smoking cessation Smoke-free policy reinforcement Smoke-free policy reinforcement The schools will be invited to adopt a smoke-free environment, implement the school’s own rules, and establish rule-compliance indicators Page 5 of 8 Leiva et al. BMC Public Health 2014, 14:373 http://www.biomedcentral.com/1471-2458/14/373 Leiva et al. BMC Public Health 2014, 14:373 http://www.biomedcentral.com/1471-2458/14/373 intervention, tips on how families can help prevent chil- dren from smoking, and advice on smoking cessation in adolescents. including visits by the research team and periodic mail- ings to each school informing them of the progress of the study and recognizing their crucial role. The import- ance of the control group in the study will also be emphasized. Teachers and the school’ The final follow-up evaluation will occur six years after inclusion, when the participants will have left the partici- pating school. At least three telephone contact numbers and the addresses of parents and tutors will be obtained early in the study. If a student is not easily located for the final evaluation, additional efforts will be made to contact them by telephone and letter. Efforts will be made to minimize the rate of loss to follow-up to reduce any possible bias resulting from loss to follow-up and to adequately assess the impact of the intervention. Prior to the start of the intervention, the teachers of the enrolled schools will participate in training course. Dur- ing this course, the needs of the teaching staff regarding smoking prevention and health themes will be identified. This course has been recognized by local government boards of education. Each school will designate a coord- inator to carry out the intervention. Classroom lessons will be subject to approval accord- ing to the school’s internal rules, and a communication system involving the coordinator, the teachers and the project researchers will be established. Annual meetings will be held with the school management team to reach agreements on the application of current legislation and the organizational structure of each smoke-free school. Teachers will participate in an initial on-line 20-hour training workshop. They will be instructed on skills re- lated to smoking prevention in adolescents and man- agement of conflict situations. This workshop will also address the teachers’ attitudes towards drug use and in- struct them on preventive activities that will promote health. Outcome assessments Th i bl The measures, variables, and timeline are summarized in Table 1. The baseline prevalence of smoking in each school will be assessed by an external evaluator who will be blinded to the group (intervention or control) to which the school has been allocated. All outcome asses- sors and data analysts will also be blinded to school allo- cation. To evaluate the effectiveness of blinding, these individuals will be asked to choose the arm to which they believe each school was assigned (possible answers: intervention, control, or unknown). Individuals who re- spond “intervention” or “control group” will be asked to indicate what led to the formation of that belief. The pri- mary outcome measure will be the prevalence of smok- ing at years three and six from enrollment. This will be assessed by self reports of tobacco consumption, where smokers are defined as those who consume more than one cigarette per week. The participating schools will be invited to adopt a smoke-free environment policy. The teachers at each school will hold a meeting to establish the school’s rules with respect to tobacco use by pupils and teachers. Each school will: implement its own rules; evaluate compli- ance; inform all teachers, students and parents of the changes being made to the rules; and establish a means to assess and record rule-compliance indicators, includ- ing incidents involving students and others smoking in the school. Strategies to ensure implementation of the intervention and avoid loss to follow-up For the project to be successful, we will need to main- tain our collaborations with the schools for at least four years. Therefore, all activities associated with the project must conform to the needs and interests of each school. Specific activities will be designed to promote continuity, Page 6 of 8 Leiva et al. BMC Public Health 2014, 14:373 http://www.biomedcentral.com/1471-2458/14/373 size, students in the first year of ESO (aged 12-13 years) will be recruited over two consecutive years at 22 schools in the Balearic Islands. selection bias, with higher rates of non-participation by students attending the intervention schools com- pared to the control schools. However, in our pilot study implementing the intervention, the response rate to the first evaluation survey of tobacco use in 14 year olds was 85%. Analytic strategy We will test for significant differ- ences in baseline characteristics between the control and intervention groups. We will perform a descriptive and cluster analysis with continuous variables summa- rized using means and standard deviations for normal distributions, and by medians and 25th and 75th per- centiles for non-normal distributions. All data analyses will involve intention-to-treat populations (i.e., all ran- domized patients, regardless of participation in any treatment session). This approach will reduce the bias that may occur when participants that fail to receive the assigned treatments are excluded from the analysis. All tests will be two-sided, and α-values of 0.05 will be considered statistically significant. Long-term follow-up of former students can be diffi- cult because many youths move away from home and have life experiences that complicate follow-up. This may result in selection biases that could compromise the internal validity of the study. Consequently, efforts will be made to collect as many contact details as possible before students leave the school (e.g., the physical ad- dresses of their parents, telephone numbers, e-mail ad- dresses, and social network information). Multiple efforts will be made to maintain contact with former students and their families, thus minimizing the rate of loss to follow-up. We will compare the smoking prevalence of adolescents in each group at 12 months against the usual null hy- pothesis of no difference between groups. We will use the Chi-squared test, taking into account the “variance inflation factor” of the adolescent cluster and intraclass correlation coefficient. We will also calculate 95% confi- dence intervals to assess the clinical significance of our intervention. Ethical approval During adolescence, young people consider and ex- plore a wide variety of options, contemplate an ideal world, and reflect on alternative political, religious, fam- ily and moral systems. The discrepancies between an ad- olescent’s ideal world and the real world are traditionally understood to form the basis for conflicts with the sys- tem, family, and/or moral imperatives. However, it is possible to take advantage of these capacities of adoles- cents and provide them with the necessary tools to effect changes in their environment. Our study protocol has been approved by the Primary Care Research Committee of Majorca and the Balearic Island Clinical Research Ethical Committee (IB 1146/09 PI). Strategies to ensure implementation of the intervention and avoid loss to follow-up In our multivariate analysis, we will adjust for potential confounders, if any, using a multilevel lo- gistic regression model. We will estimate the relative and absolute risk reductions and the number needed to treat (i.e., the estimated number of adolescents who must be treated with our intervention rather than the existing school interventions for one additional adoles- cent to be prevented from smoking), which will be cal- culated as the reciprocal of the difference between the prevalence of adolescent smokers in the intervention and control groups. All estimates will include 95% confi- dence intervals. To guarantee the comparability of the control and intervention groups, the municipalities will be stratified according to the baseline prevalence of smoking in the school prior to the intervention. Discussion In the European Union, the percentage of regular smokers quadruples (from 5% to 21%) between the ages of 12 and 16 years, with the smoking rate in the latter group being similar to that in adults [13]. The factors related to smok- ing include tobacco use among friends and older siblings, gender, availability of money, the intention to smoke in the future, low self-control in resisting the pressure to smoke, spending free time in bars, and low self-esteem [15-18]. Experts on smoking have therefore proposed in- terventions in which adolescents are given information about tobacco use and taught to identify and resist social influences. Most of these interventions, however, have not demonstrated the expected positive results in randomized clinical trials [19-25]. Authors’ contributions Authors’ contributions AY, AE, MT, AC and AL collectively drafted the study protocol and sought funding and ethical approval. AE and AY designed the intervention and are responsible for managing the trial. All authors have critically commented on a draft manuscript and have approved the final manuscript. 16. Ariza-Cardenal C, Nebot-Adell M: Factors associated with smoking progression among Spanish adolescents. Health Educ Res 2002, 17:750–760. 17. Ariza-Cardenal C, Nebot-Adell M: Predictors of the onset of tobacco consumption among secondary school students of Barcelona and Lleida. Rev Esp Salud Publica 2002, 76:227–238. Received: 22 January 2014 Accepted: 27 March 2014 Published: 16 April 2014 Received: 22 January 2014 Accepted: 27 March 2014 Published: 16 April 2014 Acknowledgments 18. Tomas Z, Ariza C, Valmayor S, Mudde A, Nebot M: [Factors associated with smoking and the intention to smoke in secondary school pupils: results of the ESFA project in Barcelona, Spain]. Gac Sanit 2002, 16:131–138. This study is being supported by a grant from the Health Research Funds (Fondos de Investigación Sanitaria; FIS) of the Carlos III Health Institute (PI10/00517). The work is also being supported by the Health Promotion and Preventive Activities-Primary Health Care Network, which is sustained by the Ministry of Health of Spain (ISCIII-RETCI G03/170 and RD06/0018). We are extremely grateful to the teachers and students of the participating schools for collaborating with us and supporting this study. 19. Peterson AV Jr, Kealey KA, Mann SL, Marek PM, Sarason IG: Hutchinson Smoking Prevention Project: long-term randomized trial in school-based tobacco use prevention–results on smoking. J Natl Cancer Inst 2000, 92:1979–1991. 20. Perry CL, Kelder SH, Murray DM, Klepp KI: Communitywide smoking prevention: long-term outcomes of the Minnesota Heart Health Program and the Class of 1989 Study. Am J Public Health 1992, 82:1210–1216. References 1. Peto R, Lopez AD, Boreham J, Thun M, Heath C Jr, Doll R: Mortality from smoking worldwide. Br Med Bull 1996, 52:12–21. 1. Peto R, Lopez AD, Boreham J, Thun M, Heath C Jr, Doll R: Mortality from smoking worldwide. Br Med Bull 1996, 52:12–21. 2. The world health report 2002 - Reducing risks, promoting healthy life: The world health report 2002 - Reducing risks, promoting healthy life. Geneva: World Health Organization; 2002. http://www.who.int/whr/2002/en/, accessed 10 May 2010. 3. Hublet A, De BD, Valimaa R, Godeau E, Schmid H, Rahav G, Maes L: Smoking trends among adolescents from 1990 to 2002 in ten European countries and Canada. BMC Public Health 2006, 6:280. 3. Hublet A, De BD, Valimaa R, Godeau E, Schmid H, Rahav G, Maes L: Smoking trends among adolescents from 1990 to 2002 in ten European countries and Canada. BMC Public Health 2006, 6:280. The effect of the wider environment on smoking has also been analyzed. For example, several transverse stud- ies have shown that the percentage of smokers varies widely among educational centers [5-7]. Furthermore, a longitudinal study involving 166 secondary schools in England [6] found that the school influenced the per- centage of students that smoked, independent of the students’ characteristics. Thus, it appears that the social environment can explain a large part of the variability among schools. 4. Ali SM, Chaix B, Merlo J, Rosvall M, Wamala S, Lindstrom M: Gender differences in daily smoking prevalence in different age strata: a population-based study in southern Sweden. Scand J Pub Health 2009, 37:146–152. 4. Ali SM, Chaix B, Merlo J, Rosvall M, Wamala S, Lindstrom M: Gender differences in daily smoking prevalence in different age strata: a population-based study in southern Sweden. Scand J Pub Health 2009, 37:146–152. 5. Azevedo A, Machado AP, Barros H: Tobacco smoking among Portuguese high-school students. Bull World Health Organ 1999, 77:509–514. 5. Azevedo A, Machado AP, Barros H: Tobacco smoking among Portuguese high-school students. Bull World Health Organ 1999, 77:509–514. 6. Aveyard P, Markham WA, Cheng KK: A methodological and substantive review of the evidence that schools cause pupils to smoke. Soc Sci Med 2004, 58:2253–2265. 6. Aveyard P, Markham WA, Cheng KK: A methodological and substantive review of the evidence that schools cause pupils to smoke. Soc Sci Med 2004, 58:2253–2265. 7. References Yanez AM, Lopez R, Serra-Batlles J, Roger N, Arnau A, Roura P: Smoking among adolescents: population study on parental and school influences. Arch Bronconeumol 2006, 42:21–24. 7. Yanez AM, Lopez R, Serra-Batlles J, Roger N, Arnau A, Roura P: Smoking among adolescents: population study on parental and school influences. Arch Bronconeumol 2006, 42:21–24. The evidence indicates that school-based preventive programs are not effective alone, but rather should be community-based, and should involve the classroom, the school environment, and the community [11]. However, few studies have rigorously analyzed the effect of such broader interventions. Most of the existing evidence comes from direct classroom interventions, with only anecdotal evidence of the intervention and its effect on individuals and the school environment [10]. Therefore, we herein propose a multifactorial intervention aimed at adolescents through their environment (school, teachers and parents). We believe that modifying the students’ immediate environment could have a marked effect on smoking rates among young people. 8. Flay BR: Understanding environmental, situational and intrapersonal risk and protective factors for youth tobacco use: the Theory of Triadic Influence. Nicotine Tob Res 1999, 1(Suppl 2):S111–S114. pp 9. Khuder SA, Dayal HH, Mutgi AB: Age at smoking onset and its effect on smoking cessation. Addict Behav 1999, 24:673–677. 10. Muller-Riemenschneider F, Bockelbrink A, Reinhold T, Rasch A, Greiner W, Willich SN: Long-term effectiveness of behavioural interventions to prevent smoking among children and youth. Tob Control 2008, 17:301–302. 11. Ariza C, Nebot M: La prevención del tabaquismo en los jóvenes: realidades y retos para el futuro. Adicciones 2004, 16:359–378. 12. Comin BE, Torrubia BR, Mor SJ, Villalbi H Jr, Nebot AM: The reliability of a self-administered questionnaire for investigation of the level of exercise, smoking habit and alcohol intake in school children. Med Clin (Barc ) 1997, 108:293–298. 13. Yañez A, Leiva A, Gorreto L, Estela A, Tejera E, Torrent M: School, family and adolescent smoking. Adicciones 2013, 25:37–44. 14. Siddiqui O, Hedeker D, Flay BR, Hu FB: Intraclass correlation estimates in a school-based smoking prevention study. Outcome and mediating variables, by sex and ethnicity. Am J Epidemiol 1996, 144:425–433. Competing interests The authors declare that there is no competing interest. Limitations One of the main limitations of community studies is the possibility of contamination, such as would occur if teachers from neighboring schools met and compared notes regarding the intervention and/or control programs. To avoid this type of contamination, the randomization unit will be the municipality. Another possible limitation is that the intervention will be carried out among students who attend the same school. This could reduce the vari- ability among study subjects; however, we will solve this by using a sufficiently large sample. There may also be a Various studies have analyzed how adolescents are in- fluenced by their immediate social contexts and socio- cultural environments. For example, teacher behavior, the educational center, and the attitudes of parents have all been associated with the initiation of tobacco use [7,26]. In addition, the prevalence of smoking among adolescents is Page 7 of 8 Page 7 of 8 Leiva et al. BMC Public Health 2014, 14:373 http://www.biomedcentral.com/1471-2458/14/373 lower when smoking is not permitted at home, especially if there is a complete ban; interestingly, this effect is inde- pendent of whether the parents are smokers [15]. Similarly, a cohort study [21] found that strong parental disapproval helped prevent smoking among adolescents, even after ad- justment for parental smoking. These findings confirm that parental attitudes towards smoking are more important than the parents’ actual behavior. Similarly, the attitude of parents towards, and emphasis on compliance with, smoking rules at school are related to smoking by adoles- cents [26,27]. The ITACA program is designed to reach the family through its adolescent members. Mallorca, Spain. 5Research Group on Evidence, Lifestyles and Health, Universitat Illes Balears, Palma, Spain. 6Instituto de Investigación Sanitaria de Palma (IdISPa) & Research Group on Evidence, Lifestyles and Health, Universitat Illes Balears, Mallorca, Spain. Mallorca, Spain. 5Research Group on Evidence, Lifestyles and Health, Universitat Illes Balears, Palma, Spain. 6Instituto de Investigación Sanitaria de Palma (IdISPa) & Research Group on Evidence, Lifestyles and Health, Universitat Illes Balears, Mallorca, Spain. Received: 22 January 2014 Accepted: 27 March 2014 Published: 16 April 2014 Competing interests The authors declare that there is no competing interest. 15. Nebot M, Tomas Z, Ariza C, Valmayor S, Lopez MJ, Juarez O: Factors Associated With Smoking Onset: 3-Year Cohort Study of Schoolchildren. Arch Bronconeumol 2004, 40:495–501. 21. Vartiainen E, Paavola M, McAlister A, Puska P: Fifteen-year follow-up of smoking prevention effects in the North Karelia youth project. Am J Public Health 1998, 88:81–85. Author details 1 1Primary Care Research Unit of Mallorca, Baleares Health services-IbSalut, Mallorca, Spain. 2Dalt Sant Joan Health Centre, Baleares Health services-IbSalut, Menorca, Spain. 3Àrea de Salut de Menorca, IB-SALUT, Menorca, Spain. 4European Institute of Studies on Prevention (Irefrea), 21. Vartiainen E, Paavola M, McAlister A, Puska P: Fifteen-year follow-up of smoking prevention effects in the North Karelia youth project. Am J Public Health 1998, 88:81–85. Page 8 of 8 Page 8 of 8 Leiva et al. BMC Public Health 2014, 14:373 http://www.biomedcentral.com/1471-2458/14/373 Leiva et al. BMC Public Health 2014, 14:373 http://www.biomedcentral.com/1471-2458/14/373 Leiva et al. BMC Public Health 2014, 14:373 http://www.biomedcentral.com/1471-2458/14/373 22. Aveyard P, Cheng KK, Almond J, Sherratt E, Lancashire R, Lawrence T, Griffin C, Evans O: Cluster randomised controlled trial of expert system based on the transtheoretical (“stages of change”) model for smoking prevention and cessation in schools. BMJ 1999, 319:948–953. 23. Ellickson PL, Bird CE, Orlando M, Klein DJ, McCaffrey DF: Social context and adolescent health behavior: does school-level smoking prevalence affect students’ subsequent smoking behavior? J Health Soc Behav 2003, 44:525–535. 24. Armstrong BK, de Klerk NH, Shean RE, Dunn DA, Dolin PJ: Influence of education and advertising on the uptake of smoking by children, 19. Med J Aust 1990, 152:117–124. 25. Brown KS, Cameron R, Madill C, Payne ME, Filsinger S, Manske SR, Best JA: Outcome evaluation of a high school smoking reduction intervention based on extracurricular activities. Prev Med 2002, 35:506–510. 26. Wen X, Chen W, Muscat JE, Qian Z, Lu C, Zhang C, Luo Y, Liang C, Han K, Deng X, Ou Y, Ling W: Modifiable family and school environmental factors associated with smoking status among adolescents in Guangzhou, China. Prev Med 2007, 45:189–197. 27. Wakefield MA, Chaloupka FJ, Kaufman NJ, Orleans CT, Barker DC, Ruel EE: Effect of restrictions on smoking at home, at school, and in public places on teenage smoking: cross sectional study. BMJ 2000, 321:333–337. doi:10.1186/1471-2458-14-373 Cite this article as: Leiva et al.: Effectiveness of a complex intervention in reducing the prevalence of smoking among adolescents: study design of a cluster-randomized controlled trial. BMC Public Health 2014 14:373. 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https://openalex.org/W4385381894	https://bmcvetres.biomedcentral.com/counter/pdf/10.1186/s12917-023-03662-9	English	null	Blood metabolomics reveals the therapeutic effect of Pueraria polysaccharide on calf diarrhea	BMC veterinary research	2,023	cc-by	8,804	© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background Neonatal calf diarrhea (NCD) is typically treated with antibiotics, while long-term application of antibi‑ otics induces drug resistance and antibiotic residues, ultimately decreasing feed efficiency. Pueraria polysaccharide (PPL) is a versatile antimicrobial, immunomodulatory, and antioxidative compound. This study aimed to compare the therapeutic efficacy of different doses of PPL (0.2, 0.4, 0.8 g/kg body weight (BW)) and explore the effect of plasma metabolites in diarrheal calves by the best dose of PPL. Results PPL could effectively improve the daily weight gain, fecal score, and dehydration score, and the dosage of 0.4 g/kg BW could reach curative efficacy against calf diarrhea (with effective rates 100.00%). Metabolomic analy‑ sis suggested that diarrhea mainly affect the levels of taurocholate, DL-lactate, LysoPCs, and intestinal flora-related metabolites, trimethylamine N-oxide; however, PPL improved liver function and intestinal barrier integrity by modu‑ lating the levels of DL-lactate, LysoPC (18:0/0:0) and bilirubin, which eventually attenuated neonatal calf diarrhea. It also suggested that the therapeutic effect of PPL is related to those differential metabolites in diarrheal calves. Conclusions The results showed that 0.4 g/kg BW PPL could restore the clinical score of diarrhea calves by improving the blood indexes, biochemical indexes, and blood metabolites. And it is a potential medicine for the treatment of calf diarrhea. Keywords Pueraria polysaccharide, Calf diarrhea, Therapeutic efficacy, Metabolomic BMC Veterinary Research BMC Veterinary Research Shen et al. BMC Veterinary Research (2023) 19:98 https://doi.org/10.1186/s12917-023-03662-9 BMC Veterinary Research Open Access Blood metabolomics reveals the therapeutic effect of Pueraria polysaccharide on calf diarrhea Liuhong Shen1†, Yu Shen1†, Liuchao You1, Yue Zhang1,2, Zhetong Su2, Guangneng Peng1, Jun‑liang Deng1, Zhijun Zhong1, Shumin Yu1, Xiaolan Zong1, Xiaofeng Wu1, Yingkun Zhu3* and Suizhong Cao1* Background Neonatal calf diarrhea (NCD) is a common disease of calves within four weeks old; the morbidity and mortal- ity were about 55% and 15%, respectively [1]. NCD usu- ally was caused by the interaction of environments (poor hygiene and high density), infectious factors (viruses, bacteria, parasites) [2], or non-infectious factors (stress, poor management) [3]. NCD is non-seasonal and prev- alent worldwide, with primary symptoms including anorexia, elevated body temperature, depression, and diarrhea [4], affecting pastures’ economic benefits. Anti- biotics are often used to treat or prevent diarrhea in ruminants that are known to cause antibiotic-resistant †Liuhong Shen and Yu Shen contributed equally to this work. †Liuhong Shen and Yu Shen contributed equally to this work. 1 The Key Laboratory of Animal Disease and Human Health of Sichuan Province, The Medical Research Center for Cow Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, Sichuan, China 2 Guangxi Innovates Medical Technology Co., Ltd. Lipu, Guangxi 546600, China 3 School of Agriculture & Food Science, University College Dublin, Belfield, Dublin D04 V1W8, Ireland © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Serum collection for biochemical indexes detection PPL with 50.00% purity and average molecular weight (Mw) of 1.09 × 105 Da [16], supplied by Guangxi Innovate Pharmaceutical Co., Ltd. Similarly, selected the venous blood of the optimal dose group of PPL and control group on D0, D3, D5, and D7 and centrifuged at 3000 r/min for 5 min to obtain the serum and stored at -20℃ for further biochemical indexes detection. Blood samples were collected via the jugular vein using the non-anticoagulation tube. Open Access T d d Shen et al. BMC Veterinary Research (2023) 19:98 Page 2 of 12 bacteria [5]. Therefore, it is necessary to exploit natural agents for NCD management. bacteria [5]. Therefore, it is necessary to exploit natural agents for NCD management. receive any other medications during the treatment with PPL. After the experiment, the animals returned to the herd for feeding. Bioactive compounds in plant extracts performed increasing importance in medicine development [6]. Pueraria lobata, which spreads widely in China, Japan, and Korea, has been used for thousands of years as medi- cine, fodder, and food. Polysaccharides and flavonoids are the main bioactive components of Pueraria lobata [7]. Polysaccharides like chitosan have been proven can effec- tively treat diarrhea [8]; furthermore, Panax ginseng pol- ysaccharides [9] and red algae polysaccharides [10] have been used to alleviate diarrhea in various animal models. Pueraria polysaccharide (PPL), a compound of Pueraria lobata, has versatile bioactivities like antimicrobial [11], immunomodulatory [12], and antioxidant [13], and it can recover the expression and structure of tight junction protein of intestine, alleviating dextran sodium sulfate- induced colitis in mice [14]. It can also increase the ben- eficial bacteria and improve intestinal flora structure in antibiotic-induced diarrheal mice [15]. However, there is a lack of research focused on the therapeutic efficiency of PPL on newborn calf diarrhea.hf Drug administrationh The day of enrollment was defined as D0. Thirty-six diarrheal calves were randomly divided into 3 groups (n = 12). The dose of each group was 0.2 g/kg, 0.4 g/kg, and 0.8 g/kg BW (dissolved in 100 mL 37℃ water) as low dose group, medium-dose group, and high-dose group, respectively. Administrate Pueraria polysaccharide Q.D P.O for five consecutive days. NCD was considered to end when their fecal score was ≤ 1 for two consecutive days. The whole experimental period was seven days. Therapeutic efficacies observation During the experimental period (D0-D7), weighed calves before administration and calculated the daily weight gains, observed and recorded the mental status [18], dehydration status [19], and fecal status [17] (the crite- ria of these status scores were present in Table 1), heart ratio, respiratory ratio, and body temperature of calves, during this period, calculated the therapeutic effects and ascertain the optimal dosage. This study investigated the therapeutic effect of PPL against calf diarrhea and determined the optimal dos- age. Besides, very few studies have explored metabolomic profiling in the PPL treatment of neonatal calf diarrhea. In this study, we conducted comprehensive metabo- lomics profiling of plasma from diarrheal calves before and after treatment with PPL using liquid chromatogra- phy (LC)-TOF–MS. Animalsh The study was conducted on an intensively managed dairy farm in Sichuan, China. Dairy calves were offered 4 L colostrum within two h after birth, then housed indi- vidually with bedding material to avoid physical contact with each other. After the first day of life, the calves were fed milk from a bucket twice a day at 8:00 and 16:00, with free access to concentrate and water for the next seven days. Blood collection for complete blood count At the same time, the venous blood of the optimal dose group of PPL and control group were selected on D0, D3, D5, and D7 for complete blood count (CBC). Blood sam- ples were collected via the jugular vein using the EDTA anticoagulation tube. Preparation of plasma samples for metabolomic analysis Preparation of plasma samples for metabolomic analysis Blood samples of healthy calves in the control group on D5 were collected and centrifuged at 3000r/min for 5 min to obtain the plasma and noted as group HS. Besides, the plasma collected from calves in optimal dos- age groups on D0 and D7 was noted as group DS and TS, respectively. Blood samples were collected via the jugular vein using the EDTA anticoagulation tube. All the plasma was stored at -80℃ for further metabolomic analysis and comparison. The 3 sets of samples were mixed in equal amounts to prepare quality control (QC) samples, and 7 replicates were set up to evaluate system stability over the entire experiment before testing. The samples were Thirty-six naturally infected Chinese Holstein dairy calves with typical diarrheal symptoms, and 12 healthy calves were selected from a unified pasture in Sichuan, China (calves with 5–7-day ages and 45–55 kg weights). The enrollment criteria for diarrheal calves were as fol- lows: (1) calves with fecal scores > 2, (2) calves aged 1–30 days [17]. The calves enrolled in this study did not Shen et al. BMC Veterinary Research (2023) 19:98 Page 3 of 12 Table 1 The scoring criteria of clinical examination Items Scoring Basis Scores Fecal status Feces with normal shape and consistency 0 Semi-formed or pasty feces 1 Feces are not formed but can adhere to ground or bedding 2 Watery feces 3 Mental status Normal, respond quickly to external stimuli and vigorously 0 Mild depressive retains sucking reflex without vitality 1 Moderate depressive, stand after stimulation with a weak or irregular sucking reflex 2 Severe depressive, unable to stand or without sucking reflex 3 Dehydration status The hydration state is normal, tent test time of upper eyelid skin < 2 s 0 Eyeball slightly sunken, tent test time of upper eyelid skin is > 2 s but < 4 s 1 Sunken eyeball, dry nose, tent test time of upper eyelid skin > 5 s 2 Severe sunken eyeball, cold ears, limbs and mouth, dry nose 3 ion peak data for which > 50% of the missing data within a group were deleted. After the data had been pre-pro- cessed by pareto-scaling, pattern recognition was per- formed using SIMCA-P software (version 14.1, Umetrics, Umea, Sweden), consisting of supervised orthogonal partial least squares discriminant analysis (OPLS-DA). Preparation of plasma samples for metabolomic analysis The variable importance in the projection (VIP) value of each variable in the OPLS-DA model was calculated to indicate its contribution to the classification. Metabolites with the VIP value > 1 were further applied to Student’s t-test at the univariate level to measure the significance of each metabolite. The P-values less than 0.1 were con- sidered statistically significant [22]. Compound iden- tification of metabolites was performed by comparing accuracy m/z value (< 25 ppm) and MS/MS spectra with an in-house database established with available authentic standards. Random forest analysis, enrichment pathway analysis, and pathway impact analysis of metabolites that differed among groups were performed using Metabo- Analyst 5.0 (https://www.metaboanalyst.ca/) and Kyoto Encyclopedia of Genes and Genomes (KEGG, https:// www.kegg.jp/kegg/). sent to Shanghai Applied Protein Technology Co., Ltd. (Shanghai, China) for liquid chromatography-tandem mass spectrometry (MS/MS) analysis. Metabolomics analysis of blood samplesh The samples were separated by UHPLC (1290 infinite LC, Agilent Technologies) HILIC column with 25℃ column temperature and 0.3 mL/min velocity. The mobile phase was A: 25 mM ammonium acetate and 25 mM ammo- nia in water, B: acetonitrile. The gradient was 85% B for 1 min, linearly reduced to 65% in 11 min, then reduced to 40% in 0.1 min and kept for 4 min, and then increased to 85% in 0.1 min, with a 5 min re-equilibration period employed. p y After detection, the AB triple TOF 6600 mass spec- trometer was used to obtain the samples’ primary and secondary spectra. The ESI source conditions after HILIC chromatographic separation were as follows, Ion Source Gas1: 60, Ion Source Gas2: 60, Curtain gas: 30, source temperature: 600 ℃, Ion Spray Voltage Float- ing ± 5500 V, TOM MS scan m/z range: 60–1000 Da, product ion scan m/z range: 25–1000 Da, TOF MS scan accumulation time 0.20 s/spectra, product ion scan accumulation time: 0.05 s/spectra, the secondary mass spectrum was obtained by information-dependent acqui- sition (IDA) with high sensitivity mode selected, declus- tering potential (DP): ± 60 V, collision energy was fixed at 35 V ± 15 eV, IDA was set as follows: exclude isotopes within 4 Da, Candidate ions to monitor per cycle: 10. Software of SPSS 19.0 was applied to check whether the data conform to the normal distribution, all meas- urement data were expressed by means ± SEM, one-way ANOVA analysis of Variance was used for compari- son between groups, and P < 0.05 indicated a significant difference. The effects of Pueraria polysaccharide on the scores of clinical symptoms Similarly, we investigated the effects of the optimal dose of PPL on the biochemical blood indexes of diarrheal calves, and the results were present in Fig. 2. The levels of total protein (TP), globulin (GLOB), alkaline phosphatase (ALP) of diarrheal calves were significantly higher than the control group. The level of glucose (GLU) of diarrheal calves was significantly lower than the control group (P < 0.05), while all these indexes recovered to normal levels after treatment (P > 0.05), there were no signifi- cant differences of the albumin (ALB), blood urea nitro- gen (BUN), creatinine (CREA), alanine aminotransferase (ALT) between medium-dose group and control groups. As shown in Table 3, the fecal scores and dehydration scores of diarrheal calves were significantly higher than group C (P < 0.05), while after PPL treatment. The men- tal scores in the medium dose group on D0 were signifi- cantly higher than in other groups. After PPL treatment, mental and dehydration scores showed a decreasing trend and no significant differences with other groups on D7 (P > 0.05). The effects of Pueraria polysaccharide on the basic physiological indexes The raw data were converted into mzXML format by Pro- teo Wizard [20], and then the XCMS program was used for peak alignment, retention time correction, and peak area extraction [21]. For the data extracted using XCMS, As shown in Table 2, when diarrhea occurred, the daily gains of diarrheal calves decreased (P < 0.05), while after PPL treatment, the daily gains of all dose groups showed Shen et al. BMC Veterinary Research (2023) 19:98 Page 4 of 12 Table 2 Effects of PPL on basic physiological indexes For each indicator, different superscripted letters represent significant differences between different groups at thesame time point (P < 0.05), while different subscripted letters represent significant differences with in one groupbetween different time points (P < 0.05) Items Treatment Group SEM Low dose Medium dose High dose Control Daily gain D1 0.33e b 0.32d b 0.32e b 0.84a 0.027 D2 0.47d b 0.50c b 0.50d b 0.87a 0.031 D3 0.51d c 0.63b b 0.64c b 0.86a 0.022 D4 0.55c,d c 0.65b b 0.69b,c b 0.86a 0.019 D5 0.62b,c c 0.71a,b b 0.70b,c b 0.82a 0.021 D6 0.64a,b c 0.76a b 0.75a,b b 0.84a 0.024 D7 0.71a b 0.79a a,b 0.80a a,b 0.85a 0.030 P (dose) < 0.001 P (time) < 0.001 P (dose × time) < 0.001 Table 2 Effects of PPL on basic physiological indexes For each indicator, different superscripted letters represent significant differences between different groups at thesame time point (P < 0.05), while different subscripted letters represent significant differences with in one groupbetween different time points (P < 0.05) For each indicator, different superscripted letters represent significant differences between different groups at thesame time point (P < 0 subscripted letters represent significant differences with in one groupbetween different time points (P < 0.05) the control group (P > 0.05). In contrast, there were no significant differences in red blood cell (RBC), lympho- cyte counts (LYMPH), monocyte count (MONO), eosin- ophil (ESO), or basophil (BASO) with the control group (P > 0.05) on D0-D7. an increasing trend and showed no significant differences (P > 0.05) with control group except for low dose group on D7, suggesting a dose–effect relationship. As shown in Table S1, the body temperatures, respiratory rates, and heart rates of diarrheal calves were no different from the control group (P > 0.05), and the PPL treatment showed no significant effect on those indexes (P > 0.05). UHPLC‑Q‑TOF–MS method of validation As shown in Table 4, the effective rate (The ratio of cured calves to the total number of diarrhea calves within seven days) of the medium dose group was the highest among the three treatment groups, thus ascertaining 0.4 g/ kg BW (medium dose) as optimal dosage against calf diarrhea. We compared the total ion chromatograms (TIC) of 7 samples in positive or negative ion modes, including the retention time, peak, intensity, and degree of sep- aration. Overlap of the TIC of QC samples was good, indicating that the method used was robust, with high repeatability and stability. The sample TIC showed that the peak shape was intact and that adjacent peaks were well separated, indicating that the chromatographic and mass spectrometric conditions were suitable for sample identification (Fig S1). The smaller the relative standard deviation (RSD) of the ion peak abundance of QC samples is, the better the stability of the instrument is, which is an important index to reflect the quality of The effects of Pueraria polysaccharide on the CBChf In this experiment, the number of RSD ≤ 30% Peak in QC samples accounts for more than 80% of the total Peak number of QC samples, as shown in Fig S2, indicating that the stability of the instrument analysis system is good, and the data can be used for follow-up analysis. Identification of differential metabolites Potential biomarkers were analyzed using the multivar- iate analysis of OPLS-DA. Each point on the OPLS-DA score map represented a sample, and the position of each sample was determined by the type and content of its metabolites (Table S2). Compounds with a VIP > 1, Table 4 The therapeutic effects of Pueraria polysaccharide on diarrheal calves Items Low dose group Medium dose group High dose group Cured number within 7 days 8 12 11 Effective rate (%) 75.00% 100.00% 91.67% Table 4 The therapeutic effects of Pueraria polysaccharide on diarrheal calves Identification of differential metabolites The effects of Pueraria polysaccharide on the CBChf The effects of the optimal dose of PPL on the CBC of diarrheal calves, as shown in Fig. 1, the white blood cell (WBC), neutrophil count (NEUT), and hematocrit (HCT) of diarrheal calves were significantly higher than the control group on D0 and showed decreasing trends after treatment. There was no significant difference with Shen et al. BMC Veterinary Research (2023) 19:98 Page 5 of 12 Table 3 Effects of PPL on scores of clinical symptoms For each indicator, different superscripted letters represent significant differences between different groups at the same time point (P < 0.05), while different subscripted letters represent significant differences with in one group between different time points (P < 0.05) Items Treatment Group SEM Low dose Medium dose High dose Control Fecal scores D0 2.17a a 2.33a a 2.08a a 0.00b 0.114 D1 1.33b,c a 1.58b a 1.50b a 0.25b 0.165 D2 1.42b a 1.25b,c a 1.08b,c a 0.08b 0.139 D3 1.08b,c a 1.08b a 1.33b a 0.17b 0.169 D4 1.17b,c a 1.00b a 1.00b,c a 0.17b 0.211 D5 1.25b,c a 1.00b a 1.17b a 0.08b 0.209 D6 0.75b,c a 0.42c a,b 0.58c,d a 0.00b 0.148 D7 0.58c a 0.25c a,b 0.33d a,b 0.08b 0.165 P (dose) < 0.001 P (time) < 0.001 P (dose × time) < 0.001 Dehydration scores D0 1.17a a 1.25a a 1.25a a 0.00b 0.093 D1 0.42b a 0.50b a 0.58b a 0.00b 0.112 D2 0.25b 0.25b,c 0.17c 0.00 0.093 D3 0.33b 0.08c 0.09c 0.00 0.089 D4 0.30b 0.00c 0.00c 0.00 0.036 D5 0.38b a 0.00c b 0.00c b 0.00b 0.058 D6 0.25b 0.00c 0.00c 0.00 0.045 D7 0.17b 0.00c 0.00c 0.00 0.042 P (dose) < 0.001 P (time) < 0.001 P (dose × time) < 0.001 Mental scores D0 0.25a 0.58a b 0.17a 0.00a 0.109 D1 0.25 0.17b 0.25 0.00 0.093 D2 0.08 0.17b 0.17 0.00 0.077 D3 0.08 0.17b 0.25 0.00 0.082 D4 0.33a 0.00b b 0.08b 0.00b 0.056 D5 0.17 0.17b 0.25 0.00 0.089 D6 0.08 0.08b 0.17 0.00 0.070 D7 0.08 0.08b 0.17 0.00 0.070 P (dose) 0.250 P (time) 0.0002 P (dose × time) 0.338 SEM For each indicator, different superscripted letters represent significant differences between different groups at the same time point (P < 0.05), while different subscripted letters represent significant differences with in one group between different time points (P < 0.05) the data. Identification of differential metabolites the data. In this experiment, the number of RSD ≤ 30% Peak in QC samples accounts for more than 80% of the total Peak number of QC samples, as shown in Fig S2, indicating that the stability of the instrument analysis system is good, and the data can be used for follow-up analysis. Potential biomarkers were analyzed using the multivar- iate analysis of OPLS-DA. Each point on the OPLS-DA score map represented a sample, and the position of each sample was determined by the type and content of its metabolites (Table S2). Compounds with a VIP > 1, Shen et al. BMC Veterinary Research (2023) 19:98 Page 6 of 12 Shen et al. BMC Veterinary Research (2023) 19:98 Fig. 1 The effects of PPL on the CBC. Green box stands for control group; pink box stands for treatment group. D0 means the results of diarrheal calves before treatment with PPL. * P < 0.05, ** P < 0.01, ns = no significant Fig. 2 The effects of PPL on the serum biochemical indexes. Green box stands for control group; pink box stands for treatment group. D0 means the results of diarrheal calves before treatment with PPL. * P < 0.05, ** P < 0.01, ns = no significant Page 6 of 12 Fig. 1 The effects of PPL on the CBC. Green box stands for control group; pink box stands for treatment group. D0 means the results of diarrheal calves before treatment with PPL. * P < 0.05, ** P < 0.01, ns = no significant Fig. 1 The effects of PPL on the CBC. Green box stands for control group; pink box stands for treatment group. D0 means the results of diarrheal calves before treatment with PPL. * P < 0.05, ** P < 0.01, ns = no significant Fig. 1 The effects of PPL on the CBC. Green box stands for control group; pink box stands for treatment group. D0 means the results of diarrheal calves before treatment with PPL. * P < 0.05, ** P < 0.01, ns = no significant ig. 1 The effects of PPL on the CBC. Green box stands for control group; pink box stands for treatment group. D0 means the r alves before treatment with PPL. * P < 0.05, ** P < 0.01, ns = no significant Fig. 2 The effects of PPL on the serum biochemical indexes. Identification of differential metabolites Green box stands for control group; pink box stands for treatment group. D0 means the results of diarrheal calves before treatment with PPL. * P < 0.05, ** P < 0.01, ns = no significant Fig. 2 The effects of PPL on the serum biochemical indexes. Green box stands for control group; pink box stands for treatment group. D0 means the results of diarrheal calves before treatment with PPL. * P < 0.05, ** P < 0.01, ns = no significant differential plasma metabolites were shown by the FC barplot (Fig. 4). fold change (FC) > 1, or < 1, and an independent t-test with P < 0.1 were initially screened as potential differ- ential metabolites. The score plots of OPLS-DA and permutation tests shown in Fig. 3 indicated that the plasma metabolic profile of diarrheal calves was differ- ent from the control group, and there were also altera- tions of the metabolites profile in diarrheal calves after treatment with PPL. The intercepts of Q2 were < 0, indi- cating no overfittings of the model, and the differential metabolites can be identified according to it. There was a total of 22 differential metabolites identified between diarrheal calves and healthy calves (Table S3), with 14 metabolites found to increase and 8 observed to decrease. Similarly, 45 metabolites were found to sig- nificantly differ between PPL-treated calves and diar- rheal calves (Table S4), with 38 metabolites found to increase and 7 observed to decrease. To compare the change of differential metabolites in each group, the The change of key metabolites induced by PPL on diarrheal calves To further investigate the potential information of dif- ferential metabolites, the random forest supervised machine learning algorithm showed that 15 prominent metabolites contributed to the discrimination power of calf health status, including trimethylamine N-oxide (TMAO) and taurocholate; and 15 prominent metabo- lites related to the therapy of PPL, including bilirubin and LysoPC (18:0/0:0) (Fig. 5A, B). The relative rank of the relative abundance of metabolites biomarkers was plotted against the effect by the score of Mean Decrease Accuracy. Then the correlation analysis of significantly altered metabolites with CBC and serum biochemis- try indexes was further revealed. As shown in Fig. 5, Shen et al. BMC Veterinary Research (2023) 19:98 Page 7 of 12 Fig. 3 The scores plot of OPLS-DA and permutation tests. A, (E) and (C), (G) Orthogonal partial least square discriminant analysis (OPLS-DA) of scores and permutation test plots for the DS vs HS and TS vs DS samples analyzed in the positive ion mode, respectively. (B), F and (D), (H) Orthogonal partial least square discriminant analysis of scores and permutation test plots for the DS vs HS and TS vs DS samples analyzed in the negative ion mode, respectively. T [1] = first principal component. To [1] = second orthogonal component. The intercept limit of Q2, calculated by regression line, is the plot of Q.2 from permutation test in the OPLS-DA model. HS = healthy calf plasma sample; DS = diarrheal calf plasma sample on D0; TS = PLP treatment diarrheal calf plasma sample on D5 Orthogonal partial least square discriminant analysis (OPLS-DA Fig. 3 The scores plot of OPLS-DA and permutation tests. A, (E) and (C), (G) Orthogonal partial least square discriminant analysis (OPLS-DA) of scores and permutation test plots for the DS vs HS and TS vs DS samples analyzed in the positive ion mode, respectively. (B), F and (D), (H) Orthogonal partial least square discriminant analysis of scores and permutation test plots for the DS vs HS and TS vs DS samples analyzed in the negative ion mode, respectively. T [1] = first principal component. To [1] = second orthogonal component. The intercept limit of Q2, calculated by regression line, is the plot of Q.2 from permutation test in the OPLS-DA model. HS = healthy calf plasma sample; DS = diarrheal calf plasma sample on D0; TS = PLP treatment diarrheal calf plasma sample on D5 Fig. Global metabolic pathways of metabolites [26]. After PPL administration, serum ALP showed a decreasing trend, indicating that PPL can promote intes- tinal integrity recovery. Globulin, an important compo- nent of the inflammatory microenvironment, is closely related to inflammatory status [27]. The recovery of globulin after PPL treatment also suggested the recov- ery of inflammatory and infectious status in diarrheal calves. After PPL treatment, serum glucose increased and exhibited no significant difference with the control group (P > 0.05) in diarrheal calves, demonstrating that PPL could recover the nutrition absorption and energy metabolism of diarrheal calves. Due to the limitation of the experimental conditions, the dehydration and ion dis- turbance of diarrhea calves were not analyzed in detail. Further study is needed. [26]. After PPL administration, serum ALP showed a decreasing trend, indicating that PPL can promote intes- tinal integrity recovery. Globulin, an important compo- nent of the inflammatory microenvironment, is closely related to inflammatory status [27]. The recovery of globulin after PPL treatment also suggested the recov- ery of inflammatory and infectious status in diarrheal calves. After PPL treatment, serum glucose increased and exhibited no significant difference with the control group (P > 0.05) in diarrheal calves, demonstrating that PPL could recover the nutrition absorption and energy metabolism of diarrheal calves. Due to the limitation of the experimental conditions, the dehydration and ion dis- turbance of diarrhea calves were not analyzed in detail. Further study is needed. Sketched the global metabolic pathways by the com- bination of KEGG pathway information. As shown in Fig. 6, the key metabolites mentioned above were mainly involved in 2 metabolic pathways, glycerol phospholipid choline metabolism and primary bile acid synthesis. The change of key metabolites induced by PPL on diarrheal calves 4 The FC barplot of differential metabolites in each group. A, B are the FC barplot of differential metabolites of DS vs HS, TS vs DS; red means up-regulation, blue means down-regulation Shen et al. BMC Veterinary Research (2023) 19:98 Page 8 of 12 Fig. 5 Potential information of differential metabolites. A, B the random forest supervised machine learning algorithm; C-F the correlation analysis of significantly altered metabolites with CBC and serum biochemistry indexes; G change of the DL-lactate concentration. ‘’ indicates significant difference (P < 0.005). The red color and green color stand for different group, respectively Fig. 5 Potential information of differential metabolites. A, B the random forest supervised machine learning algorithm; C-F the correlation analysis of significantly altered metabolites with CBC and serum biochemistry indexes; G change of the DL-lactate concentration. ‘’ indicates significant ff Fig. 5 Potential information of differential metabolites. A, B the random forest supervised machine learning algorithm; C-F the correlation analysis of significantly altered metabolites with CBC and serum biochemistry indexes; G change of the DL-lactate concentration. ‘*’ indicates significant difference (P < 0.005). The red color and green color stand for different group, respectively positively correlated with NEUT (r = 0.71, P < 0.001) and WBC (r = 0.64, P < 0.001). Notably, GLU was closely associated with DL-lactate (r = -0.54, P < 0.01) and stearidonic acid (r = 0.63, P < 0.01) after PPL treatment. It should be noted that the concentration of DL-lactate was altered in diarrheal calves before and after being treated with PPL(Fig. 5G). Our findings suggested that PPL administration could ameliorate diarrhea by mod- ulating key plasma metabolites in diarrheal calves. spearman rank correlation analysis indicated a strong positive correlation between TMAO and ALP (r = 0.64, P < 0.001), GLOB (r = 0.57, P < 0.01), TP (r = 0.55, P < 0.01), WBC (r = 0.52, P < 0.01) and NEUT (r = 0.65, P < 0.001) in the diarrheal calves. After being treated with PPL, bilirubin was positively correlated with ALP (r = 0.56, P < 0.01), WBC (r = 0.61, P < 0.01) and NEUT (r = 0.57, P < 0.01); similarly, LysoPC (18:0/0:0) was Shen et al. BMC Veterinary Research (2023) 19:98 Page 9 of 12 Discussion Apart from diluted soft faces, calf diarrhea also showed symptoms, including dehydration, weight loss, anorexia, and mental depression. Besides, diarrheal calves usually exhibited changes in complete blood count indexes and biochemical indexes [23]. In this study, PPL can allevi- ate the daily weight gain, fecal score, dehydration score, and mental score of diarrheal calves and reach curative efficacy (with a 75% cure rate). As signs of infection, the increased WBC and NEUT in diarrheal calves was coincided with another research about diarrheal calves [24], while after treatment of PPL, the WBC and NEUT returned and showed no significant difference with the control group (P > 0.05), indicating the amelioration of infectious and inflammatory degrees of diarrheal calves. Serum biochemical parameters are important indexes that reflect the health status of animals [25]. In the study, diarrheal calves showed increased serum ALP, globulin and decreased glucose. ALP was a marker of intestinal injury and increased obviously when intestinal damage As well known, diarrhea was an important factor lead- ing to intestinal microbial imbalance [28]. Gut micro- bial dysbiosis can increase TMAO concentrations [29]. TMAO is the metabolite of choline and phosphatidyl- choline from gut microbiota and hepatic flavin monooxy- genases [30]. And in this study, the positive correlation between TMAO and ALP also indicated that diarrhea- induced intestinal damage in neonatal calves. We also found the level of taurocholate was decreased in diar- rheal calves. The primary bile acid synthesis pathway, in which taurocholate takes part, is also downregulated. Bile acids secreted into the small intestinal are conjugated to Fig. 6 The global metabolic pathway of metabolites. The trisection rectangle represents the differential metabolites; the box on the top is DS vs HS group, and the box at the bottom is DS vs TS group; green represents downregulation, white represents no significant difference, red represents upregulation Fig. 6 The global metabolic pathway of metabolites. The trisection rectangle represents the differential metabolites; the box on the top is DS vs HS group, and the box at the bottom is DS vs TS group; green represents downregulation, white represents no significant difference, red represents upregulation Shen et al. BMC Veterinary Research (2023) 19:98 Page 10 of 12 Page 10 of 12 of the LysoPC family members, including LysoPC 16:0, LysoPC 18:0, and LysoPC 18:1(9Z), increased in diar- rheal calves. Discussion LysoPCs were regarded as proinflammatory mediators, LysoPC 18:0 can induce the initiation of neu- trophils [42], and stimulate the adhesion of eosinophils [43]. Diarrheal symptoms are usually related to systemic inflammatory responses, such as abnormal blood rou- tine changes and biochemistry parameters [44], which could explain the increase of WBC and NEUT in diar- rheal calves. Simultaneously, the down-regulation of LysoPC (18:0/0:0) also suggested that PPL attenuated system inflammatory response in diarrhea calves. We also found that the level of LysoPE (16:0) was increased in treated calves. The study indicated that LysoPE levels were reduced in the liver of mice with liver damage [45]. We speculated that the liver is a possible source of lipid perturbations in calves with diarrhea, which the follow- ing experiment could explore. taurine to form taurocholate [31]. Intestinal floras can modify the conjugated amino acids by secreting bile salt hydrolases [32]. Moreover, calves that suffer from diar- rhea usually have weakened bile acid hepatointestinal circulation [33], which causes primary bile acid synthesis downregulation. Hence, we speculated that the down- regulation of taurocholate might relate to the increas- ing concentration of bile salt hydrolases, which reflected the intestinal flora disturbance, but more research was needed. Usually, maintaining normal intestinal function archi- tecture and functioning is essential for alleviating diar- rhea. Our study noticed that PPL decreased the fecal score, which might be related to the DL-lactate change. The increases in the serum D-lactate have been reported to correlate with the extent of intestinal barrier dysfunc- tion and diarrhea [34, 35]. D-lactate is produced only by colonic bacteria as a normal byproduct of bacterial fer- mentation. As the normal mucosal barrier is damaged and permeability increases, a large amount of D-lactate is released through the damaged intestinal mucosa into the peripheral blood, leading to increased blood levels of D-lactate. Because the liver cannot metabolize D-lactate, a rise in the serum concentration occurs [36]. Consist- ent with these findings, we noted a decrease in the serum DL-lactate contents following PPL treatment. However, it is a pity that our technology can only detect DL-lactate and cannot accurately distinguish its chiral structure, so subsequent experiments need specific detection of lac- tate to verify the experimental results. Conclusionh The PPL showed a significant effect on calf diarrhea, and it can reach optimal therapeutic efficacy with a dose of 0.4 mg/kg/day. Diarrhea was associated with meta- bolic disorders in neonatal calves. Specifically, we found associations of diarrhea with shifts in taurocholate, DL- lactate, LysoPCs and intestinal flora-related metabolites, TMAO; while PPL improved liver function and intestinal barrier integrity by modulating the levels of DL-lactate, LysoPC (18:0/0:0) and bilirubin, which eventually amelio- rated neonatal calf diarrhea. The only catch: the effects of PPL on the phenotype index of diarrheal calves, such as inflammatory factors, need further investigation. Studies showed that diarrhea is usually followed by a negative energy balance [37]. Negative energy bal- ance is characterized biochemically by the reduction in GLU concentrations [38]. Concurrently, we found DL- lactate was negatively correlated with GLU, then gradu- ally recovered after PPL was treated. It may be due to the anorexia of diarrhea calves, resulting in poor nutri- tion absorption. However, PPL can improve the appetite of diarrhea calves by restoring their symptoms. The level of GLU was decreased in diarrheal calves. Increases in ALP and bilirubin levels were thought to be liver dam- age developed in calves with diarrhea [39]. Similarly, in this study, the levels of bilirubin and ALP were down- regulated after PPL was treated. Therefore, we speculated that PPL could attenuate calf diarrhea by improving liver function. Abbreviation NCD Neonatal calf diarrhea PPL Pueraria polysaccharide CBC Complete blood count TOF/MS Time-of-flight/ mass spectrometer IDA Information-dependent acquisition OPLS-DA Orthogonal partial least squares-discrimination analysis VIP Variable importance in the projection WBC White blood cell NEUT Neutrophil count HCT Hematocrit RBC Red blood cell LYMPH Lymphocyte counts MONO Monocyte count ESO Eosinophil BASO Basophil TP Total protein GLOB Globulin ALP Alkaline phosphatase GLU Glucose ALB Albumin BUN Blood urea nitrogen CREA Creatinine ALT Alanine aminotransferase TIC Total ion chromatograms RSD Standard deviation In addition to damaging the intestinal integrity and liver function in neonatal calves, the induction of diar- rhea triggers the inflammation response in the calf with diarrhea. LysoPC is generated by the enzyme phospho- lipase A2, which hydrolyses phosphatidylcholine at the sn-2 position [40], and participates in the component of biological membranes in animal cells [41]. The levels Page 11 of 12 Shen et al. BMC Veterinary Research (2023) 19:98 Shen et al. BMC Veterinary Research FC Fold change TMAO Trimethylamine N-oxide 4. Hart BL. Acknowledgements We thank Shanghai Applied Protein Technology Co. Ltd. (Shanghai, China) for UHPLCTOF/MS analysis. 11. Ekachai C. Antimicrobial property and antioxidant composition of crude extracts of Pueraria mirifica, Butea superba and Mucuna macrocarpa. Maejo Int J Sci Tech. 2009;3(1):212–21. Consent for publication 19. Boccardo A, Sala G, Ferrulli V, Pravettoni D. Cut-off values for predictors associated with outcome in dairy calves suffering from neonatal calf diar‑ rhea. A retrospective study of 605 cases. Livest Sci. 2021;245:104407. Declarations 16. 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Plasma metabolomic study in Chinese patients with wet age-related macular degeneration. BMC Ophthalmol. 2017;17(1):165.ll Supplementary Information 6. Zeng P, Li J, Chen Y, Zhang L. The structures and biological functions of polysaccharides from traditional Chinese herbs. Prog Mol Biol Transl Sci. 2019;163:423–44. The online version contains supplementary material available at https://doi. org/10.1186/s12917-023-03662-9. The online version contains supplementary material available at https://doi. org/10.1186/s12917-023-03662-9. 7. She S, Liu W, Li T, Hong Y. Effects of puerarin in STZ-induced diabetic rats by oxidative stress and the TGF-beta1/Smad2 pathway. Food Funct. 2014;5(5):944–50. Additional file 1: Table S1. The scoring criteria of clinical examina‑ tion. Table S2. Validation results of OPLS model. Table S3. Differential metabolites identified of C vs D groups in the positive or negative mode. Table S4. Differential metabolites identified of D vs T groups in the positive or negative mode. Figure S1. TIC of QC sample in positive and negative modes respectively. Figure S2. RSD of QC sample in positive and negative modes respectively. Additional file 1: Table S1. The scoring criteria of clinical examina‑ tion. Table S2. Validation results of OPLS model. Table S3. Differential metabolites identified of C vs D groups in the positive or negative mode. Table S4. Differential metabolites identified of D vs T groups in the positive or negative mode. Figure S1. TIC of QC sample in positive and negative modes respectively. Figure S2. RSD of QC sample in positive and negative modes respectively. 8. Alam MR, Kim WI, Kim JW, Na CS, Kim NS. Effects of Chitosan-oligo‑ saccharide on diarrhoea in Hanwoo calves. Veterinární Medicína. 2012;57(8):385–93. 9. Shanshan L, Yuli Q, Lixue C. Di Qu, Zhiman L, Kun G, Jianbo C, Yinshi S: Effects of Panax ginseng polysaccharides on the gut microbiota in mice with antibiotic-associated diarrhea. Int J Biol Macromol. 2019;124:931–7. 10. Bo L, Qing-Mei L, Gui-Ling L, Le-Chang S, Yuan-Yuan G, Ya-Fen Z, Hong L, Min-Jie C, Guang-Ming L. The anti-diarrhea activity of red algae-orig‑ inated sulphated polysaccharides on ETEC-K88 infected mice. RSC Adv. 2019;9(5):2360–70. Author’s contributions LS, YS, YZ and LY designed the study. ZS, GP, ZZ and JD conducted the experi‑ ment. YS performed lab analysis and wrote the manuscript. SY, XW and XZ performed statistics and analyzed the data. YZ and SC revised the article. All authors carefully read and approved the final revision of the manuscript. 12. Zhou D, Mengmeng Z, Huixian L, Qiping Z, Furao L, Hui W. Structural characterization and immunomodulatory activity of a novel polysac‑ charide from Pueraria lobata (Willd.) Ohwi root. Int J Biol Macromol. 2020;154:1556–64. 13. Zhen-Bin W, Bing-Bing C, Lina L, Jing-Kun Y. Fractionation, physicochemi‑ cal characteristics and biological activities of polysaccharides from Pueraria lobata roots. J Taiwan Inst Chem E. 2016;67:54–60. Ethics approval and consent to participate The permissions for conducting the study in the dairy farm in Sichuan, China was obtained. Sample collection was performed in strict accordance with the guidelines of the Care and Use of Laboratory Animals of China, and all procedures were approved by the Animal Care and Use Committee of Sichuan Agricultural University (No.2013–028). This study was carried out in compliance with the ARRIVE guidelines. This study has obtained the informed consent from the farm owner. 17. McGuirk SM. Disease management of dairy calves and heifers. Vet Clin North Am Food Anim Pract. 2008;24(1):139–53. 18. Walker PG, Constable PD, Morin DE, Drackley JK, Foreman JH, Thur‑ mon JC. 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https://openalex.org/W2587698565	http://pdf.blucher.com.br/engineeringproceedings/1enei/044.pdf	Portuguese	null	INOVAÇÃO SECUNDÁRIA NA CONSTRUÇÃO DE CAPACIDADES TECNOLÓGICAS NO SETOR AUTOMOBILÍSTICO CHINÊS	Blucher Engineering Proceedings	2,016	cc-by	7,245	1 Mestre em Política Científica e Tecnológica. Este trabalho procede da minha dissertação de Mestrado intitulada “O Estado e as Empresas Multinacionais no desenvolvimento produtivo e tecnológico da indústria automobilística chinesa”. Resumo Este trabalho se mostra importante colaboração na gestão pública da inovação tecnológica setorial para os Países em desenvolvimento que desejam evolução das capacidades tecnológicas e/ou construção desses conjuntos para setores da indústria de alta intensidade de P&D. Quanto a nosso material e métodos empregados, foi realizada uma pesquisa empírica, descritiva por levantamento de dados em fontes secundárias. Todos os dados após serem tabulados foram tratados com o uso da estatística descritiva. Ficou demonstrado o período da formação das empresas automobilísticas chinesas bem como as origens das capacidades tecnológicas. Concluímos que o desenvolvimento das capacidades de aprendizagem institucional apoiou as construções das outras capacidades, como as produtivas e as tecnológicas, e isto crescentemente evoluiu para aumento a partir dos anos de 1990, nas montadoras chinesas engajadas nas políticas públicas para o desenvolvimento do setor. Além disso as relações em Joint Ventures Internacionais (JVIs) elevou os níveis de aprendizagem das firmas nesses conjuntos de capacidades observadas. Os efeitos de transbordamento causados pelo dinâmico processo da aprendizagem das firmas promoveram rápida capacitação para inovar nas empresas independentes de capital privado que se instalaram na concorrência local a partir de 1990. Como resultado destes fatores, empresas multinacionais, transferência de avançada tecnologia, estrutura de absorção de inovações tecnológicas, a indústria automobilística chinesa se tornou líder mundial em produção e vendas no setor. INOVAÇÃO SECUNDÁRIA NA CONSTRUÇÃO DE CAPACIDADES TECNOLÓGICAS NO SETOR AUTOMOBILÍSTICO CHINÊS Rodrigo Diogo Teixeira1 Sérgio Reis Robles de Queiroz2 Ana Cristina Andrade3 Flávia Luciene Consoni; 2 Eng. Civil (USP, 1978); Mestre (Unicamp, 1987); Doutor (Unicamp, 1993); Livre-Docente (Unicamp, 2004). Credenciado no Mestrado e Doutorado em Política Científica e Tecnológica. 3 Psicóloga, compositora e cantora, palestrante em Saúde Mental. 3 Psicóloga, compositora e cantora, palestrante em Saúde Mental. 2 Eng. Civil (USP, 1978); Mestre (Unicamp, 1987); Doutor (Unicamp, 1993); Livre-Docente (Unicamp, 2004). Credenciado no Mestrado e Doutorado em Política Científica e Tecnológica. Introdução Este trabalho está vinculado à linha de pesquisa de Economia da Inovação tecnológica e tem como eixo principal mostrar a complexa organização do Estado chinês na promoção de sua industrialização para setores de alta intensidade de uso de P&D. Ele serve como base para estudos sobre o papel do Estado na gestão de políticas públicas envolvendo um multidimensionamento para esferas educacionais de formação de recursos humanos para o sistema de unidades criativas, que transformam e criam novos conhecimentos e informações tecnológicas que são insumos para inovar. E por outro eixo, ele demonstra como as empresas estrangeiras multinacionais foram importante no processo de construção das capacidades tecnológicas nas empresas do setor automobilístico chinês. A competitividade é um fenômeno da maior importância nos estudos sobre organização industrial, pois empresas e regiões competitivas viabilizam maiores taxas de crescimento econômico e de desenvolvimento social. O seu entendimento, porém, não é unânime. Focando as organizações industriais, alguns especialistas costumam associar a competitividade à maior eficiência técnica dos processos produtivos adotados. A comparação de indicadores técnicos, do tipo insumo-produto, produtividade dos fatores, entre outros, com as best-practices verificadas na indústria, fornece um ranking e aponta as organizações mais eficientes. A análise por envoltória desenvolvida por Charnes; Cooper; Rhodes (1978) é freqüentemente utilizada para o estudo da eficiência técnica. Outros preferem associar a competitividade ao desempenho de mercado, isto é, ao crescimento da participação no mercado ou marketshare (COUTINHO; FERRAZ, 1995). Neste artigo, adota-se o segundo entendimento de competitividade. A inovação secundária tende-se a ser difundida mais rapidamente do que as inovações primarias que despendem maiores níveis de organizações da P&D das empresas multinacionais com conhecimento avançado em linha de fronteira tecnológica. O conceito de capacidade é entendido neste trabalho como aquele relativo ao indivíduo ou à organização e que engloba recursos tais como habilidades, conhecimentos e experiência que permitem, a tais agentes, avançarem para estágios superiores de desenvolvimento. Trata-se de uma bagagem que, ao permitir o acúmulo de capacidades, a partir de processos contínuos de aprendizagem, possibilita que as organizações de uma forma geral desenvolvam sempre melhores e diferenciados processos, produtos, práticas organizacionais, etc (LALL, 1992; BELL; PAVITT, 1993). Ou seja, a partir de processo de aprendizagem a empresa consegue acumular capacidades ao longo do tempo, de níveis mais simples aos mais evoluídos e complexos. Autores como Katz (1987), Dahlman et al. Introdução (1987) e Lall (1987; 1992; 1994) afirmam que este processo de aprendizagem nas organizações é contínuo, evolutivo e cumulativo com o tempo, culminando no processo de construção de capacidades. Segundo Figueiredo (2004), o produto final do processo de aprendizagem revela o nível da tecnologia aplicada no conjunto do processo produtivo e deste modo o grau de complexidade tecnológica será igual à capacitação técnica em nível operacional que reflete a qualidade e experiência do estoque de trabalhadores operacionais trabalhando em seus departamentos internos. Demais trabalhos sobre as capacidades tecnológicas se baseiam em várias taxonomias que avaliam seu crescimento nas empresas e/ou setores da indústria de países desenvolvidos e em desenvolvimento Lall (1992), Bell, Pavitt (1993), Bell (1982; 1996; 2007), Figueiredo (2003; 2004; 2007), Consoni (2004), Queiroz, Quadros (2005), Quadros (2009), Dutrénit (2004), Amsden (2001), Bell (2007) e Bell, Figueiredo (2013). Este artigo utiliza-se destes autores para evidenciar a importância dos atores públicos e privados nas mudanças de trajetórias tecnológicas para a construção de capacidades produtivas, tecnológicas e institucionais. A ascensão do setor automobilístico chinês de 1980 a 2014 comprova tais teorias do desenvolvimento das forças produtivas. Interessa-nos estudar a relação entre a mudança estrutural do setor automobilístico chinês ocorrida nos anos 1980 e o comportamento das empresas do setor em aumento progressivo do conjunto de capacidades tecnológicas, produtivas e de aprendizagem institucional. Este setor, porém, apresenta um parque industrial com significativa presença de empresas transnacionais, diversificado e representativo distribuído nas 31 Províncias chinesas. Segundo a Agência especializada no setor Fourin, em 2012, existia 179 plantas no setor automobilístico na China. A maior quantidade de plantas produtivas está localizada na região Oeste, 90, seguida respectivamente, pelas regiões Central 48, Leste 25 e Noroeste 16 plantas. A indústria automobilística chinesa responde pela primeira colocação em produção e vendas no setor na concorrência direta com as empresas transnacionais no mercado interno e externo. Cabe ressaltar que a capacidade de produção e adequação a novos produtos como veículos elétricos hodiernamente é bastante significativo para a perpetuação dos chineses na liderança automobilística mundial, segundo dados da OICA e CAAM, órgãos oficiais internacionais. O artigo está dividido em quatro seções além desta introdução. Na segunda explicita-se a metodologia empregada. Na terceira é desenvolvida o referencial conceitual que serviu de base para o desenvolvimento do estudo empírico. Na quarta são apresentados os resultados da pesquisa. Na última seção são apresentadas as considerações finais. Material e método A pesquisa se pautou numa amostra de empresas retirada do China Association of Automobile Manufacturers (CAAM) órgão oficial do setor. Este trabalho é um estudo qualitativo, apoiado em pesquisa empírica, descritiva, documental e histórica. Para sua execução, foram utilizados essencialmente dados secundários. A análise se baseou na literatura internacional sobre a indústria automobilística chinesa, leitura das informações coletadas e interpretação dessas informações. Para o estudo do desenvolvimento dessas capacidades na indústria automobilística chinesa, foram selecionadas 6 empresas do setor que juntas em 2012 representavam 64,7% das vendas de automóveis. Dentre essas empresas, três são do Estado (SAIC, DFM, Changan) e as demais independentes de capital privado (CHERY, GEELY e BYD). Após tal seleção, foram definidas seis variáveis para medição das capacidades produtivas e tecnológicas por empresa selecionada, sendo três variáveis para cada um dos grupos de capacidades. Para capacidades produtivas, as variáveis compreendem: volume de produção anual; número de plantas produtivas; e, número de modelos. Para as tecnológicas, as variáveis são: número de patentes; número de pessoal ocupado em tempo integral nos laboratórios internos de P&D; e, número de plantas de P&D. O levantamento dos dados sobre patentes de empresas deste setor foi realizado no State Intelectual Property Office da R.P.C entre 2011-2013. Para a condução dessas análises, foram acessados livros e artigos científicos sobre a indústria automobilística chinesa, além de consultas a internet, sítios das empresas chinesas do setor, fontes documentais e bancos de dados estatísticos de órgãos oficiais chineses como Ministério da Ciência e Tecnologia (C&T) da China, Ministério da Educação da China, Agência Oficial de Propriedade Intelectual da China, China Statistical Yearbook, Fourin China Automotive Intelligence, Associação dos Fabricantes de Automóvel da China (CAAM em inglês), além de esferas de coordenação da indústria automobilística mundial, como a Organização Internacional dos Fabricantes de Automóveis (OICA em inglês) e o GERPISA. Os dados colhidos na pesquisa foram analisados por intermédio da estatística descritiva. Referencial teórico ou conceitual A cooperação para este comportamento especificamente para a obtenção de aprendizado tecnológico para que os chineses – como vemos hoje em dia, na sua indústria automobilística – chegassem ao primeiro lugar em vendas e produção se deu por intermédio de parcerias com empresas estrangeiras, a primeira parceira deles foram as empresas russas. Da indústria automobilística russa se originou as primeiras capacidades produtivas e tecnológicas para os chineses para a produção de caminhões em meados de 1950 (HOLWEG; LUO; OLIVER, 2005, p.5). Em 1950 como conta a história estes parceiros chineses já possuíam know how para produção de veículos pois pela lógica de guerras por mercados, a produção automobilística tinha alta demanda – falo do período de 1910 a 1950 -, ao que a história aponta houve a primeira transferência de capacidades de produzir automóveis para os chineses em 1950. Os primeiros automóveis chineses foram caminhões pesados de 5 Toneladas para a indústria militar. As montadoras russas em 1950 já eram reconhecidas no mundo pela qualidade de seus caminhões. Os caminhões chineses herdaram essa qualidade. Em 1953, após a chegada de 39 engenheiros enviados a Rússia para treinamento a mando de Mao Tsé, é inaugurada a First Automobile Works. Em seguida a Shanguai Automobile Corporation, em 1955. E assim por diante dados em Wang (2009) apontam que em 1979 havia 56 montadoras na China e entre o período de 1966-1979 a evolução do número de montadoras foi de 42,86 %, ou 24 novas plantas (WANG, 2009, p. 385).Como afirma Nam (2010, p.3), “a indústria automobilística chinesa seguiu na estratégia Exchange-market-for-technology”. As JVIs aliadas à gestão estatal obrigaram as EMNs a realizar a transferência de tecnologia mais avançada às montadoras chinesas por meio dessas parcerias. Através do Ministério da Ciência e Tecnologia o governo guiou o desenvolvimento produtivo e tecnológico e o avanço das capacidades de produção e tecnologia deste setor. Vários ministérios integrados coordenaram por instrumento de política industrial no setor a construção das capacidades produtivas, tecnológicas e institucionais, sendo que a última deu se maior ênfase para desenvolver as demais. Na década de 1980 iniciou-se a abertura comercial chinesa com a execução do plano de metas de evolução dos níveis gerais de conhecimentos tecnológicos através dos planos organizados pelas estruturas institucionais chinesas, responsáveis por a longo prazo formar tais níveis elevados de capacidades no setor automobilístico. Em 1982, foram executados três planos envolvendo várias esferas de coordenação ministeriais. Referencial teórico ou conceitual O aprendizado tecnológico e organizacional como um todo é considerado elemento-chave dentre os fatores condicionantes do desevolvimento produtivo- tecnológico (Bell, 1984; Bell & Pavitt, 1993; Kim, 1997). Bell define o aprendizado tecnológico como “the acquisition of additional technical skill and knowledge by individuals and through them by organisations” (Bell, 1984, p. 188). De acordo com Bell, Figueiredo (2013, p.8) sobre as firmas de países em desenvolvimento têm se destacado na construção de capacidades tecnológicas por diversas vias, tais como: a) uso das fontes internas e externas de conhecimentos tecnológicos, b) subcontratações, c) joint ventures, d) licenciamentos, e) treinamento de talentos no exterior, f) imitação, g) transferência de conhecimento incorporado em normas, h) aprendizado interativo “face- to-face interactions”, i) aprendizagem através de pressões para atender aos padrões internacionais, j) treinamento com líderes e empresas estrangeiras, e, k) efeito transbordamento de conhecimentos (spillovers). Estas empresas conseguem desenvolver in-house capacidades de marketing e designer independentes atualizando suas capacidades produtivas e tecnológicas, gerando através de aprendizado tecnológico com diversas fontes, o que neste trabalho é chamado de capacidades institucionais, as quais permitem as condições necessárias ao bom desenvolvimento das forças produtivas no espaço para que se alcance o desenvolvimento socioeconômico. A literatura sobre aprendizado tecnológico se desenvolve sobre a capacidade de absorção de conhecimento e informações tácitas e não tácitas a partir de indivíduos em organizações produtivas ou unidades criativas ligadas as organizações industriais. Estas aplicam conhecimentos dessas unidades criativas no âmbito do processo produtivo e geram as inovações tecnológicas, que são largamente utilizadas na promoção da competitividade das firmas por mercados. Os pilares analíticos desta tal função da produção internalizada nas firmas industriais considerando as unidades criativas – indivíduos – pode ser entendida em duas dimensões do conhecimento intangível e tangível, onde, o primeiro esta nas experiências vivenciadas pelos indivíduos ligados as áreas estratégicas da produção interna a firma, como Marketing, Pesquisa & Desenvolvimento e outros, e a segunda dimensão, esta na distribuição normalizada dos saberes nos veículos da comunicação de nossa sociedade cientifica e cultural, e sofre variações do ambiente educacional e nível de civilidade locais presentes no Mundo. Há inúmeros trabalhos internacionais sobre o desenvolvimento da indústria automobilística asiática em especial, a chinesa (HE, 2008; WANG, 2009; HOLWEG; LUO; OLIVER, 2005; NAM, 2010). Estes trabalhos mostram uma interação quanto a transferência de tecnologia de empresas transnacionais as empresas locais. Referencial teórico ou conceitual Os planos foram o Programa Nacional de Pesquisa em Tecnologias Chave (PNPTC) e Programa Nacional de Tecnologias Chaves em P&D (PNTC P&D), e o Programa Nacional de P&D em Indústrias de Alta Tecnologia (PNPD). Tinham o prazo de 1982-1997 para serem concluídos. O Programa Nacional de P&D em indústrias de média-alta intensidade tecnológica, por exemplo, apoiou a política de atração das alianças entre empresas multinacionais e chinesas na estrutura legal de joint ventures produtivas ea fim de transferir para essas últimas, conhecimentos tecnológicos avançados e desenvolve-los localmente a posteriori. De acordo como Wang (2009) registra, a transferência de tecnologia da FAW para a SAW. O autor demonstra que enquanto a FAW importou 95% dos equipamentos e máquinas usados em sua primeira base produtiva, a SAW comprou em 1969 a mesma porcentagem dos equipamentos de empresas de origem nacional, no mercado interno, sem importar do exterior, em contraste com as maciças importações realizadas pela FAW. e máquinas usados em sua primeira base produtiva, a SAW comprou em 1969 a mesma porcentagem dos equipamentos de empresas de origem nacional, no mercado interno, sem importar do exterior, em contraste com as maciças importações realizadas pela FAW. Também durante a década de 1980 o governo chinês criou instituições de ciência e tecnologia visando apoiar o sistema nacional de inovação. Como observa em economias desenvolvidas este sistema está estritamente ligado com o bom nível de qualidade da educação destas economias, capazes de atender a demanda interna e externa em todos os níveis de exigência da sociedade. No ano de 1985 foi fundado o China Automotive Technology & Research Center (CATARC) após a aprovação do National Science and Technology Commission. Este Centro de Tecnologia e Pesquisa automotiva faz o papel da administração técnica da indústria, na regulação, planejamento de políticas governamentais e encontra-se subordinado ao State-owned Assets Supervision and Administration Commission of the State Council (SASAC) que é o órgão regulador máximo do Governo Central. Dentre suas responsabilidades, é o responsável pela seleção dos negócios e investimentos das empresas estrangeiras que, de 1985 a 2013, realizaram investimentos diretos estrangeiros no mercado chinês, em especial, na indústria automobilística. O governo chinês criou ainda em 1988 o Departamento de Política Industrial, que foi o responsável pela primeira política automotiva de 1994 – que citada aqui será mais detalhada na seguinte seção. Esta irá fortalecer ainda mais a competitividade no mercado interno do setor. 4 Operações de knock-down (KD) na indústria automobilística referem-se à montagem e transporte de kits de veículos desmontados para serem montados em mercados estrangeiros. Em muitos casos, foi utilizada Referencial teórico ou conceitual O planejamento adotado para o desenvolvimento das capacidades de alto valor agregado que estão nas engrenagens do setor automobilistico chinês sempre foram bem sinalizadas nas empresas que existiam na China de 1950 até 2014. Na medida em que apareciam os bons resultados no setor aumentavam-se os níveis de capacidades institucionais, trazendo dinamismo de aprendizagem nas esferas administrativas e também governamentais. As parcerias de chinesas com estrangeiras em multiplas JVIs – usadas como escolas para aprendizado tecnológico – promove a atualização produtiva e tecnológica das empresas chinesas. Além de ser a base de seu rápido progresso de aquisição de capacidades produtivas e tecnológicas, vistos no setor, a partir de 1990. O Estado decidia sobre a localização e a forma da partilha da tecnologia nas JVIs. E também distribuia os projetos de desenvolvimento de modelos de automóveis entre suas montadoras nestas parcerias. A VW nos anos de 1980 celebrou uma JVI com duas de suas maiores empresas no setor, a SAIC e FAW, e delegou à FAW-VW um novo projeto a ser desenvolvido em 1988 (que teve a parceria da subsidiaria da VW no Brasil), tendo por objetivo produzir o primeiro automóvel de passageiro com peças 100% chinesas e motor alemão, VW-Jetta, no inicio dos anos de 1990 para o mercado local. Estes contratos entre empresas complementam os ativos e podem gerar um dinâmico processo de aprendizagem sobre métodos de produção e criação de inovações tecnológicas de processos e/ou produtos. Segundo Nam (2010, p.3) as JVIs são redes de empresas que acoplam suas capacidades tecnológicas compartilhadas entre si. Este tipo de arranjo foi empregado pelos chineses na evolução das firmas chinesas da indústria automobilística. Aos poucos a China foi substituindo a importação de automóveis baseada em Completely Knock-Down4 (CKD) pela produção programada. Ao final do período analisado, como afirma He (2008, p.8), “o governo chinês decidiu transformar sua indústria automobilística concentrada na produção de caminhões em automóveis de passageiros, a partir de 1987”. Referencial teórico ou conceitual Seguindo as contribuições de autores consagrados sobre o fundamento desta obra, como Lall (1992), Bell, Pavitt (1993), Bell (1982; 1996; 2007), Figueiredo (2003; 2004; 2007), Consoni (2004), Queiroz, Quadros (2005), Quadros (2009), Dutrénit (2004), Amsden (2001), Bell (2007) e Bell, Figueiredo (2013) é possível qualquer País do Mundo obter capacidades produtivas, tecnológicas e institucionais em um período de 30 anos se houver as congruências necessárias no diálogo entre os principais atores setoriais da inovação, ou seja, no âmbito público e privado uma união de investimentos estáveis e no âmbito cientifico uma harmonia fina com os empresários locais ligados a administração das unidades produtivas, onde se dá a inovação tecnológica e a capitalização responsável pela distribuição de riquezas a sociedade via bem e serviços finais e salários. Resultados O núcleo central das capacidades institucionais iniciada com a criação de esferas de planejamento socioeconômico – Conselho do Estado, Ministérios, órgãos setoriais e outras instituições - vai culminar com a criação da primeira lei chinesa sobre JVIs, fundamental para a atualização das capacidades produtivas e tecnológicas industriais. Em fins deste período abordado surge a necessidade de progresso técnico na indústria automobilística e demais setores chineses da indústria e as JVIs foram escolhidas para obtê-lo. O início do setor o primeiro bem a ser aprendido foi como fazer caminhões, e a escola de produção foi a empresa russa Zavod Imena Stalina – ZIS. GRÁFICO 1. Evolução da produção de carros chinesa -1949-1979 esta abordagem para contornar altos impostos incidentes sobre a importação de veículos acabados, ou para evitar os investimentos para uma instalação completa de montagem de veículos onde os volumes não justificam isso. Operações KD podem assumir várias formas, desde kits completos (CKD) a semi-KD (SKD) onde as operações de prensagem, soldagem e pintura são feitas localmente, enquanto que as peças são importadas em conjuntos (como kits) a partir do estrangeiro. GRÁFICO 1. Evolução da produção de carros chinesa -1949-1979 GRÁFICO 1. Evolução da produção de carros chinesa -1949-1979 esta abordagem para contornar altos impostos incidentes sobre a importação de veículos acabados, ou para evitar os investimentos para uma instalação completa de montagem de veículos onde os volumes não justificam isso. Operações KD podem assumir várias formas, desde kits completos (CKD) a semi-KD (SKD) onde as operações de prensagem, soldagem e pintura são feitas localmente, enquanto que as peças são importadas em conjuntos (como kits) a partir do estrangeiro. Fonte: Elaboração própria a partir de Oica-Fourin e Catarc, 2014. Fonte: Elaboração própria a partir de Oica-Fourin e Catarc, 2014. Vale notar que em 1979 havia 56 montadoras na China e entre o período de 1966-1979 a evolução do número de montadoras foi de 42,86 %, ou 24 novas plantas (WANG, 2009, p. 385). A China termina o período com a promulgação da primeira versão da lei sobre JVI. Essas construções participativas que vêm no contexto macroeconômico de abertura comercial desenvolvem capacidades produtiva e tecnológica via sistema de aprendizado e absorção de conhecimentos tecnológicos. O avanço das capacidades tecnológicas se deu no setor a partir de 1979 e o oque causou esta evolução foi o bom planejamento e restruturação organizacional e produtiva pelo qual as empresas automobilísticas foram submetidas como podemos obervar na Figura 1 a seguir. Como afirma He (2008, p.8), “o governo chinês decidiu transformar sua indústria automobilística concentrada na produção de caminhões em automóveis de passageiros, a partir de 1987. Desde então, a indústria automobilística chinesa entrou em uma era de crescimento rápido”. Figura 1. A evolução de Instituições da China Figura 1. A evolução de Instituições da China Fonte: Elaboração própria a partir de Market Analysis Report: China’s Automotive Industry, APCO (2010, p.15). Fonte: Elaboração própria a partir de Market Analysis Report: China’s Automotive Industry, APCO (2010, p.15). Como consequência dessas transformações no modo e meios de produção no setor, em 1990, cresceu o número de parceiras que transferia suas capacidades produtivas e tecnológicas avançadas as firmas chinesas, via essas esferas organizadas para captar e distribuir desenvolvimento cientifico e tecnológico as firmas chinesas que a partir de 1980 se submetem as joint ventures chinesas com as multinacionais estrangeiras. Veja a evolução da produção em 1990. GRÁFICO 2. Evolução da produção de 1980-1990 Fonte: Elaboração própria a partir de Oica-Fourin e Catarc, 2014. GRÁFICO 2. Evolução da produção de 1980-1990 GRÁFICO 2. Evolução da produção de 1980-1990 Fonte: Elaboração própria a partir de Oica-Fourin e Catarc, 2014. Observe no gráfico 2, que a produção de caminhões decresce a partir de 1994 e aumenta-se vertiginosamente a produção de carros comerciais e automóvel de passageiros. Fonte: Elaboração própria a partir de Oica-Fourin e Catarc, 2014. Provavelmente este fenômeno está ligado ao controle sobre o progresso de capacidades tecnológicas e produtivas feitos no ambiente regional pelos promotores institucionais de política setorial voltada ao progresso industrial com o apoio da primeira política para o setor datada de 1994, que criou mecanismo de demanda local e conseguiu substituir as importações de autopeças das montadoras automobilísticas produzindo na China. Em 2004 ira ser promulgada a segunda política para o setor adequando as empresas chinesas as the best-practices internacionais, se aproveitando da ascensão chinesa junto a Organização Mundial do Comércio, em dezembro de 2001. GRÁFICO 3. Desenvolvimento das capacidades da produção - 1990-2000 Fonte: Elaboração própria a partir de dados da CATARC, 2009. Fonte: Elaboração própria a partir de dados da CATARC, 2009. A chave do sucesso no setor para Richet, Ruet (2007, p.3) foi o aumento de bases produtivas dentro da China com elevação das capacidades produtivas e tecnológicas no setor com incentivos do Estado. Em seu trabalho esses autores apontam o declínio da taxa de produção industrial (em %) concluindo para três níveis medidos de concentração que os cálculos realizados para uma, para duas e para três empresas de concentração industrial passam de 19,2% para 14,7%, de 38,0% para 25,4% e 43,0% para 34,4%, respectivamente, de 1985 a 1998. Tomemos uma nota, aqui, nos anos de 1990 a concorrência por mercado no setor, entre chinesas e estrangeiras, colaborou no desenvolvimento de novas tecnologias no setor. A partir de 1990 a China já se afastara dos sistemas de inovação nacional dos soviéticos e dos americanos, que se concentravam na defesa, e já se projetava para um sistema com foco estabelecido na competitividade empresarial promovida pela inovação tecnológica em suas empresas industriais. Sobre este assunto, como registrou (AMSDEN, 2009, p.482), a inflexão que proporcionou este grande passo da China nos anos de 1990 aconteceu no período anterior, mais precisamente em 1985, quando o Comitê Central do Partido Comunista Chinês decretava que o meio chinês para atingir crescimento econômico e desenvolvimento industrial para empresas de alta tecnologia deveria se basear na aquisição de ciência e tecnologia5. O desenvolvimento institucional e científico-tecnológico veio com a criação de parques científicos com projetos nacionais de P&D in-house. Os incentivos fiscais e o crédito subsidiado foram importantes em ambos os casos (AMSDEN, 2009, p.482). 5 Para maiores informações consultar: SAICH, T. Reforms of China’s Science and Technology Organizational System: Science and Technology in Post-Mao China (eds). D. Simon and M. Goldman. Cambridge: Cambridge University Press, p.69-88, 1989. Fonte: Elaboração própria a partir de Oica-Fourin e Catarc, 2014. Considere que a capacidade produtiva ilustra o desempenho na produção e diversificação dos veículos; as capacidades tecnológicas são um indicador do aperfeiçoamento destes produtos/ processos; e as capacidades institucionais são um meio de avaliar e dar destaque ao papel do Estado, da evolução jurídica-legal das suas regulações e da coordenação de suas políticas internas. A sintonia entre tais capacidades foi fato fundamental para projetar a indústria automobilística chinesa no cenário nacional e internacional. Para ilustrar o avanço da indústria automotiva chinesa em tais capacidades, elegemos um conjunto de indicadores que bem representam tal evolução. Quanto às capacidades produtivas, trabalhamos com: o volume de produção; o número de plantas; e, o número de modelos de veículos por empresa. Com relação às capacidades tecnológicas, usamos: o número de patentes por empresa; o número de laboratórios e institutos de P&D in-house; e o número de pessoal empregado internamente nos laboratórios e institutos de P&D por empresa. E em relação as capacidades de aprendizagem institucional, fazemos menção ao: número de intuições de P&D em indústrias de alta tecnologia; número de instituições do sistema educacional (ensino superior e tecnológico); e, valor total em investimentos em ativos fixos (em 100 milhões de yuans). TABELA . Taxonomia de avaliação para as capacidades produtivas, tecnológicas e aprendizado institucional (2014) categoria A categoria B categoria C capacidades produtivas volume de produção número de modelos número de plantas capacidades tecnologicas número de patentes número de pessoal ocupado em P&D in-house número de instituições próprias de P&D in-house capacidades de aprendizagem industrial número de instituições de P&D número de instituições Ensino Superior e Tecnologo valor total investimentos em ativos fixos Fonte: Elaboração própria. 5 Para maiores informações consultar: SAICH, T. Reforms of China’s Science and Technology Organizational System: Science and Technology in Post-Mao China (eds). D. Simon and M. Goldman. Cambridge: Cambridge University Press, p.69-88, 1989. categoria A categoria B categoria C capacidades produtivas volume de produção número de modelos número de plantas capacidades tecnologicas número de patentes número de pessoal ocupado em P&D in-house número de instituições próprias de P&D in-house capacidades de aprendizagem industrial número de instituições de P&D número de instituições Ensino Superior e Tecnologo valor total investimentos em ativos fixos Fonte: Elaboração própria. 5 Para maiores informações consultar: SAICH, T. Reforms of China’s Science and Technology Organizational System: Science and Technology in Post-Mao China (eds). D. Simon and M. Goldman. Cambridge: Cambridge University Press, p.69-88, 1989. Fonte: Elaboração própria a partir de Oica-Fourin e Catarc, 2014. Tabela 11. Ilustração das capacidades produtivas de plantas automotivas chinesas (1949-2013) Empresas número de plantas número de modelos volume de produção anual Total 166 531 18.208.000 SAIC 18 150 4.000.000 Geely 10 17 1.800.000 Beijing Automotive Industry Holding Co Ltd ( BAIC ) 26 40 1.600.000 Dongfeng Motor Company Limited 8 74 1.600.000 FAW Group Corporation 7 86 1.600.000 Changan Automobile Company Limited 21 25 1.500.000 Chery 16 25 1.350.000 Jianghuai Automobile Corp. 3 29 1.130.000 Huashei Auto Group (Brilliance Auto) 4 39 800.000 Great Wall 30 8 800.000 GAC Changfeng Motor Co. 2 5 728.000 BYD 19 18 700.000 Hafei 2 15 600.000 Fonte: Elaboração própria com fontes das próprias montadoras para outubro de 2013. Fonte: Elaboração própria com fontes das próprias montadoras para outubro de 2013. Quanto o aumento de conhecimentos sobre a produção de automóvel da montadoras chinesas impactou na evolução das decodificações de fontes de conhecimentos via joint ventures com as maiores empresas multinacionais do setor, pode ser analisado na ilustração abaixo que mostra as capacidades tecnológicas construídas ao longo de 30 anos como demonstra Bell; Lall (1995) quando afirmam que é possível com junção de esforços um País sair da semi industrializado a industrialização absoluta com inovações secundarias efetivas, ou seja, aprendizado continuo e interações com empresas mestras em skills para a construção de capacidades tecnológicas em todos os setores industriais, alcançando os de alta performance tecnológica, como os chineses assim bem efetuaram. Vejamos: TABELA 13. Avaliação das capacidades tecnológicas das montadoras chinesas (2013) Empresas Número de patentes concedidas 2009- 2012 Invenção Modelo de utilidade Número pessoal ocupado na P&D interno de tempo integral Número de instituições próprias de P&D interno Chery 1.166 743 423 6.000 2 Geely 1.750 366 1.384 2.808 2 BYD 156 76 80 5.200 4 Huashei Auto Group (Brilliance Auto) 31 0 31 1.500 1 Great Wall 305 83 222 8.350 3 SAIC 336 162 174 5.500 2 GAC Changfeng Motor Co., Ltd 6 0 6 1.200 2 Beijing Automotive Industry Holding Co Ltd (BAIC) 25 6 19 4.500 3 Dongfeng Motor Company Limited¹ 347 82 265 s.i. 0 FAW Group Corporation¹ 49 33 16 s.i. 0 Changan Automobile Company Limited 920 428 492 4.000 13 Hafei¹ 12 3 9 2.000 0 Jianghuai Automobile Corp. 86 12 74 s.i 3 Sub-Total 5.189 1.994 3.195 41.058 35 TOTAL DA INDÚSTRIA 37.256 15.770 21.486 s.i. s.i. ELA 13. Fonte: Elaboração própria a partir de Oica-Fourin e Catarc, 2014. Avaliação das capacidades tecnológicas das montadoras chinesas (2013) TABELA 15. Medição das capacidades institucionais (2010) Medição da capacidade de aprendizagem institucional Nº de instituições de P&D na indústria de alta tecnologia (unidades) 3.184 Nº de instituições do sistema educacional, Nível Superior e Tecnólogo (unidades) 2.101 Valor Total de investimentos em ativos fixos (100 milhões de US$) 3.836 Fonte: China Statistical Yearbook (2011) Medição da capacidade de aprendizagem institucional Nº de instituições de P&D na indústria de alta tecnologia (unidades) 3.184 Nº de instituições do sistema educacional, Nível Superior e Tecnólogo (unidades) 2.101 Valor Total de investimentos em ativos fixos (100 milhões de US$) 3.836 Empresas ¹estas empresas não realizam P&D in-house dentro de suas próprias instalações, elas usam os laboratórios de EMNs, com as quais mantém-se em joint ventures. A empresa Hafei é uma subsidiária da empresa Changan, portanto seu pessoal ocupado em realizar a P&D in-house, se utilizam dos laboratórios da Changan. s.i. significa: sem informação precisa. Fonte: Fontes das próprias montadoras para outubro de 2013 com acesso ao número de patentes e modelos de utilidade do State Intelectual Property Office da R.P.C. Essa evolução possível foi pelo constante aprendizado das esferas de coordenação criadas desde o Ministério da Ciência e Tecnologia nos anos 1950. A Tabela abaixo demonstra esse crescimento: TABELA 15. Medição das capacidades institucionais (2010) Fonte: China Statistical Yearbook, (2011) Além desses números pode-se notar os crescentes dispêndios desde 2006 até 2010, no processo da internacionalização massiva de empresas do setor no Mundo no gráfico abaixo: GRÁFICO 16. Economia de recursos internos chinesa (2006-2010) GRÁFICO 16. Economia de recursos internos chinesa (2006-2010) Fonte: China Statistical Yearbook (2011). Fonte: China Statistical Yearbook (2011). A importância das instituições chinesas organizadas pelo Governo centralizado na coordenação das forças matriciais, acumulação de capital, expansão de alianças com investimentos e parcerias com o capital estrangeiro, e, apoio de um marco regulatório especifico para os setores chaves de desenvolvimento industrial, como a indústria automobilística, levaram ao que se nota no gráfico abaico. GRÁFICO 4. Produção da indústria automobilística chinesa de 1955-2013 Fonte: CATARC (2009) e OICA (2014). GRÁFICO 4. Produção da indústria automobilística chinesa de 1955-2013 Fonte: CATARC (2009) e OICA (2014). Após a promulgação da segunda política automobilística chinesa houve muitas operações de F&A no setor, como a compra da Shanghai Maple Guorun Automobile e Volvo car, pela empresa independente Geely, compra da Xi’an Automobile pela empresa chinesa BYD, formando a BYD Automobile que se especializou na produção de automóveis elétricos, dentre outras operações semelhantes. A empresa Shanghai Maple Guorun Automobile nasceu em 2000 em Fengjing, Shanghai. Esta empresa veio da JVI entre a Dongfeng-PSA Peugeot Citroen desde 1992, e em 2002 foi adquirida uma parte de seu capital social pela empresa Geely que em 2008 integralizou-o incorporando esta empresa. Em 2013 a Shanghai Maple firmou uma JVI com uma empresa de tecnologia, Kangdi Technologies Group, Inc., para o desenvolvimento de capacidades produtivas e tecnológicas de automóveis elétricos, as quais esta empresa automobilística, localizada na mesma província da Geely, Zhejiang, O Gráfico 8 mostra a evolução da produção do setor de 2003-2012, em que se pode ver como mudaram as primeiras 4 posições do ranking da produção automobilística no mundo. GRÁFICO 5. Principais produtores de automóveis no mundo (2003-2012) GRÁFICO 5. Principais produtores de automóveis no mundo (2003-2012) GRÁFICO 5. Principais produtores de automóveis no mundo (2003-2012) Fonte: Elaboração própria a partir OICA-FOURIN, 2013; ANFAVEA, 2013. Fonte: Elaboração própria a partir OICA-FOURIN, 2013; ANFAVEA, 2013. A liderança atual chinesa na indústria automobilística mundial pode ser visualizada na Tabela 1, logo a seguir. TABELA 1. Mercado e produção de automóveis no mundo (2014) Ranking Vendas Produção (Em milhões de unidades) (Em milhões de unidades) 1 China 21.984 China 22.117 2 Estados Unidos 15.884 Estados Unidos 11.046 3 Japão 5.376 Japão 9.630 4 Brasil 3.767 Alemanha 5.718 5 Alemanha 3.257 Coréia do Sul 4.522 6 Índia 3.241 Índia 3.881 7 Rússia 2.950 Brasil 3.741 8 Reino Unido 2.596 México 3.053 9 França 2.201 Tailândia 2.457 10 Canadá 1.780 Canadá 2.380 Demais países 21.257 18.755 TOTAL 85.394 87.300 Fonte: Elaboração própria a partir OICA-FOURIN, 2013; ANFAVEA, 2014. A China continua a consolidar sua força competitiva no setor frente às montadoras de países desenvolvidos. No ranking da produção e vendas as três primeiras colocações mostram onde se concentra a maior concorrência por mercados no mundo no setor. Países como Itália, França, Espanha não aparecem no ranking da produção e países como Canadá, Tailândia e México, ocupam lugar entre os 10 mais importantes na produção automobilística, em 2014. Já com relação ao ranking de vendas de automóveis entre os 10 mais importantes mercados para o setor, se destaca a ascensão da Rússia na sétima colocação; e o Brasil aparece à frente da Alemanha. Considerações finais Em primeiro lugar, os resultados nos dão prova de que as empresas automobilísticas chinesas cresceram em capacidades produtivas e tecnológicas influenciadas pelo dinâmico processo de evolução das capacidades institucionais. Esta constatação foi confirmada pelo trabalho da pesquisa exploratória de dados empíricos que demonstram modo e o tempo das aquisições e transformações nas unidades produtivas do setor. Podemos apontar que as joint ventures foram responsáveis pela rápida evolução de capacidades produtivas e tecnológicas que levou a resultados expressivos em vendas no mercado Mundial. Em segundo lugar, a atualização tecnológica veio como consequência da estratégia chinesa de por medidas de políticas públicas como as de 1994 e 2004 criar demanda e oferta interna para substituir as importações de autopeças e estimular o desenvolvimento das empresas chinesas locais, isto aumentou a competitividade baseando a concorrência em inovação tecnológica. Elevando ainda mais a níveis cada vez mais altos as capacidades tecnológicas das montadoras de origem chinesa. Se estas se elevam concomitantemente também acompanha as capacidades produtivas, fazendo com que as chinesas se internacionalizasse para mercados antes ocupados por as maiores empresas do setor e ganhasse mercado dessas firmas, resultando em uma liderança inquestionável no setor desde 2009 onde passou os Estados Unidos em produção de automóveis, e também o mesmo desempenho competitivo se mostrou em vendas. 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https://openalex.org/W4384930835	http://journals.pan.pl/Content/124245/PDF-MASTER/aoa.2022.142009.pdf	English	null	About Unusual Diffraction and Thermal Self-Action of Magnetosonic Beam	Archives of Acoustics	2,023	cc-by-sa	5,883	About Unusual Diﬀraction and Thermal Self-Action of Magnetosonic Beam Anna PERELOMOVA Gdansk University of Technology, Faculty of Applied Physics and Mathematics Gdansk, Poland; e-mail: anna.perelomova@pg.edu.pl (received September 7, 2021; accepted April 11, 2022) The dynamics of slightly diverging two-dimensional beams whose direction forms a constant angle θ with the equilibrium straight magnetic strength is considered. The approximate dispersion relations and corresponding links which specify hydrodynamic perturbations in conﬁned beams are derived. The study is dedicated to the diﬀraction of a magnetosonic beam and nonlinear thermal self-action of a beam in a thermoconducting gaseous plasma. It is shown that the divergence of a beam and its thermal self-action is unusual in some particular cases of parallel propagation (θ = 0) and has no analogues in the dynamics of the Newtonian beams. The nonlinear attenuation of Newtonian beams leads to their defocusing in gases, whereas the unusual cases correspond to the focusing in a presence of magnetic ﬁeld. The examples of numerical calculations of thermal self-action of magnetoacoustic beams with shock fronts are considered in the usual and unusual cases of diﬀraction concerning stationary and non-stationary self-action. It is discovered that the diﬀraction is more (θ = 0) or less (θ = π/2) manifested as compared to that of the Newtonian beams. The beams which propagate oblique to the magnetic ﬁeld do not reveal diﬀraction. The special case, when the sound and Alfvénic speeds are equal, is discussed. This magnetosonic beams incorporate acoustic and Alfvénic properties and do not undergo diﬀraction in this particular case. Keywords: non-linear magnetoacoustics; diﬀraction of beams; acoustic thermal self-action; magnetohydrody- namics. ywords: non-linear magnetoacoustics; diﬀraction of beams; acoustic thermal self-action; magnetohydrody Copyright © 2022 A. Perelomova This is an open-access article distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 Internationa (CC BY-SA 4.0 https://creativecommons.org/licenses/by-sa/4.0/) which permits use, distribution, and reproduction in an medium, provided that the article is properly cited, the use is non-commercial, and no modiﬁcations or adaptations are made Archives of Acoustics Archives of Acoustics Vol. 47, No. 3, pp. 355–362 (2022), doi: 10.24425/aoa.2022.142009 Research Paper 2. Equations of MHD ﬂow The dynamics of a gas is governed by the ideal MHD equations. That means that the spatial and tem- poral scales of perturbations in a ﬂow must be much larger than gyro-kinetic scales. The model imposes equal temperature of ions and electrons and makes use of one-ﬂuid perfectly electrically conducting gas. The set of initial partial derivatives’ equations con- sists of the continuity equation, momentum equation, energy balance equation, and electrodynamic equa- tions (Freidberg, 1987; Krall, Trivelpiece, 1973; Callen, 2003): (2) ∂ρ ∂t + ∇⋅(ρv) = 0, ρ(∂v ∂t + (v ⋅∇)v) = −∇p + 1 µ0 (∇× B) × B, (1) ∂p ∂t + (v ⋅∇)p + γp(∇⋅v) = 0, (1) ∂p ∂t + (v ⋅∇)p + γp(∇⋅v) = 0, ∂p ∂t + (v ⋅∇)p + γp(∇⋅v) = 0, where ̃ N = (N1,...,N8)T is a vector which consists of quadratically nonlinear terms. Equations (2) contain the terms of order M 1 (the left-hand side), M 2 (the right-hand side) in the series expansion in a small pa- rameter, the magnetosonic Mach number M (M equals the ratio of characteristic magnitude of velocity to the speed of magnetosonic perturbations). The dispersion relations specify all kinds of wave and non-wave motion of small magnitudes in a plasma, that is, in the case of insigniﬁcant nonlinearity. They follow from the linea- rised version of Eqs (2), if one assumes that all per- turbations are proportional to exp(iωt −ikxx −ikzz) (k = (kx,0,kz) is the wave vector). They are in fact roots of the dispersion equation: ∂B ∂t = ∇× (v × B), ∇⋅B = 0, where p, ρ, v, B, are hydrodynamic pressure and mass density of a plasma, its velocity, the magnetic ﬁeld, µ0 is the magnetic permeability of a free space, and γ denotes the ratio of speciﬁc isobaric heat capacity and speciﬁc isochoric heat capacity, γ = CP /CV . The third equation in fact incorporates the continuity and the energy balance equation and relies on the internal energy for an ideal gas, p ρ(γ−1). The fourth equation in the set is the ideal induction equation, and the ﬁfth one ensures the solenoidal character of B (the Maxwell equation). Many examples of plasmas such as Solar at- mosphere, Earth’s magnetosphere, neutron star mag- netospheres are described reasonably well by the MHD system of equations. 1. Introduction axis of a beam’s propagation because they strengthen proportionally to the squared magnitude. In particu- lar, the nonlinear transfer of energy into the entropy mode is stronger in the par-axial area. That leads to the larger heating in the par-axial area and forma- tion of thermal lenses. In turn, thermal lenses have impact on the propagation of a beam since the local sound speed depends on the equilibrium temperature. The MHD (magnetohydrodynamic) beams and the re- lated nonlinear phenomena reveal a wide variety of be- haviour in view of strong dependence on the direction of the magnetic ﬁeld, diversity of wave modes and equi- librium parameters of a plasma. Particularly, the pa- rameter of nonlinearity and damping coeﬃcient due to thermal conduction are variable (Chin et al., 2010; Nakariakov et al., 2000). The divergence is inherent to the ﬂows exceed- ing one dimension. Two- and three-dimensional beams with initially planar fronts propagate usually with in- creasing of their characteristic width. Thus, a beam’s energy and impulse spread over larger cross-sections. Diﬀraction is more pronounced in the cases when a ra- tio of the transducer characteristic length to the wave length is small. Hence, it is of great importance in medical and technical applications, where accurate evaluation of the actual focus and excess tempera- ture in the focal zone is the key issue (Yufeng, 2015; Duck, 2002). Position of the actual focus diﬀers from the geometric one due to diﬀraction and nonlinear ef- fects following a beam. The nonlinear shift of the focus has been explained and described for the ﬁrst time in (Rudenko, Sapozhnikov, 2004). The nonlinear phe- nomena are also associated with inhomogeneous distri- bution of magnitude of wave perturbations across the This study considers diﬀraction of a magnetosonic beam in dependence of orientation of the axis of prop- agation and the equilibrium parameters of a plasma. It turns out that the obliquely propagating beam al- Archives of Acoustics – Volume 47, Number 3, 2022 356 turbations are superscripted by ′. The constant equi- librium magnetic ﬁeld is aligned along axis z, so that B0 = (0,0,B0). The bulk ﬂow is absent, v0 = 0. The MHD equations may be written in the following form (McLaughlin et al., 2011): most does not diverge. As for the case of parallel pro- pagation, the diﬀraction term may take an unusual sign. 1. Introduction These features probably have no analogues in the wave theory of conﬁned beams. As for the perpendi- cular propagation, it occurs commonly but with weaker divergence compared to that in the Newtonian beams. ∂ρ′ ∂t + ρ0 (∂vx ∂x + ∂vz ∂z ) = N1, ∂vx ∂t + 1 ρ0 ∂p ∂x −B0 ρ0µ0 (∂Bx ∂z −∂Bz ∂x ) = N2, ∂vy ∂t −B0 ρ0µ0 ∂By ∂z = N3, ∂vz ∂t + 1 ρ0 ∂p′ ∂z = N4, ∂p′ ∂t + γp0 (∂vx ∂x + ∂vz ∂z ) = N5, ∂Bx ∂t −B0 ∂vx ∂z = N6, ∂By ∂t −B0 ∂vy ∂z = N7, ∂Bz ∂t + B0 ∂vx ∂x = N8, (2) 2. Equations of MHD ﬂow ∂2 ∂t2 (∂2ϕ ∂t2 −C2 A∆ϕ) −c2 0∆(∂2ϕ ∂t2 −C2 A ∂2ϕ ∂z2 ) = 0, (4) where where ∆= ( ∂2 ∂x2 + ∂2 ∂z2 ). 3. Quasi-planar dynamics Let us consider weak diﬀraction of small magnitude perturbations in a beam which is reasonably directed along axis x1 and introduce the small parameter m responsible for its divergence. Figure 1 explains the geometry of a ﬂow. This is the case of θ = 0. The leading order disper- sion relations for the magnetosonic modes are shown in Table 1. Table 1. Leading order dispersion relations. c0 ≠CA ±(CAkz + C3 Ak2 x 2(C2 A−c2 0)kz ) ±(c0kz + c3 0k2 x 2(c2 0−C2 A)kz ) c0 = CA ±(c0kz −c0kx 2 + 3c0k2 x 8kz ) ±(c0kz + c0kx 2 + 3c0k2 x 8kz ) Table 1. Leading order dispersion relations. c0 ≠CA ±(CAkz + C3 Ak2 x 2(C2 A−c2 0)kz ) ±(c0kz + c3 0k2 x 2(c2 0−C2 A)kz ) c0 = CA ±(c0kz −c0kx 2 + 3c0k2 x 8kz ) ±(c0kz + c0kx 2 + 3c0k2 x 8kz ) Table 1. Leading order dispersion relations. θ B0 x1 x2 Direction of propagation z x k k⟂ k Fig. 1. Geometry of a ﬂow. θ B0 x1 x2 Direction of propagation z x k k⟂ k Fig. 1. Geometry of a ﬂow. The modes diﬀer by the relations between speciﬁc perturbations. These relations unambiguously deter- mine modes along with the dispersion relations. For all these modes, vy = 0, B′ y = 0. Fig. 1. Geometry of a ﬂow. The component of the wave vector k parallel to the direction of propagation is k∣∣(it is of order 1), and the perpendicular component is k⊥(it is of order √m), so as: 3.1.1. Case c0 ≠CA 2. Equations of MHD ﬂow The exceptions are the problems which relate to kinetic eﬀects, magnetic reconnection, some laboratory plasmas, weakly ionised plasmas, cos- mic rays, molecular clouds, and some other cases. The equation of state for an ideal gas is valid besides ap- plications dealing with very cool and dense plasmas. ω2(ω2 −C2 A cos2(θ)k2 z)(c2 0(k2 x + k2 z)(C2 Ak2 z −ω2) +ω2(ω2 −C2 A(k2 x + k2 z))) = 0, (3) where c0 = √γp0 ρ0 = √ (γ −1)CP T0 ω2(ω2 −C2 A cos2(θ)k2 z)(c2 0(k2 x + k2 z)(C2 Ak2 z −ω2) +ω2(ω2 −C2 A(k2 x + k2 z))) = 0, (3) where c0 = √γp0 ρ0 = √ (γ −1)CP T0 denotes the acoustic speed in unmagnetised gas in equilibrium, and CA = B0 √ρ0µ0 The two-dimensional geometry of a ﬂow is con- sidered following Botha et al. (2000), McLaughlin et al. (2011). This assumes dependence of all perturba- tions on x and z. All equilibrium quantities are treated as constants and subscripted by 0, and the MHD per- is the Alfvén speed. Among roots of Eq. (3), there are two Alfvén (non-acoustic) modes of diﬀerent directions 357 A. Perelomova – About Unusual Diﬀraction and Thermal Self-Action of Magnetosonic Beam of propagation (ω2 −C2 A cos2(θ)k2 z = 0) and two sta- tionary modes (ω2 = 0). Four roots of Eq. (3) describe the magnetosonic modes, fast and slow, of diﬀerent di- rection of propagation. The magnetosonic beams are of interest in this study. The dynamic equation for any non-zero wave perturbation ϕ(x,z,t) follows from Eq. (3): where a∗= c8 0 + c4 0C4 A + C8 A −2c2 0C2 A(c4 0 + C4 A)cos(2θ) + c4 0C4 A cos(4θ). The case of the parallel propagation (θ = 0, k∣∣= kz, k⊥= kx) imposes two roots of Eq. (6), C = CA and C = c0. If CA = c0, the roots of dispersion relation (3) are degenerate and the denominator C4 −c2 0C2 A equals zero. This case should be considered individually. We start with the cases of wave vector parallel or perpen- dicular to the equilibrium magnetic ﬁeld. These cases impose zero G. ∂2 ∂t2 (∂2ϕ ∂t2 −C2 A∆ϕ) −c2 0∆(∂2ϕ ∂t2 −C2 A ∂2ϕ ∂z2 ) = 0, (4) where ∆= ( ∂2 ∂x2 + ∂2 ∂z2 ). 3.1.2. Case c0 = CA The links of perturbations inherent to the disper- sion relation ω = ±(c0kz −c0kx 2 + 3c0k2 x 8kz ) (this is the case CA = c0), take the forms: where where ε∣∣= γ + 1 2 is the parameter of nonlinearity, is the parameter of nonlinearity, vx = ±(−c0ρ′ ρ0 + c0 2ρ0 ∫dz ∂ρ′ ∂x + c0 4ρ0 ∫dz∫dz ∂2ρ′ ∂x2 ), vz = ±(c0ρ′ ρ0 + c0 2ρ0 ∫dz ∂ρ′ ∂x −c0 8ρ0 ∫dz∫dz ∂2ρ′ ∂x2 ), p′ = c2 0ρ′, (11) B′ x = B0 ρ0 ρ′ −5B0 8ρ0 ∫dz∫dz ∂2ρ′ ∂x2 , B′ z = −B0 ρ0 ∫dz ∂ρ′ ∂x . vx = ±(−c0ρ′ ρ0 + c0 2ρ0 ∫dz ∂ρ′ ∂x + c0 4ρ0 ∫dz∫dz ∂2ρ′ ∂x2 ), vz = ±(c0ρ′ ρ0 + c0 2ρ0 ∫dz ∂ρ′ ∂x −c0 8ρ0 ∫dz∫dz ∂2ρ′ ∂x2 ), ′ 2 ′ (11) D2 ∣∣,0 = c2 0 ∣c2 0 −C2 A∣= βγ ∣βγ −2∣ is the squared parameter responsible for diﬀraction, and 2 is the squared parameter responsible for diﬀraction, and 2 (11) β = 2 γ c2 0 C2 A B′ x = B0 ρ0 ρ′ −5B0 8ρ0 ∫dz∫dz ∂2ρ′ ∂x2 , is the plasma-β which reﬂects the ratio of hydrostatic and magnetic pressures. Equation (8) recalls the fa- mous Khokhlov-Zabolotskaya (KZ) equation for an acous- tic pressure in a slightly diverging beam in an ideal gas (Kuznetsov, 1971): is the plasma-β which reﬂects the ratio of hydrostatic and magnetic pressures. Equation (8) recalls the fa- mous Khokhlov-Zabolotskaya (KZ) equation for an acous- tic pressure in a slightly diverging beam in an ideal gas (Kuznetsov, 1971): B′ z = −B0 ρ0 ∫dz ∂ρ′ ∂x . The case c0 = CA is especial, it is not a limit of the general case and represents the type of motion com- bining Alfvén and acoustic properties. In particular, vx and vz are of the same order. Operating on the variables τ = t −z/c0, mz, √mx, one concludes that there are no physically meaningful perturbations in this form. Any perturbation of the form ϕ(τ,mz,mx) is a solution to Eq. (4). The conclusion is that there is no leading order diﬀraction in this case and the dy- namic equation for the vx is as follows: ∂ ∂τ (∂p′ ∂z − ε∣∣ ρ0c3 0 p′ ∂p′ ∂τ ) = c0 2 ∂2p′ ∂x2 . 3.1.1. Case c0 ≠CA This case is purely 358 Archives of Acoustics – Volume 47, Number 3, 2022 acoustic in the absence of the magnetic ﬁeld with the non-zero leading order perturbations as follows: transform to the only link recalling that for the Alfvén modes (B′ y = ∓B0 CA vy): B′ x = ∓B0 CA vx. p′ = c2 0ρ′ = c0ρ0vz. The relations of perturbations specify modes uniquely. The relations of perturbations specify modes uniquely. They reveal the diﬀerence between modes and may in- dicate the type of a motion on a pair of the disper- sion relations. To be speciﬁc, a wave propagating in the positive direction of axis z is considered. Any non- zero perturbation inherent to this mode is supposed to be a function of the retarded time τ = t −z/c0, mz, √mx. This choice of slow scale suggests that the spa- tial variations occur more slowly along the axis z of a beam than across the beam from the point of view of an observer who moves at the speed c0 along the axis of a beam (Rudenko, Soluyan, 1977; Hamil- ton, Blackstock, 1988). Assuming the Mach num- ber M and m of the same order, taking into account nonlinear terms N1,...,N8, discarding terms of the high order in smallness and transforming equation from the slow scale back to x, z yields an equation governing the magnetosonic pressure: It has a property which diﬀerentiates it from the Alfvén modes. Namely, this mode reveals divergence while the Alfvén mode refers to exact dispersion relations ω = ±CAkz and behaves like a planar wave. It has a property which diﬀerentiates it from the Alfvén modes. Namely, this mode reveals divergence while the Alfvén mode refers to exact dispersion relations ω = ±CAkz and behaves like a planar wave. The non-acoustic wave may be extracted from Eq. (4) by means of making use of the set of variables τ = t −z/CA, mz, √mx. One arrives at the dynamic equation: ∂ ∂τ (∂vx ∂z − 3 2C2 A vx ∂vx ∂τ ) = ⎧⎪⎪⎪⎪⎪⎪⎪⎨⎪⎪⎪⎪⎪⎪⎪⎩ D2 ∣∣,ACA 2 ∂2vx ∂x2 , c0 < CA, − D2 ∣∣,ACA 2 ∂2p′ ∂x2 , c0 > CA, (10) where (10) where D2 ∣∣,A = C2 A ∣c2 0 −C2 A∣= 2 ∣βγ −2∣. 3.1.1. Case c0 ≠CA In particular, the links inherent to the dispersion relations for acoustic beams, ω = ±(c0kz + c3 0k2 x 2(c2 0 −C2 A)kz ), kz = k∣∣cos(θ) −k⊥sin(θ), kx = k∣∣sin(θ) + k⊥cos(θ). All subsequent formulas are leading order; they include the terms up to m1 in the power series. By expanding Eq. (3) in the series and collecting the leading order terms, one arrives at the dispersion relation: All subsequent formulas are leading order; they include the terms up to m1 in the power series. By expanding Eq. (3) in the series and collecting the leading order terms, one arrives at the dispersion relation: take the form: vx = ± c3 0 (c2 0 −C2 A)ρ0 ∫dz ∂ρ′ ∂x , vz = ±(c0ρ′ ρ0 − c3 0 2(c2 0 −C2 A)ρ0 ∫dz∫dz ∂2ρ′ ∂x2 ), p′ = c2 0ρ′, B′ x = − c2 0B0 (c2 0 −C2 A)ρ0 ∫dz ∂ρ′ ∂x , B′ z = c2 0B0 (c2 0 −C2 A)ρ0 ∫dz∫dz ∂2ρ′ ∂x2 . (7) vx = ± c3 0 (c2 0 −C2 A)ρ0 ∫dz ∂ρ′ ∂x , vz = ±(c0ρ′ ρ0 − c3 0 2(c2 0 −C2 A)ρ0 ∫dz∫dz ∂2ρ′ ∂x2 ), ω = ±(Ck∣∣+ Gk⊥+ D2C 2 k2 ⊥), (5) (5) where C is the positive root of the equation: p′ = c2 0ρ′, (7) C4 −C2(c2 0 + C2 A) + c2 0C2 A cos2(θ) = 0. (6) (6) B′ x = − c2 0B0 (c2 0 −C2 A)ρ0 ∫dz ∂ρ′ ∂x , B′ z = c2 0B0 (c2 0 −C2 A)ρ0 ∫dz∫dz ∂2ρ′ ∂x2 . C is a speed of propagation of both fast or slow modes, and G = c2 0C2 AC cos(θ)sin(θ) C4 −c2 0C2 A cos2(θ) , D2 = 2(c2 0 + C2 A+ a∗ (C4−c2 0C2 A cos2(θ))3 C6)− C2c4 0C4 A sin2(2θ) (C4−c2 0C2 A cos2(θ))2 4C2k∣∣ , G = c2 0C2 AC cos(θ)sin(θ) C4 −c2 0C2 A cos2(θ) , C c0CA cos (θ) D2 = 2(c2 0 + C2 A+ a∗ (C4−c2 0C2 A cos2(θ))3 C6)− C2c4 0C4 A sin2(2θ) (C4−c2 0C2 A cos2(θ))2 4C2k∣∣ , The terms including partial derivative with respect to x are of order √m, and these including the second order derivative are of order m1. 3.1.1. Case c0 ≠CA Hence, the divergence may be anomalous in depen- dence on the ratio of CA and c0 and the kind of a wave mode. This relates to the signs minus in the right-hand sides of Eqs (8) and (10) which reﬂect diﬀraction. ∂ ∂τ (∂p′ ∂z − ε∣∣ ρ0c3 0 p′ ∂p′ ∂τ )= ⎧⎪⎪⎪⎪⎪⎪⎪⎨⎪⎪⎪⎪⎪⎪⎪⎩ D2 ∣∣,0c0 2 ∂2p′ ∂x2 , c0 > CA, − D2 ∣∣,0c0 2 ∂2p′ ∂x2 , c0 < CA, (8) (8) 3.1.2. Case c0 = CA 3.2. Perpendicular wave vector and the equilibrium magnetic ﬁeld which equals 1 2T0 (T0 designates the equilibrium tem- perature). Some damping mechanism along with non- linearity is a necessary condition for nonlinear transfer of wave energy into the non-wave entropy mode. We consider exclusively thermal conduction χ among these mechanisms. The system of equations which describes nonlinear thermal self-action of a beam, is as follows: In the case θ = π/2, k∣∣= kx, k⊥= kz, one arrives at the leading order dispersion relations: ω = ±(C⊥kx + (C4 ⊥−c2 0C2 A)k2 z 2C3⊥kx ), C⊥= √ c2 0 + C2 A. (13) (13) C⊥= √ c2 0 + C2 A. ∂ ∂τ (∂p′ ∂z −δT ′ c0 ∂p′ ∂τ −γ + 1 2ρ0c3 0 p′ ∂p′ ∂τ −(γ −1)χ 2CP c3 0ρ0 ∂2p′ ∂τ 2 ) = ± D2 ∣∣,0c0 2 ∂2p′ ∂x2 , (15) ∂T ′ ∂t − χ ρ0CP ∂2T ′ ∂x2 = c0 Cp Fms, (16) A perturbation in pressure in the mode propagating in the positive direction of axis x is described by the dynamic equation (we seek perturbations as functions of τ = t −x/C⊥, mx, √mz and account for nonlinear distortion of a wave): A perturbation in pressure in the mode propagating in the positive direction of axis x is described by the dynamic equation (we seek perturbations as functions of τ = t −x/C⊥, mx, √mz and account for nonlinear distortion of a wave): (15) ∂T ′ ∂t − χ ρ0CP ∂2T ′ ∂x2 = c0 Cp Fms, (16) (16) ∂ ∂τ (∂p′ ∂x − ε⊥ ρ0C3⊥ p′ ∂p′ ∂τ ) = D2 ⊥C⊥ 2 ∂2p′ ∂z2 , (14) where Fms designates the averaged over period mag- netosonic force: (14) Fms = (γ −1)χ CP c5 0ρ3 0 ⟨(∂p′ ∂τ ) 2 ⟩. (17) where where (17) D2 ⊥= c4 0 + C4 A + c2 0C2 A (c4 0 + C4 A)2 = 1 − 2βγ (βγ + 2)2 . T ′ denotes perturbation of temperature which in the case of periodic excitation satisﬁes the inhomogeneous diﬀusion Eq. (16). The acoustic force which is valid for all kinds of exciters has been derived by (Leble, Perelomova, 2018). Equation (15) resembles Eq. (1) (Rudenko, Sapozhnikov, 2004) but diﬀers by the right-hand part which may be negative and includes the scaling coeﬃcient D2 ∣∣,0. 3.2. Perpendicular wave vector and the equilibrium magnetic ﬁeld The parameter of nonlinearity ε⊥depends on the equi- librium parameters of a plasma (Chin et al., 2010; Nakariakov et al., 2000): ε⊥= 3C2 A + (γ + 1)c2 0 2C2⊥ = 9 + 4γβ 8 + 4γβ ε∣∣. ∣∣,0 In the cases when nonlinear eﬀects dominate over diﬀraction, the “fast time” may be eliminated in the frames of geometric acoustics. The subsequent analysis which shows the pecularities of anomalous divergence will be done for the example of cylindrically symmetric beam, since the theory is well developed in this partic- ular case (Rudenko, Sapozhnikov, 2004). Introduc- ing new variables R = r D∣∣,0 and the eikonal ψ(x,r) (r is the transversal to the direction of a beam coordinate) and substituting: Equation (14) may be rearranged into the KZ equa- tion by substitution X = x D⊥. Since 3 4 < D2 ⊥< 1, the divergence is usual but less manifested in the case of a plasma aﬀected by a magnetic ﬁeld. All conclusions concerning linear and nonlinear dynamics of perturba- tions in an acoustic beam can be generalised in the case of a plasma aﬀected by a magnetic ﬁeld in view of the transversal scaling. Equation (14) may be rearranged into the KZ equa- tion by substitution X = x D⊥. Since 3 4 < D2 ⊥< 1, the divergence is usual but less manifested in the case of a plasma aﬀected by a magnetic ﬁeld. All conclusions concerning linear and nonlinear dynamics of perturba- tions in an acoustic beam can be generalised in the case of a plasma aﬀected by a magnetic ﬁeld in view of the transversal scaling. 3.1.2. Case c0 = CA (9) (9) Equation in the upper row of set (8) may be rearranged into the KZ equation by the substitution X = x D∣∣,0 . Since D∣∣,0 > 1, the divergence is more pronounced in the case of a plasma aﬀected by a magnetic ﬁeld. In the planar case ( ∂ ∂x ≡0), the links ω = ±(CAkz + C3 Ak2 x 2(C2 A −c2 0)kz ) ∂vx ∂z −γ 2c2 0 vx ∂vx ∂τ = 0. (12) (12) 359 A. Perelomova – About Unusual Diﬀraction and Thermal Self-Action of Magnetosonic Beam It describes a perturbation in a planar wave and has unusual parameter of nonlinearity diﬀerent from that in the pure acoustic case. thermal self-defocusing speciﬁc for beams in the gases due to the positive thermal coeﬃcient: δ = 1 c0 ∂c0 ∂T ∣ p , 3.2. Perpendicular wave vector and the equilibrium magnetic ﬁeld 4.1. Non-stationary self-focusing In the frames of geometrical acoustics, one sets the right-hand side of Eq. (18) zero and arrives at the sys- tem: In the case of small thermal conductivity, when the diﬀusion term in Eq. (16) may be neglected, a variation in temperature takes the form: ∂p′ ∂z −γ + 1 2ρ0c3 0 p′ ∂p′ ∂θ −(γ −1)χ 2CP c3 0ρ0 ∂2p′ ∂θ2 ± ∂p′ ∂R ∂ψ ∂R ±p′ ∂2ψ ∂R2 = 0, (19) 2 (19) ∂T ′ ∂t = (γ + 1)ω 3πc4 0ρ2 0CP A3. ∂ψ ∂z ± 1 2 ( ∂ψ ∂R) 2 + δT ′ = 0. The function f follows from Eqs (22) and (23): The upper sign in the previous and next formulas de- signates the normal diﬀraction, and the lower sign con- cerns the unusual case. Equation (19) describes among other formation and propagation of a sawtooth wave with a ﬁnite shock front with the magnitude A and the frequency ω, so as: f 5 ⎛ ⎜ ⎝ 1 + Z ∫ 0 dy f(y,Θ) ⎞ ⎟ ⎠ 4 ∂ ∂Θ ( 1 f ∂2f ∂Z2 ) = ±1, (24) (24) where the following dimensionless quantities are intro- duced: p′(z,R,θ) = A(z,R) ⋅(−ωθ π + tanh((γ + 1)CP 2(γ −1)χ A(z,R)θ)), Z = z zs , Θ = t t0 , t0 = (γ + 1)ωρ0CP a2 0T0 4πc2 0P0 . −π ω ≤θ ≤π ω . (20) −π ω ≤θ ≤π ω . (20) The boundary and initial conditions for initially non- focused beam are as follows: The boundary and initial conditions for initially non- focused beam are as follows: Substituting Eq. (20) into (19) and (17) and letting χ →0 (this makes the wave saw-tooth shaped) results in equations: f(Z = 0,Θ) = f(Z,Θ = 0) = 1, ∂f ∂Z (Z = 0,Θ) = 0. ∂A ∂z + A2 P0zs ± ∂A ∂R ∂ψ ∂R ± A ∂2ψ ∂R2 = 0, Equation (24) recalls the dynamic equation (Eq. (22) in Rudenko, Sapozhnikov, 2004) but has a diﬀerent meaning. The sign plus corresponds to the normal de- focusing in gases, and the sign minus corresponds to anomalous divergence. Hence, the unusual divergence corresponds to the focusing which speciﬁes majority of liquids (apart from water). (21) ∂R2 Fms = (γ + 1)ω 3πc5 0ρ3 0 A3, (21) where where zs = 2πc3 0ρ0 ωP0 4. Thermal self-action of sound beams p′ = (x,r,θ = τ −ψ(x,r)/c0), The magnitude of a magnetosonic pressure is larger in the vicinity of a beam’s axis than on the perip- hery. That leads to more eﬀective nonlinear excitation of the entropy mode in this area, that is, to magne- toacoustic heating. Enlargement in temperature en- tails a change in the local speed of sound propagation. Some kind of a thermal lense forms in the vicinity of axis of a beam. In turn, these uneven variations dis- tort the wave front. In order to study unusual features of perturbations which associate with the anomalous divergence, we focus on the parallel propagation with the speed c0. Usually, a magnetosonic beam undergoes one arrives at the limit of short wavelengths small in comparison with the scale of thermal inhomogeneties to the equation: ∂ ∂θ (∂p′ ∂z −γ + 1 2ρ0c3 0 p′ ∂p′ ∂θ −(γ −1)χ 2CP c3 0ρ0 ∂2p′ ∂θ2 ± ∂p′ ∂R ∂ψ ∂R ± p′ ∂2ψ ∂R2 −1 c0 ∂p′ ∂θ (∂ψ ∂z ± 1 2 ( ∂ψ ∂R) 2 + δT ′)) = ±c0 ∂2p′ ∂R2 . (18) ∂ ∂θ (∂p′ ∂z −γ + 1 2ρ0c3 0 p′ ∂p′ ∂θ −(γ −1)χ 2CP c3 0ρ0 ∂2p′ ∂θ2 ± ∂p′ ∂R ∂ψ ∂R ± p′ ∂2ψ ∂R2 −1 c0 ∂p′ ∂θ (∂ψ ∂z ± 1 2 ( ∂ψ ∂R) 2 + δT ′)) = ±c0 ∂2p′ ∂R2 . (18) −1 c0 ∂p′ ∂θ (∂ψ ∂z ± 1 2 ( ∂ψ ∂R) 2 + δT ′)) = ±c0 ∂2p′ ∂R2 . (18) (18) 360 Archives of Acoustics – Volume 47, Number 3, 2022 4.2. Stationary self-focusing is the shock formation distance, and P0 is the ini- tial magnitude of magnetosonic pressure at the axis of a beam. Equation (21) may be resolved by assuming the parabolic wave form (where f(z,t) is some func- tion): is the shock formation distance, and P0 is the ini- tial magnitude of magnetosonic pressure at the axis of a beam. Equation (21) may be resolved by assuming the parabolic wave form (where f(z,t) is some func- tion): We consider stationary temperature ﬁeld in Eq. (16), so that ∂T ′ ∂t = 0. An equation for the unknown function f takes the form: ⎛ ⎜ ⎝ 1 + Π Z ∫ 0 dy f(y) ⎞ ⎟ ⎠ 3 f 2 d2f dZ2 = ±Π3, (25) ψ(z,R,t) = φ(z,t) ± R2 2 ∂ ∂z lnf(z,t) (25) with the solution: with the solution: A = P0 f(z,t)Φ( R a0f(z,t)) ⋅⎛ ⎝1 + 1 zs Φ( R a0f(z,t)) z ∫ 0 dy f(y,t) ⎞ ⎠, where where z0 = π2c5 0ρ0 3T0χ(γ + 1)2ω2 , Z = z z0 , Π = z0 zs . The boundary conditions for an initially unfocused beam are: where a0 is the initial beam radius, and Φ is responsible for the pressure transversal distribution, A(z = 0,R) = P0Φ(R/a0). The eikonal equation takes the form: f(Z = 0) = 1, df dZ (Z = 0) = 0. ± ∂2f ∂z2 = δT2f, (22) (22) Hence, the case of anomalous divergence may be con- sidered as usual with the normal divergence but with the negative coeﬃcient, −δ (Eq. (20) in (Rudenko, Sapozhnikov, 2004)). In this case, unusual focusing of a beam in a gaseous plasma occurs. This corresponds where T2 is the coeﬃcient in the transversal expansion: where T2 is the coeﬃcient in the transversal expansion: T ′ = T0 −R2 2 T2(z,t). (23) (23) (23) A. Perelomova – About Unusual Diﬀraction and Thermal Self-Action of Magnetosonic Beam 361 to the sign minus on the right of Eq. (25). For a Gaus- sian at Z = 0 beam, Φ(ξ) = exp(−ξ2), and the charac- teristic width of a beam is deﬁned as the transversal distance at which the peak pressure is less e times its value at the axis: counterparts in the wave theory. The links of pertur- bations which specify every mode on par with disper- sion relations in the cases of parallel or perpendicular propagation are derived. 4.2. Stationary self-focusing They depend on the equilib- rium parameters of a plasma. In general, these links include integral operators and may be used as indica- tors of individual kinds of plasma motion. The magne- tosonic modes are p-modes, that is, a perturbation in pressure is not zero for these modes. It is discovered that the case C = c0 = CA is very special. The links of perturbations combine properties of acoustic and magnetic modes, Eqs (11). A beam does not undergo diﬀraction and behaves as a planar wave with especial parameter of nonlinearity γ/2. a a0 = f ¿ Á Á Á Á Àlog ⎛ ⎜ ⎝ e + (e −1) Z ∫ 0 dy f(y) ⎞ ⎟ ⎠ . Figure 2 shows the characteristic width of a beam in the case of stationary and non-stationary thermal self- action in the normal (defocusing) and unusual cases (focusing). Numerical calculations of Eqs (24) and (25) with the appropriate initial and boundary conditions have been undertaken in Mathematica. The nonlinear thermal self-action is also adjusted by transversal scaling. One may suppose that the ther- mal self-action may occur unusually if a beam diverges unusually. It turns out that the anomalous diﬀraction operates in such a way that it changes the sign of ther- mal coeﬃcient δ. This corresponds to the thermal fo- cusing of a beam instead of defocusing which normally takes place in all gases, that is, to the negative ther- mal coeﬃcient δ. The stationary and non-stationary self-action is considered. The only damping mechanism which is taken into account, is the thermal conduction of a plasma. The thermal self-action leads to forma- tion of thermal lenses with the exception of the case C = c0 = CA when it leads to formation of a heated ﬂat layer. In this case, diﬀraction is not of importance in concerning to a beam itself and to the thermal eﬀects in its ﬁeld. The non-acoustic beams propagate with the speed CA, which also depends on the temperature. But it is not a leading order coupling of these modes with the entropy mode. Hence, this is not a case of thermal self-action. a) Z Π Π b) θ θ θ θ Z Fig. 2. Dimensionless width of a beam which is planar at a transducer: a) stationary self-action, b) non-stationary self-action. a) Z Π Π b) Fig. 2. 4.2. Stationary self-focusing Dimensionless width of a beam which is planar at a transducer: a) stationary self-action, b) non-stationary self-action. References 1. Botha G.J.J., Arber T.D., Nakariakov V.M., Keenan F.P. (2000), A developed stage of Alfvén wave phase mixing, Astronomy and Astrophysics, 363(3): 1186–1194. 5. Concluding remarks The unusual diﬀraction behaviour of magnetohy- drodynamic beams in a plasma is discovered. The beams oblique to the magnetic ﬁeld do not reveal diﬀraction. The beams directed along the magnetic ﬁeld (this is the case θ = 0) may behave unusually in dependence to the ratio of the sound speed c0 and the speed of Alfvénic mode CA. In particular, the un- usual diﬀraction (that means the minus sign by the diﬀraction term) speciﬁes a sound beam which propa- gates with the speed c0 if CA > c0. 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https://openalex.org/W2233858256	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0146364&type=printable	English	null	Spectroscopic Evidence of the Improvement of Reactive Iron Mineral Content in Red Soil by Long-Term Application of Swine Manure	PloS one	2,016	cc-by	8,585	Spectroscopic Evidence of the Improvement of Reactive Iron Mineral Content in Red Soil by Long-Term Application of Swine Manure Chichao Huang1, Sha Liu1, Ruizhi Li1, Fusheng Sun1, Ying Zhou2, Guanghui Yu1 1 National Engineering Research Center for Organic-based Fertilizers, Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Jiangsu Provincial Key Lab for Organic Solid Waste Utilization, Nanjing Agricultural University, Nanjing 210095, PR China, 2 Shanghai Institute of Measurement and Testing Technology, Shanghai 201203, China a1111 * yuguanghui@njau.edu.cn (GY); henan223@163.com (FS) Abstract Mineral elements in soil solutions are thought to be the precursor of the formation of reactive minerals, which play an important role in global carbon (C) cycling. However, information regarding the regulation of mineral elements release in soil is scarce. Here, we examined the long-term (i.e., 23 yrs) effects of fertilisation practices on Fe minerals in a red soil in Southern China. The results from chemical analysis and Fourier-transform infrared spec- troscopy showed that long-term swine manure (M) treatment released greater amounts of minerals into soil solutions than chemical fertilisers (NPK) treatment, and Fe played a domi- nant role in the preservation of dissolved organic C. Furthermore, Fe K-edge X-ray absorp- tion near-edge fine structure spectroscopy demonstrated that reactive Fe minerals were mainly composed of less crystalline ferrihydrite in the M-treated soil and more crystalline goethite in the NPK-treated soil. In conclusion, this study reported spectroscopic evidence of the improvement of reactive Femineral content in the M-treated soil colloids when com- pared to NPK-treated soil colloids. OPEN ACCESS Citation: Huang C, Liu S, Li R, Sun F, Zhou Y, Yu G (2016) Spectroscopic Evidence of the Improvement of Reactive Iron Mineral Content in Red Soil by Long- Term Application of Swine Manure. PLoS ONE 11(1): e0146364. doi:10.1371/journal.pone.0146364 Editor: Gang Yang, Southwest University, CHINA Editor: Gang Yang, Southwest University, CHINA Received: June 3, 2015 Accepted: December 16, 2015 Published: January 11, 2016 Copyright: © 2016 Huang et al. This is an open access article distributed under the terms of the Copyright: © 2016 Huang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. RESEARCH ARTICLE Improvement of Reactive Iron Minerals in Red Soil by Manure Amendment soils. By evaluating soil C accumulation for 3 years across a 7-year chronosequence of three farms converted to management-intensive grazing, Machmuller et al. [5] showed that soil cat- ion exchange capacity linearly increased with soil carbon accumulation within a decade of management-intensive grazing practices. This result suggested mineral elements (i.e., soil cat- ions) could be affected by agricultural practices. Furthermore, Keiluweit et al. [6] indicated that root exudates might affect the formation of reactive minerals. Also, our recent investigations demonstrated that long-term organic fertilisation treatments could increase the concentrations of reactive minerals (i.e., non-crystalline Fe [7] and allophane [8,9]) in red soils in Southern China. However, non-crystalline Fe contributed only a portion of reactive Fe minerals present in the soil, which were usually defined as Fe extracted with oxalate or citrate-bicarbonate- dithionite (CBD) and might not serve to characterise the total amount of reactive Fe minerals. A better understanding of the effect of fertilisation practices on reactive iron oxides is impor- tant for predicting and managing C preservation in soils. no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. X-ray absorption near-edge fine structure (XANES) spectroscopy is an element specific technique that is sensitive to the oxidation state and to the local structure of the absorber ele- ment [10]. Using hard X-rays at the Fe K-edge, this technique provided a powerful tool to not only identify but also quantify the various Fe phases present in soils, which could be very com- plex and might mask magnetically weak phases when examined by Mössbauer spectroscopy or X-ray magnetic circular dichroism [11]. However, the composition of reactive Fe minerals in long-term fertilised soils remains poorly understood. The objectives of this study were 1) to examine the effect of long-term fertlisation regimes on the composition of reactive Fe minerals in these soils, 2) to test which functional groups in soil C were preferentially binding with reactive iron oxides, and 3) to mimic Fe mineral trans- formation with the addition of organic acids. For these purposes, four contrasting fertilisation regimes that each supplied 300 kg N/ha/year (two crops, wheat and corn) were examined dur- ing a long-term (i.e., 23 yrs) fertilisation experiment: i) no fertiliser (Control), ii) inorganic chemical fertilisers of nitrogen, phosphorus and potassium only (NPK or chemical fertilisation hereafter), iii) swine manure only (M), and iv) a combination of swine manure and NPK fertili- sers (MNPK) (M and MNPK are collectively called organic fertilisation hereafter). Introduction Funding: This work was financially jointly supported by National Natural Science Foundation of China (41371248 and 41371299), National Basic Research Program of China (2011CB100503), Natural Science Foundation of Jiangsu Province of China (BK20131321), the Qing Lan Project, the Innovative Research Team Development Plan of the Ministry of China (IRT1256), the 111 Project (B12009), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and Research Project of Shanghai Municipal Bureau of Quality and Technical Supervision (I00RJ1414). The funders had Globally, soil organic matter (SOM) contained greater than three-fold more carbon (C) than the atmosphere and terrestrial vegetation [1]. The biogeochemical cycles of organic C and iron (Fe) were strongly interlinked [2,3]. Lalonde et al. [2] suggested that approximately 21.5% of the organic carbon in soil or sediment was directly bound to reactive Fe minerals. Therefore, reactive Fe minerals could play an important role in the long-term storage of organic C and thus the dynamics of the global C cycle. However, information concerning the mechanisms that regulate reactive Fe minerals in soils is scarce. Recently, a study of two-century land use changes on soil iron crystallinity and accumula- tion [4] suggested that land use changes (agriculture and reforestation) play an important role in transforming the iron crystallinity and its interaction with organic matter decomposition in PLOS ONE \| DOI:10.1371/journal.pone.0146364 January 11, 2016 1 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0146364 January 11, 2016 2.3 Fourier-transform infrared (FTIR) spectroscopy analysis The soil samples and the CBD extracts were collected and freeze-dried for FTIR analysis. FTIR spectra were measured for 1 mg of freeze-dried sample added to 100 mg of potassium bromide (KBr, IR grade) and collected using a Nicolet iS10 FTIR spectrometer (Thermo Nicolet, USA) at 4 cm-1 of resolution after 200 scans over the range from 4,000–400 cm-1. Improvement of Reactive Iron Minerals in Red Soil by Manure Amendment single superphosphate at 53 kg P ha−1, and K as KCl at 100 kg K ha−1 before crop planting. The N content of the manure was 16.7 g kg−1 in dry weight. The ratio of organic N in manure com- pared to that added as inorganic N in fertilizer was 2.3:1. Thirty percent of the total amount of individual fertilizer applied each year was used for wheat and 70% for corn. The experimental plots were completely dependent on precipitation. Fresh soil was thoroughly mixed, air-dried, and sieved through a 5-mm screen for further analysis. The air-dried samples were passed through a 2-mm screen prior to sample storage, passed through a 0.25-mm screen before pH, SOC, and C/N measurements, and passed through a 0.15-mm screen before extraction experiments. Soil colloids were isolated using the following procedure [15]. Briefly, air-dried soil was sus- pended in deionised water at the ratio of 1:5 (w/v), shaken for 8 h at 25°C, and centrifuged for 6 min at 2,500 g. Aliquots of the supernatant suspensions containing the soil colloids were transferred into 50-mL glass vials, stored in the dark at 4°C, and analysed within a few days. 2.2 Chemical analysis DOC was measured using a TOC/TN analyser (multi N/C 3000, Analytik Jena AG, Germany). The main metal ions in the colloids were quantified after digestion. The following procedure was performed: first, the soil colloids were mixed with 10% nitric acid at a ratio of 1:1 (v/v) on a heating plate [16]; then, the mixture was heated to 150°C and held there for 2 hrs. After diges- tion, the mixture was filtered through a filtration membrane (0.45 μm) and stored in a suitable container. The main mineral elements, namely, Fe, Al, Ca, Mg and Si, were quantified by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The extraction of reactive Fe minerals (Fed) was conducted using a 0.1 M citrate-bicarbon- ate-dithionite (CBD) solution [2]. To determine the total Fe (Fet) content in the soils, 0.5 g of powdered soil was weighed into Teflon tubes, mixed with trace metal grade acids (5 ml HNO3 and 10 ml HF), and carefully boiled for 1 hr at 120 ± 3°C. After cooling, 5 ml of HClO4 was added, and the sample was heated to 220 ± 3°C until the sample boiled to dryness; then, 5 ml of HF and 2 ml of HClO4 were added into the Teflon tubes. The sample was digested completely until the residue turned white and grey. Finally, the digest was dissolved with 3 ml of HNO3 (v/ v = 1:1) and increased to a volume of 50 ml in a polypropylene centrifuge tube with deionised water [17,18]. The concentration of Fet was analysed by ICP-AES. 2.1 Sample source and handling Soil samples were collected from a depth of 0–20 cm in September 2013 from the Qiyang Experiment using a 5-cm internal diameter auger. The long-term fertilisation experiment was established on a Ferralic Cambisol soil in September 1990 at the Qiyang Experimental Station of the Chinese Academy of Agricultural Sciences, Hunan, China (26°45’N and 111°52’E, 120 m above sea level) [8, 12]. The mean values for annual temperature, evaporation, frost-free days and sunshine hours were 18°C, 1,470 mm, 300 d and 1,610 h, respectively, and the site received 1,255 mm of mean annual precipitation, approximately 70%–80% of which falls from April to October. No specific field permits were required for this study. The land accessed is not pri- vately owned or protected. No protected species were sampled. A detailed description of the long-term fertilisation experiment site has been provided previously [8,13]. According to the Food and Agriculture Organization of the United Nations (FAO) classification, the red soil is a Ferralic Cambisol [14]. The top soil contains approximately 61.4% clay, 34.9% silt, and 3.7% sand. Each plot was 20 m long and 10 m wide with a 1.0-m deep cement barrier zone between each plot. Each plot was separated into three equal-sized regions, and 10 cores were randomly sampled from each region. We carefully selected four fertilisation treatments for this study: Control, NPK, NPKM, and M. The N fertilizer was provided as urea at 300 kg N ha−1, P as PLOS ONE \| DOI:10.1371/journal.pone.0146364 January 11, 2016 2 / 15 2.5 Fe K-edge XAFS analysis Fe K-edge absorption spectra were collected using an Si (111) double crystal monochromator at the XAFS station of the BL14W1 beamline of the Shanghai Synchrotron Radiation Facility (SSRF). The storage ring was operated at 3.5 GeV with an electron current that decreased from 210 to 150 mA within about 8 hrs. Samples were ground into a fine powder and brushed onto tape that was stacked together to approximately one X-ray-absorption length at the corre- sponding metal edges. The intensities of the incident and the transmitted X-rays were moni- tored in ionisation chambers filled with nitrogen gas. All of the spectra presented were measured at room temperature. Standard samples of ferrihydrite, goethite, lepidocrocite, maghemite, Fe(III) sulphate, Fe(II) sulphate, Fe(III) oxalate, and Fe(II) oxalate were recorded in the transmission mode (S1 Fig), while the prepared samples were measured in the fluores- cence mode. The X-ray energy scale was calibrated to the iron K-edge (7112.0 eV) using an iron metal foil before XANES measurements were performed. The incident X-ray energy was varied from 7090 to 7180 eV in 0.5-eV increments using a monochromator for a 10 s dwell, and a 19 element high-purity Ge detector was used to collect an energy scan near the iron K edge of a given iron-containing particle. The XAFS data were processed and analysed using the ATHENA software (version 2.1.1). 2.4 C 1s NEXAFS spectroscopy The colloidal particles from the soils collected from the four fertilisation treatments were char- acterised by C 1s NEXAFS spectroscopy on BL08U at the Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences. For specimen preparation, one droplet of soil colloid suspension was deposited at a 100-nm thickness onto a Si3N4 window, which was previously glued onto the detection plate of the microscope. The sample thickness is important to obtain a good signal-to-noise ratio when using NEXAFS spec- troscopy [19]. The main 1s-p and Rydberg/mixed valence transitions in the fine structure regions of the C K-edge spectra were recorded in the energy range from 284–310 eV. Back- ground measurements were collected by measuring an empty Au wafer on each sample plate 3 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0146364 January 11, 2016 Improvement of Reactive Iron Minerals in Red Soil by Manure Amendment loaded into the chamber. The normalisation current was also measured during each scan by collecting the TEY from an Au mesh. The mesh was monitored for C contamination and was periodically refreshed using an in situ Au evaporator incorporated into the beamline vacuum system [20]. All the data were normalised prior to curve fitting using the ATHENA software (version 2.1.1) [21]. Peak resonances with specific bonding environments were assigned on the basis of the spectral signatures of pure chemical standards representative of specific functional groups [19]. The details of deconvolution have been described elsewhere [15,20,22,23]. Spectral regions represented with Gaussian curves were described as being generally attributed to the functional groups from G1 to G8, details are given in data in S1 Table. 2.6 Simulation studies on the formation of SRO Fe minerals with the addition of oxalic acid The soil colloids solutions for the M treatment and oxalic acid solution were prepared. Oxalic acid solutions were added to the soil colloid solutions with stirring. The final concentrations of oxalic acid in the soil colloid suspensions were 10 and 100 mg/L, and the pH values were adjusted to 6.7 which were same as that of the raw soil colloids solution. After one day incuba- tion, the suspensions in the series of reaction solutions were lyophilized under -50°C for 2 day and then used for Fe K-edge XANES measurement. 3.1 Characteristics of soils in contrasting fertilisation treatments After 23 yrs of long-term fertilisation, a higher SOC (soil organic carbon) concentration was measured from the NPKM and M treatments (i.e., 12.37 ± 0.08 and 14.76 ± 0.04 g/kg, respec- tively) than the NPK and Control treatments (i.e., 10.62 ± 0.03 and 8.05 ± 0.05 g/kg, respec- tively) (Table 1). Meanwhile, the soil pH values for the NPKM and M treatments were greater than those for the NPK treatment, indicating that organic fertilisers could enhance the buffer- ing capacity of soils while chemical fertilisers accelerate soil acidification. Compared to raw soil (i.e., the unfertilized soil prior to long-term fertilisation treatments), both organic and chemical fertilisations increased soil C pools. However, organic fertilisation improved soil pH while chemical fertilisation decreased soil pH. These SOC and pH results were similar to those reported in our previous publications [7–9], supporting the finding that organic fertilisation could increase soil C pools and enhance soil buffering capacity, while chemical fertilisation decreases soil C pools and accelerates soil acidification. Additionally, total iron (Fet) in soils exhibited an opposite trend to SOC concentration (Table 1), suggesting that total iron was not responsible for the preservation of SOC. To deter- mine whether soluble reactive Fe played an important role in the preservation of SOC, the con- centrations of mineral elements (i.e., Fe, Al, Si, Ca, and Mg) and dissolved organic C (DOC) in soil colloids were measured (Fig 1-a). Compared to raw soil, organic fertilisation increased soil DOC pool while control and chemical fertilisations decreased it. Intriguingly, the highest con- centrations of both mineral elements and DOC were found in the NPKM treatment, followed by the M treatment, with the lowest concentrations observed in the Control and NPK treat- ments. Moreover, there are strong correlations among the mineral elements and DOC (Fig 1-b), demonstrating that all of the tested mineral elements together controlled the preser- vation of DOC. Importantly, the slopes of the relationships between mineral elements and DOC follow the order: Fe (1.17) > Si (0.85) > Al (0.83) >> Ca (0.11) > Mg (0.04), revealing that among the tested mineral elements, the per unit Fe was bound with more DOC than other mineral elements. Therefore, Fe played an important role in the preservation of DOC in red soils in Southern China. Improvement of Reactive Iron Minerals in Red Soil by Manure Amendment Table 1. Characteristics of soils from the various long-term fertilisation treatmentsa. Treatments SOC (g/kg) Bulk soil pH (H2O) Fet (g/kg) Fed (mg/g) Fed/Fet DOC (mg/L) Raw soil 8.54 ± 0.03 5.7± 0.09 53.60 ± 0.68 41.36 ± 0.74 0.77 ± 0.06 34.52 ± 1.61 Control 8.05 ± 0.05 5.47 ± 0.07 50.70 ± 1.32 40.17 ± 0.78 0.79 ± 0.03 26.17 ± 13.32 NPK 10.62 ± 0.03 4.15 ± 0.02 55.40 ± 1.89 45.18 ± 3.36 0.82 ± 0.03 22.19 ± 1.43 NPKM 12.37 ± 0.08 5.84 ± 0.02 49.31 ± 0.59 41.52 ± 5.31 0.84 ± 0.11 217.08 ± 82.95 M 14.76 ± 0.04 6.63 ± 0.05 46.72 ± 1.94 39.84 ± 1.86 0.85 ± 0.01 101.39 ± 14.15 aControl, no fertilisation; NPK, chemical fertilisation; NPKM, chemical plus swine manure fertilisation; M, swine manure fertilisation. SOC, soil organic carbon. DOC, dissolved organic carbon. Fed, the CBD extracted reactive Fe. Fet, the total Fe. Fed/Fet, this ratio represents the iron freeness index. Table 1. Characteristics of soils from the various long-term fertilisation treatmentsa. aControl, no fertilisation; emical plus swine manure fertilisation; M, swine manure fertilisation. SOC, soil organic carbon. DOC, dissolved ed reactive Fe. Fet, the total Fe. Fed/Fet, this ratio represents the iron freeness index. , ; NPK, chemical fertilisation; NPKM, chemical plus swine manure fertilisation; M, swine manure fertilisation. SOC, soil organic carbon. DOC, dissolved organic carbon. Fed, the CBD extracted reactive Fe. Fet, the total Fe. Fed/Fet, this ratio represents the iron freeness index. 2.7 Statistical analysis One-way analysis of variance (ANOVA) was employed to test the effects of long-term fertilisa- tion on soil reactive Fe minerals. Significance was determined by performing one-way ANO- VA’s followed by Tukey’s HSD post hoc tests. Conditions of normality and homogeneity of variance were met. Means ± SE (n = 3) followed by different letters indicate significant differ- ences between treatments at P < 0.01. Values of Pearson’s correlation coefficient (R) were used to evaluate the linear correlations among reactive Fe minerals and C functional groups. The Pearson’s coefficient is always a number between –1 and +1, where –1 denotes a perfect nega- tive correlation, +1 denotes a perfect positive correlation, and 0 denotes the absence of a rela- tionship. The correlations were considered to be statistically significant at a 95% confidence interval (P < 0.05). 4 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0146364 January 11, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0146364 January 11, 2016 Improvement of Reactive Iron Minerals in Red Soil by Manure Amendment Fig 1. Concentrations of mineral elements in soil colloids under contrasting fertilisation treatments (a) and their correlations with dissolved organic carbon (DOC) (b). Control, no fertilisation; NPK, chemical fertilisation; NPKM, chemical plus swine manure fertilisation; M, swine manure fertilisation. Significant differences among fertilisation treatments were determined using one-way ANOVA followed by Tukey’s HSD post hoc test at P < 0.01; conditions of normality and homogeneity of variance were met. The slopes of the relationships between mineral elements and DOC shown in (b) clearly demonstrate that DOC is dominated by Fe, Si, and Al rather than by Ca and Mg in red soils in Southern China. Fig 1. Concentrations of mineral elements in soil colloids under contrasting fertilisation treatments (a) and their correlations with dissolved organic carbon (DOC) (b). Control, no fertilisation; NPK, chemical fertilisation; NPKM, chemical plus swine manure fertilisation; M, swine manure fertilisation. Significant differences among fertilisation treatments were determined using one-way ANOVA followed by Tukey’s HSD post hoc test at P < 0.01; conditions of normality and homogeneity of variance were met. The slopes of the relationships between mineral elements and DOC shown in (b) clearly demonstrate that DOC is dominated by Fe, Si, and Al rather than by Ca and Mg in red soils in Southern China. Fig 1. Concentrations of mineral elements in soil colloids under contrasting fertilisation treatments (a) and their correlations with dissolved organic carbon (DOC) (b). Control, no fertilisation; NPK, chemical fertilisation; NPKM, chemical plus swine manure fertilisation; M, swine manure fertilisation. Significant differences among fertilisation treatments were determined using one-way ANOVA followed by Tukey’s HSD post hoc test at P < 0.01; conditions of normality and homogeneity of variance were met. The slopes of the relationships between mineral elements and DOC shown in (b) clearly demonstrate that DOC is dominated by Fe, Si, and Al rather than by Ca and Mg in red soils in Southern China. doi:10.1371/journal.pone.0146364.g001 with SOC (Table 1). However, the iron freeness index, indicated by the Fed/Fet ratio, had a sim- ilar relationship to SOC, revealing that Fed/Fet rather than Fed may be the critical factor in the determination of SOC preservation. An increase in the Fed/Fet ratio suggests a high degree of soil weathering [24]. Compared to raw soil, all of the fertilisations increased the Fed/Fet ratio. Therefore, organic fertilisation increased the rate of soil weathering much more than control and chemical fertilisations. 3.1 Characteristics of soils in contrasting fertilisation treatments The higher concentrations of DOC and mineral elements in the NPKM treatment in relation to the NPK or M treatment may be due to the better growth of plants in the NPKM treatment. This would result in increased root exudate production in the NPKM treatment, which simulated higher concentration of DOC and mineral element production. To quantify the concentration of reactive Fe, the CBD method was used to extract Fe (i.e., Fed) from soils. The results demonstrated that the Fed in soils exhibited a distinct relationship 5 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0146364 January 11, 2016 3.2 FTIR spectroscopy of reactive minerals under contrasting fertilisation treatments FTIR spectroscopy was used to examine the difference of organic C and minerals in the CBD extracts and bulk soils (Fig 2). For the Control and NPK treatments, the band intensity between the CBD extracts and bulk soils was similar, except for 3465 cm-1 (O-H network). Interestingly, for the M and NPKM treatments, the band intensity of the CBD extracts was more stronger at 6 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0146364 January 11, 2016 Improvement of Reactive Iron Minerals in Red Soil by Manure Amendment Fig 2. Fourier-transform IR spectra of the CBD extracts and the bulk soils from the variouslong-term fertilisation treatments. Control, no fertilisation; NPK, chemical fertilisation; NPKM, chemical plus swine manure fertilisation; M, swine manure fertilisation. CBD, citrate-bicarbonate-dithionite. The grey region indicates the extracted reactive minerals and organic C pool, suggesting that considerably greater amounts of reactive mineral components (Fe-O, Al- O, and Si-O) and organic C were extracted from the long-term organic treatments (i.e., M and NPKM) using the CBD method than were extracted from the Control and NPK treatments. Fig 2. Fourier-transform IR spectra of the CBD extracts and the bulk soils from the variouslong-term fertilisation treatments. Control, no fertilisation; NPK, chemical fertilisation; NPKM, chemical plus swine manure fertilisation; M, swine manure fertilisation. CBD, citrate-bicarbonate-dithionite. The grey region indicates the extracted reactive minerals and organic C pool, suggesting that considerably greater amounts of reactive mineral components (Fe-O, Al- O, and Si-O) and organic C were extracted from the long-term organic treatments (i.e., M and NPKM) using the CBD method than were extracted from the Control and NPK treatments doi:10.1371/journal.pone.0146364.g002 1640 cm-1 (aromatic C), 1100 cm-1 (alkyl C), 1020 cm-1 (alkyl C), 790 cm-1 (Fe-O), 690 cm-1 (Al-O), 520 cm-1 (Al-O-Si) and 480 cm-1 (Si-O) than that of bulk soils. Therefore, FTIR of the CBD extracts and bulk soils confirmed that substantially greater amounts of reactive mineral components (i.e., Fe-O, Al-O, and Si-O) and organic C were extracted from the soils following the M and NPKM treatments using the CBD method than from the Control and NPK treat- ments (Fig 2). When the CBD method and the FTIR spectra were combined, it was concluded that the Fed/Fet ratio, rather than Fed, provides a suitable index for the characterisation of the amount of reactive Fe that is related to the preservation of SOC. PLOS ONE \| DOI:10.1371/journal.pone.0146364 January 11, 2016 3.3 Composition of reactive minerals as studied by Fe K-edge XANES spectra To determine the composition of the reactive Fe minerals, the Fe K-edge XANES spectra with the linear combination fitting (LCF) were analysed using eight reference materials (Fig 3). The eight reference materials were ferrihydrite, goethite, lepidocrocite, maghemite, Fe(III) sulphate, Fe(II) sulphate, Fe(III) oxalate, and Fe(II) oxalate (S1 Fig). Of these, ferrihydrite, goethite, lepi- docrocite, and magnetite represent the main reactive minerals; Fe(III) sulphate and Fe(II) sul- phate represent the primary forms of tri- and bi-valent inorganic irons, while Fe(III) oxalate and Fe(II) oxalate represent the primary forms of tri- and bi-valent organic irons. The LCF results for the soil colloids (Fig 3 and Table 2) revealed that Fe(III) was predominant (71%- 84.2%) following both the organic and the chemical fertilisation treatments. The other Fe phases were composed of less crystalline ferrihydrite (15.8%-25.9%) in the organic (i.e., NPKM and M) treatments and more crystalline maghemite (29.0 ± 1.5%) in the chemical (i.e., NPK) treatments. For the Control treatment, the reactive Fe minerals in the soil colloids were 7 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0146364 January 11, 2016 Improvement of Reactive Iron Minerals in Red Soil by Manure Amendment composed of 47.5% inorganic Fe(III) and 52.5 ± 2.5% ferrihydrite. Given the greater C preser- vation capability of ferrihydrite than maghemite [25–27], it is reasonable to conclude that the Fig 3. Fe K-edge XANES spectra of soil colloids from contrasting fertilisation treatments. (a) Control, no fertilisation; (b) NPK, chemical fertilisation; (c) NPKM, chemical plus swine manure fertilisation; (d) M, swine manure fertilisation. The scattered circles represent the linear combination fitting (LCF) results of the sample spectra. doi:10.1371/journal.pone.0146364.g003 Fig 3. Fe K-edge XANES spectra of soil colloids from contrasting fertilisation treatments. (a) Control, no fertilisation; (b) NPK, chemical fertilisation; (c) NPKM, chemical plus swine manure fertilisation; (d) M, swine manure fertilisation. The scattered circles represent the linear combination fitting (LCF) results of the sample spectra. doi:10.1371/journal.pone.0146364.g003 composed of 47.5% inorganic Fe(III) and 52.5 ± 2.5% ferrihydrite. Given the greater C preser- vation capability of ferrihydrite than maghemite [25–27], it is reasonable to conclude that the Fe minerals present in soil colloids after long-term organic fertilisation are more reactive than those present after long-term chemical fertilisation. Improvement of Reactive Iron Minerals in Red Soil by Manure Amendment Table 2. Linear combination fit (LCF) results of the Fe K-edge XANES spectra of soil colloids from various fertilisation treatments and the simu- lated studies a. Samples LCF results (%) LCF parameters Ferrihydrite Goethite Magnetite Ferrous sulfate Ferric oxalate R-factor Chi-square fertilisation treatment Control 23.0 ± 2.3 71.7 ± 1.2 5.3 ± 1.4 ND ND 0.000042 3.71 × 10−5 NPK ND 77.6 ± 1.6 13.8 ± 0.7 ND 8.7 ± 0.7 0.000061 5.34 × 10−5 NPKM 43.4 ± 1.4 53.2 ± 1.1 ND 3.4 ± 0 ND 0.000054 4.68 × 10−5 M 30.3 ± 2.5 64.3 ± 1.2 5.4 ± 1.6 ND ND 0.000048 4.09 × 10−5 Simulated studies M+10 mg/L oxalic acid 53.1±0.013 47.8±1.4 ND ND 11.3±0.9 0.000713 1.08×10−4 M+100 mg/L oxalic acid 49.7±3.1 40.9±2.5 ND ND 14.4±0.6 0.000644 1.23×10−4 aControl, no fertilisation; NPK, chemical fertilisation; NPKM, chemical plus swine manure fertilisation; M, swine manure fertilisation. ND, not detected. The values of the ﬁtting parameters (i.e., R-factor and chi-square) indicate that the ﬁtting results are satisfactory. sults of the Fe K-edge XANES spectra of soil colloids from various fertilisation treatments and the simu- Samples Control, no fertilisation; NPK, chemical fertilisation; NPKM, chemical plus swine manure fertilisation; M, swine manure fertilisation. ND, not detected. The values of the ﬁtting parameters (i.e., R-factor and chi-square) indicate that the ﬁtting results are satisfactory. emical plus swine manure fertilisation; M, swine manure fertilisation. ND, not detected. The values of the ﬁtting are) indicate that the ﬁtting results are satisfactory. NPK, chemical fertilisation; NPKM, chemical plus swine manure fertilisation; M, swine manure fertilisation. ND, no parameters (i.e., R-factor and chi-square) indicate that the ﬁtting results are satisfactory. 53.1 ± 0.0% and 49.7 ± 3.1%, respectively. Furthermore, the extended X-ray absorption fine structure (EXAFS) spectra and the radial structure functions (RSFs, uncorrected for phase shift) of those data (Fig 4-b and 4-c) demonstrated that the addition of oxalic acid caused struc- tural changes to Fe minerals, indicative of the occurrence of both co-precipitation and absorp- tion [28] with the addition of oxalic acid to soil colloids in the M treatment. The above results suggest that oxalic acid can promote the transformation from Fe(III) to ferrihydrite, which is consistent with aprevious report that the low-molecular-weight (LMW) organic acid may incorporate into the network structure of SRO minerals [29], inhibiting fur- ther growth of SRO minerals [3]. Ferrihydrite was indicative of recent Fe weathering and forms by the rapid oxidation of Fe (II) in solution [30]. Therefore, its weathering was an important step in the transformation process [27]. Considering the large amounts of Fe in the organic treatment (Table 1), it is sug- gested that Fe in bulk soils might be reduced to Fe(II) first, and then oxidized to Fe(III) with the assistance of oxidizing substances, and lastly, the Fe(III) is incorporated into the network structure of organic acids to form ferrihydrite. 3.4 Simulation of reactive mineral formation by adding organic acid The factors that affect the formation of reactive minerals in the natural system were compli- cated [27]. To confirm the role of organic inputs in the formation process, a simulated study, i.e., adding oxalic acid to soil colloids from the M treatment, was conducted. The LCF results of Fe K-edge XANES spectra (Fig 4-a and Table 2) demonstrated that incu- bation of soil colloids with oxalic acid at a concentration of 10 and 100 mg/L for 1 day could decrease the percentage of goethite from 64.3 ± 1.2% to 47.8 ± 1.4% and 40.9 ± 2.5%, respec- tively, and simultaneously increase the percentage of ferrihydrite from 30.3 ± 2.5% to 8 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0146364 January 11, 2016 3.5 Organic groups preserved by reactive minerals To date, it remains unclear which components of soil organic C are preserved by reactive Fe minerals. Synchrotron-based C 1s near-edge X-ray fine structure (NEXAFS) spectroscopy can offer valuable insights into the composition of organic C [31]. Using C 1s NEXAFS spectros- copy combined with deconvolution analysis [20], it was found that compared to the NPK treat- ment, the NPKM and M treatments markedly increased the proportion of carboxylic groups (288.4–289.1 eV) from 24.2% to 33.2% and increased the percentages of both the aromatic (283.0–286.1 eV) and phenolic (286.2–287.5 eV) groups by greater than 2.8-fold (Fig 5 and Table 3). These results demonstrated that organic fertilisation treatments enhanced the reten- tion of carboxylic and aromatic C in soils. To determine whether the retention of C is directly correlated with the content of reactive Fe minerals, the Pearson correlations between C functional groups and Fet as well as Fed were evaluated (Table 4). The results showed that total Fe was significantly correlated with the car- boxylic C groups (P < 0.001, R2 > 0.69) but was not significantly correlated with the other C groups. However, the CBD extracted Fe was significantly correlated with aromatic C and O- 9 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0146364 January 11, 2016 Improvement of Reactive Iron Minerals in Red Soil by Manure Amendment Fig 4. Linear combination fitting (LCF) results of XANES Fe K-edge normalized spectra (a), Fe K-edge EXAFS (b), and radial structure function (RSFs, uncorrected for phase shift) (c) in simulated studies. The scattered circles represent the LCF results of the sample spectra. Fitting parameters (i.e., R-factor and chi-square, Table 2) indicated that the fitting results are convincible. Fig 4. Linear combination fitting (LCF) results of XANES Fe K-edge normalized spectra (a), Fe K-edge EXAFS (b), and radial structure function (RSFs, uncorrected for phase shift) (c) in simulated studies. The scattered circles represent the LCF results of the sample spectra. Fitting parameters (i.e., R-factor and chi-square, Table 2) indicated that the fitting results are convincible. doi:10.1371/journal.pone.0146364.g004 alkyl C functional groups (P < 0.001, R2 > 0.77). Therefore, it is reasonable to conclude that reactive Fe minerals are responsible for the retention of aromatic C and O-alkyl C in soils. alkyl C functional groups (P < 0.001, R2 > 0.77). Therefore, it is reasonable to conclude that reactive Fe minerals are responsible for the retention of aromatic C and O-alkyl C in soils. Improvement of Reactive Iron Minerals in Red Soil by Manure Amendment Fig 5. C 1s NEXAFS spectra and their deconvolution results for soil colloids from the various long-term fertilisation treatments. (a) Control, no fertilisation; (b) NPK, chemical fertilisation; (c) NPKM, chemical plus swine manure fertilisation; (d) M, swine manure fertilisation. G1–G8 representeight Gaussian curves. The specific C forms of G1–G8 are given in S1 Table. Artan represents an arctangent step function. been suggested that the weathering of soil was driven by microorganisms [34], the diversity and function of which were affected by nutrient availability [35]. Here, it is inferred that the organic fertilisation treatments not only provided a high availability of nutrients (i.e., manure) to microorganisms but also created a suitable environment (e.g., correct pH) for microbial Fig 5. C 1s NEXAFS spectra and their deconvolution results for soil colloids from the various long-term fertilisation treatments. (a) Control, no fertilisation; (b) NPK, chemical fertilisation; (c) NPKM, chemical plus swine manure fertilisation; (d) M, swine manure fertilisation. G1–G8 representeight Gaussian curves. The specific C forms of G1–G8 are given in S1 Table. Artan represents an arctangent step function. doi:10.1371/journal.pone.0146364.g005 Fig 5. C 1s NEXAFS spectra and their deconvolution results for soil colloids from the various long-term fertilisation treatments. (a) Control, no fertilisation; (b) NPK, chemical fertilisation; (c) NPKM, chemical plus swine manure fertilisation; (d) M, swine manure fertilisation. G1–G8 representeight Gaussian curves. The specific C forms of G1–G8 are given in S1 Table. Artan represents an arctangent step function. been suggested that the weathering of soil was driven by microorganisms [34], the diversity and function of which were affected by nutrient availability [35]. Here, it is inferred that the organic fertilisation treatments not only provided a high availability of nutrients (i.e., manure) to microorganisms but also created a suitable environment (e.g., correct pH) for microbial Table 3. Deconvolution results for using C 1s NEXAFS on soil colloids from the various long-term fertilisation treatmentsa. Treatment Proportion of absorption regions (%) Table 3. Deconvolution results for using C 1s NEXAFS on soil colloids from the various long-term fertilisation treatmentsa. Discussion It is well known that, compared to chemical fertilisation treatments, long-term organic fertilisa- tion treatments can increase soil C pools [8,9,32]. However, the effects of long-term organic fertilisation treatments on the release of mineral elements are poorly understood. The release of mineral elements in soils was considered a precursor to the formation of reactive minerals [33]. The results in this study demonstrated that organic fertilisation treatments resulted in the release of considerable amounts of mineral elements into soil colloids, which were significantly (P < 0.01) correlated with the concentration of DOC. This result was also consistent with the higher degree of soil weathering that received organic fertilisation treatments (Table 1). It had PLOS ONE \| DOI:10.1371/journal.pone.0146364 January 11, 2016 10 / 15 , ; NPK, chemical fertilisation; NPKM, chemical plus swine manure fertilisation; M, swine manure fertilisation. Improvement of Reactive Iron Minerals in Red Soil by Manure Amendment Table 4. Pearson correlation coefficients amonghighly reactive Fe fractions (i.e., Fed) and C functional groups (n = 12)a. Functional group Energy level (eV) Fed Fet P R2 P R2 Aromatic C 283.0–286.1 <0.001 0.77 0.062 0.23 Phenolic C 286.2–287.5 <0.05 0.28 0.065 0.23 Alkyl C 287.6–288.3 <0.05 0.40 0.111 0.15 Carboxylic C 288.4–289.1 <0.01 0.48 <0.001 0.69 O-alkyl C 289.2–289.8 <0.00001 0.99 <0.01 0.61 Carbonyl C 289.9–290.2 0.16 0.10 <0.05 0.34 aFed, the citrate-bicarbonate-dithionite extracted Fe. Fet, the total Fe. d i 10 1371/j l 0146364 004 son correlation coefficients amonghighly reactive Fe fractions (i.e., Fed) and C functional groups (n = 12)a. Table 4. Pearson correlation coefficients amonghighly reactive Fe fractions (i.e., Fed) and C functional g aFed, the citrate-bicarbonate-dithionite extracted Fe. Fet, the total Fe. activity. Thus, on the one hand, microbes were present in high abundance, which promotes the weathering of soil and on the other hand, more rapid weathering provided a greater availability of nutrients for the formation of reactive minerals. Analysis of the concentrations of ferrihydrite in the applied fertilisers and the XRF spectros- copy results [36] indicated that ferrihydrite introduced by fertilisers was negligible, supporting that fertilisers had a minor effect on the concentration of reactive minerals in soil. Further- more, the results of simulated studies demonstrated that the addition of organic acid could transfer goethite to ferrihydrite (Fig 4), providing direct evidence that organic inputs promoted the formation of reactive minerals. The presence of organic matter was critical to the formation of reactive minerals because organic matter could become incorporated into the network struc- ture of short-range ordered (i.e., SRO) minerals and thus prevent the formation of sheets or inter-layer H-bonds that were essential components of crystalline minerals [8,37,38]. More- over, high concentrations of organic matter might promote the formation of reactive Fe miner- als by inhibiting further growth of reactive Fe minerals to their crystalline counterpart [3]. Therefore, soils that receive long-term swine manure inputs contained high concentrations of organic matters (mainly as carboxylic C, alkyl C, carbonyl C, and O-alkyl C, Table 3) that facilitated the rapid formation of reactive Fe minerals. The formation of reactive Fe minerals facilitated the storage of SOM, which was perhaps the most important determinant of soil qual- ity and soil sustainability [39]. Treatment Proportion of absorption regions (%) Aromatic C Phenolic C Alkyl C Carboxylic C O-alkyl C Carbonyl C (283–286.1 eV) (286.2–287.5 eV) (287.6–288.3 eV) (288.4–289.1 eV) (289.2–289.8 eV) (289.9–290.2 eV) Control 2.6 0.7 19.4 29.6 12.1 35.6 NPK 0.5 0.1 25.7 24.2 16.3 33.2 NPKM 1.4 1.1 18.9 33.2 13.2 32.2 M 1.8 0.5 22.9 46.6 12.3 15.9 aControl, no fertilisation; NPK, chemical fertilisation; NPKM, chemical plus swine manure fertilisation; M, swine manure fertilisation. doi:10 1371/journal pone 0146364 t003 nvolution results for using C 1s NEXAFS on soil colloids from the various long-term fertilisation treatmentsa Table 3. Deconvolution results for using C 1s NEXAFS on soil colloids from the various long-term fertilisati PLOS ONE \| DOI:10.1371/journal.pone.0146364 January 11, 2016 11 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0146364 January 11, 2016 Acknowledgments The authors thank B.R. Wang for his assistance in soil sampling in the Qiyang Long-term Fer- tilization Experiment, technical assistances from J.Y. Ma to use the Fe K-edge XANES facility of the BL14W1 beamline and from X.Z. Zhang as well as L.J. Zhang to use the C 1s NEXAFS facility of the 08U beamline in the Shanghai Synchrotron Radiation Facility. S1 Fig. Fe K-edge XANES spectra of reference materials. (PDF) S1 Table. Peak assignment for C forms obtained from C 1s NEXAFS and corresponding FTIR spectroscopy [12,17,40]a. (PDF) Author Contributions Conceived and designed the experiments: GY. Performed the experiments: CH SL RL FS. Ana- lyzed the data: CH SL RL FS YZ GY. Contributed reagents/materials/analysis tools: CH GY. Wrote the paper: GY CH SL RL FS YZ. Conceived and designed the experiments: GY. Performed the experiments: CH SL RL FS. Ana- lyzed the data: CH SL RL FS YZ GY. Contributed reagents/materials/analysis tools: CH GY. Wrote the paper: GY CH SL RL FS YZ. Improvement of Reactive Iron Minerals in Red Soil by Manure Amendment the quantities of Fe minerals were similar, the forms of Fe minerals present in organic fertilised soils played a more important role in the preservation of soil C than those present in inorganic fertilised soils. A better understanding of the effects of fertilisation practices on reactive iron oxides is important for predicting and managing C preservation in soils. Conclusion In summary, these findings demonstrated that long-term organic fertilizer treatment increased the mobilization of Fe minerals. Moreover, the interaction between organic materials and Fe minerals may facilitate the rapid formation of reactive Fe minerals in soil colloids. These find- ings raise new possibilities for investigating the regulation of reactive soil minerals and their roles in efficient C preservation, in addition to providing a pathway for predicting and manag- ing the global C cycle. PLOS ONE \| DOI:10.1371/journal.pone.0146364 January 11, 2016 Supporting Information S1 Fig. Fe K-edge XANES spectra of reference materials. (PDF) It had been shown that the quality of the land and so its suitabil- ity in China had decreased, based on the second National Land Resource Survey of China [40]. Therefore, it is urgent for China to improve the quality of low- and medium-grade arable land. One of the effective ways to improve the quality of the land and its suitability of the land was adding manure to soil [41]. Moreover, the comparison between the FTIR spectra of the CBD extracts and those of the bulk soils has demonstrated, for the first time, that FTIR spectroscopy is a suitable method for the characterisation of reactive Fe minerals related to the preservation of SOC. Reactive Fe minerals had been suggested to play an important role in preserving soil C. However, it is unclear as to which Fe minerals are responsible for the preservation of soil C. Therefore, the identification of reactive Fe minerals is critical for improving our understanding of the mechanisms underlying the preservation of soil C by reactive Fe minerals. The identifica- tion of reactive Fe minerals is still a complicated issue, owing to the limitations of traditional techniques (i.e., X-ray diffraction) that had been used to identify SRO minerals as the main components of reactive Fe minerals [42]. Fe K-edge XANES spectroscopy clearly demonstrated that organic fertilisation treatments promoted the formation of reactive Fe minerals, i.e., ferri- hydrite, compared to chemical fertilisation treatments. These results suggested that even when 12 / 15 References Luisa Fdez-Gubieda M, Muela A, Alonso J, Garcia-Prieto A, Olivi L, Fernandez-Pacheco R, et al. (2013) Magnetite biomineralization in magnetospirillum gryphiswaldense: time-resolved magnetic and structural studies. ACS Nano 7: 3297–3305. doi: 10.1021/nn3059983 PMID: 23530668 12. 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https://openalex.org/W2076953438	https://europepmc.org/articles/pmc3237608?pdf=render	English	null	Craniectomy for Malignant Cerebral Infarction: Prevalence and Outcomes in US Hospitals	PloS one	2,011	cc-by	5,932	Abstract doi:10.1371/journal.pone.0029193 Editor: Andreas Meisel, Charite´ Universitaetsmedizin Berlin, Germany Received October 20, 2011; Accepted November 22, 2011; Published December 14, 2011 Copyright: 2011 Walcott et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: walcott.brian@mgh.harvard.edu , , y Received October 20, 2011; Accepted November 22, 2011; Published December 14, 2011 Copyright: 2011 Walcott et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. ceived October 20, 2011; Accepted November 22, 2011; Published December 14, 2011 Received October 20, 2011; Accepted November 22, 2011; Published December 14, 2011 Copyright: 2011 Walcott et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2011 Walcott et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2011 Walcott et al. This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction infarction using the Nationwide Inpatient Sample (NIS), the largest all-payer representative sample of the US medical community. The surgical treatment of life-threatening, space-occupying cerebral edema following massive middle cerebral artery infarc- tion, so-called ‘‘malignant’’ infarction, remains a controversial issue. Historically, there has been a reluctance to perform this operation given high rate of mortality and profound morbidity associated with survivors. Until recently, only case series and nonrandomized case control studies suggested any benefit of decompressive craniectomy (DC).[1–8] Several recent randomized controlled trials have demonstrated improved survival and functional outcome after DC in certain populations. [9–11] The findings from these randomized controlled trials are recapitulated in several recent reviews.[3] [12–13] We sought to identify trends in the prevalence and outcomes of DC for malignant cerebral Brian P. Walcott1, Elena V. Kuklina2, Brian V. Nahed1, Mary G. George2, Kristopher T. Kahle1, J. Marc Simard3, Wael F. Asaad4, Jean-Valery C. E. Coumans1 Brian P. Walcott1, Elena V. Kuklina2, Brian V. Nahed1, Mary G. George2, Kristopher T. Kahle1, J. Marc Simard3, Wael F. Asaad4, Jean-Valery C. E. Coumans1 1 Department of Neurosurgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, United States of America, 2 Division for Heart Disease and Stroke Prevention, National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 3 Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 4 Department of Neurosurgery, Brown University School of Medicine, Providence, Rhode Island, United States of America PLoS ONE \| www.plosone.org Abstract Object: Randomized trials have demonstrated the efficacy of craniectomy for the treatment of malignant cerebral edema following ischemic stroke. We sought to determine the prevalence and outcomes related to this by using a national database. Methods: Patient discharges with ischemic stroke as the primary diagnosis undergoing craniectomy were queried from the US Nationwide Inpatient Sample from 1999 to 2008. A subpopulation of patients was identified that underwent thrombolysis. Two primary end points were examined: in-hospital mortality and discharge to home/routine care. To facilitate interpretations, adjusted prevalence was calculated from the overall prevalence and two age-specific logistic regression models. The predictive margin was then generated using a multivariate logistic regression model to estimate the probability of in-hospital mortality after adjustment for admission type, admission source, length of stay, total hospital charges, chronic comorbidities, and medical complications. Results: After excluding 71,996 patients with the diagnosis of intracranial hemorrhage and posterior intracranial circulation occlusion, we identified 4,248,955 adult hospitalizations with ischemic stroke as a primary diagnosis. The estimated rates of hospitalizations in craniectomy per 10,000 hospitalizations with ischemic stroke increased from 3.9 in 1999–2000 to 14.46 in 2007–2008 (p for linear trend,0.001). Patients 60+ years of age had in-hospital mortality of 44% while the 18–59 year old group was found to be 24%(p = 0.14). Outcomes were comparable if recombinant tissue plasminogen activator had been administered. Conclusions: Craniectomy is being increasingly performed for malignant cerebral edema following large territory cerebral ischemia. We suspect that the increase in the annual incidence of DC for malignant cerebral edema is directly related to the expanding collection of evidence in randomized trials that the operation is efficacious when performed in the correct patient population. In hospital mortality is high for all patients undergoing this procedure. Citation: Walcott BP, Kuklina EV, Nahed BV, George MG, Kahle KT, et al. (2011) Craniectomy for Malignant Cerebral Infarction: Prevalence and Outcomes in US Hospitals. PLoS ONE 6(12): e29193. doi:10.1371/journal.pone.0029193 Citation: Walcott BP, Kuklina EV, Nahed BV, George MG, Kahle KT, et al. (2011) Craniectomy for Malignant Cerebral Infarction: Prevalence and Outcomes in US Hospitals. PLoS ONE 6(12): e29193. doi:10.1371/journal.pone.0029193 Editor: Andreas Meisel Charite´ Universitaetsmedizin Berlin Germany Citation: Walcott BP, Kuklina EV, Nahed BV, George MG, Kahle KT, et al. (2011) Craniectomy for Malignant Cerebral Infarction: Prevalence and Outcomes in US Hospitals. PLoS ONE 6(12): e29193. Methods We studied the prevalence and outcomes of DC for malignant cerebral infarction from 1999 to 2008 using data obtained from the Healthcare Cost and Utilization Project, Agency for Healthcare Research and Quality (AHRQ) Rockville, MD.[14] The NIS is a hospital discharge database that represents approximately 20% of all inpatient admission to non-federal hospitals in the US. The NIS contains discharge data on 100% of discharges, an expanding, stratified random sample of 1,044 non- federal hospitals from 40 states in 2008. Detailed information on December 2011 \| Volume 6 \| Issue 12 \| e29193 1 PLoS ONE \| www.plosone.org Craniectomy in Stroke Figure 1. Trends in intervention procedures among hospitalizations for ischemic stroke* (n = 4,248,855). Hospitalizations with ischemic stroke listed as the primary diagnosis were identified using the first listed International Classification of Disease 9th Revision clinical modifier (ICD-9 CM) diagnostic codes 433.11, 433.31, 433.81, 433.91, 434.01, 434.11, 434.91, and 436). Patients with the diagnosis of intracranial hemorrhage (ICD-9 CM diagnostic codes 430, 431, and 432.x) and patients with the diagnosis of posterior intracranial circulation occlusion (ICD-9 CM diagnostic codes 433.01 & 433.21) were excluded. NA: estimates are not reportable due to a small sample size. doi:10.1371/journal.pone.0029193.g001 Figure 1. Trends in intervention procedures among hospitalizations for ischemic stroke (n = 4,248,855). Hospitalizations with ischemic stroke listed as the primary diagnosis were identified using the first listed International Classification of Disease 9th Revision clinical modifier (ICD-9 CM) diagnostic codes 433.11, 433.31, 433.81, 433.91, 434.01, 434.11, 434.91, and 436). Patients with the diagnosis of intracranial hemorrhage (ICD-9 CM diagnostic codes 430, 431, and 432.x) and patients with the diagnosis of posterior intracranial circulation occlusion (ICD-9 CM diagnostic codes 433.01 & 433.21) were excluded. NA: estimates are not reportable due to a small sample size. doi:10.1371/journal.pone.0029193.g001 430, 431, and 432.x were excluded. Also, patients with the diagnosis of posterior intracranial circulation occlusion (ICD-9 CM diagnostic codes 433.01 & 433.21) were excluded. Then, the core study population was established by selecting for those also undergoing craniotomy (including DC), identified by ICD-9 CM procedure codes 01.24 or 01.25. A subpopulation of the study population was created using ICD-9 CM procedure code 99.10 to identify those that received any form of thrombolysis. (As of 2008, an ICD-9 CM code exists specifically for thrombolysis given at referring, rather than admitting, hospitals. Hospitalizations with ischemic stroke listed as the primary diagnosis were identified using the first listed International Classification of Disease 9th Revision clinical modifier (ICD-9 CM) diagnostic codes 433.11, 433.31, 433.81, 433.91, 434.01, 434.11, 434.91, and 436). Patients with the diagnosis of intracranial hemorrhage (ICD-9 CM diagnostic codes 430, 431, and 432.x) and patients with the diagnosis of posterior intracranial circulation occlusion (ICD-9 CM diagnostic codes 433.01 & 433.21) were excluded. NR: estimates are not reportable due to a small sample size. doi:10.1371/journal.pone.0029193.t001 Methods However, we specifi- cally did not analyze this due to the absence of the code in the the design of the NIS is available at http://www.hcup-us.ahrq. gov. The NIS includes .100 clinical and non-clinical variables for each hospital stay. These include diagnoses, procedures, admission and discharge status, demographics, charges, and lengths of stay. Patients registered in the NIS from 1999 to 2008 were included in the analysis. Those with ischemic stroke listed as the primary diagnosis were identified using the first listed International Classification of Disease 9th Revision clinical modifier (ICD-9 CM) diagnostic codes 433.11, 433.31, 433.81, 433.91, 434.01, 434.11, 434.91, and 436). Patients with the diagnosis of intracranial hemorrhage identified by ICD-9 CM diagnostic codes Table 1. Trends in intervention procedures among hospitalizations for ischemic stroke* (n = 4,248,855), Nationwide Inpatient Sample, 1999-2008. 1999-00 2001-02 2003-04 2005-06 2007-08 Hospitalizations with ischemic stroke (weighted) Total hospitalizations 909,064 883,209 830,751 803,241 822,691 Intervention group DC + rtPA NR 27 36 97 174 DC only 346 316 435 669 1,016 rtPA only 8,374 8,887 10,509 16,850 24,619 DC, overall 351 343 471 766 1,190 Prevalence, per 10,000 hospitalizations with ischemic stroke DC + rtPA 0.05 (0.00) 0.30 (0.00) 0.43 (0.00) 1.21 (0.00) 2.11 (0.00) DC only 3.81 (0.01) 3.58 (0.00) 5.24 (0.01) 8.33 (0.01) 12.35 (0.01) rtPA only 92.11 (0.04) 100.6 (0.05) 126.5 (0.08) 209.8 (0.10) 299.3 (0.11) DC, overall 3.86 (0.01) 3.88 (0.00) 5.67 (0.01) 9.54 (0.01) 14.46 (0.01) Hospitalizations with ischemic stroke listed as the primary diagnosis were identified using the first listed International Classification of Disease 9th Revision clinical modifier (ICD-9 CM) diagnostic codes 433.11, 433.31, 433.81, 433.91, 434.01, 434.11, 434.91, and 436). Patients with the diagnosis of intracranial hemorrhage (ICD-9 CM diagnostic codes 430, 431, and 432.x) and patients with the diagnosis of posterior intracranial circulation occlusion (ICD-9 CM diagnostic codes 433.01 & 433.21) were excluded. NR: estimates are not reportable due to a small sample size. doi:10.1371/journal.pone.0029193.t001 Table 1. Trends in intervention procedures among hospitalizations for ischemic stroke (n = 4,248,855) Sample, 1999-2008. rvention procedures among hospitalizations for ischemic stroke* (n = 4,248,855), Nationwide Inpatient Hospitalizations with ischemic stroke listed as the primary diagnosis were identified using the first listed International Classification of Disease 9th Revision clinical modifier (ICD-9 CM) diagnostic codes 433.11, 433.31, 433.81, 433.91, 434.01, 434.11, 434.91, and 436). Methods Patients with the diagnosis of intracranial hemorrhage (ICD-9 CM diagnostic codes 430, 431, and 432.x) and patients with the diagnosis of posterior intracranial circulation occlusion (ICD-9 CM diagnostic codes 433.01 & 433.21) were excluded. NR: estimates are not reportable due to a small sample size. doi:10.1371/journal.pone.0029193.t001 December 2011 \| Volume 6 \| Issue 12 \| e29193 PLoS ONE \| www.plosone.org 2 Craniectomy in Stroke Table 2. Numbers and prevalence of decompressive craniectomy with and without rtPA among hospitalizations for ischemic stroke by age (n = 4,248,955), Nationwide Inpatient Sample, 1999–2008. Intervention group All 18–59 years 60+ years Hospitalizations with ischemic stroke (weighted) DC + rtPA 338 237 101 DC only 2,783 1,784 999 DC, overall 3,121 2,021 1,099 Prevalence, per 10,000 hospitalizations with ischemic stroke DC + rtPA 0.80 (0.00) 2.99 (0.00) 0.29 (0.00) DC only 6.55 (0.00) 22.44 (0.01) 2.89 (0.00) DC, overall 7.35 (0.00) 25.43 (0.02) 3.18 (0.00) Hospitalizations with ischemic stroke listed as the primary diagnosis were identified using the first listed International Classification of Disease 9th Revision clinical modifier (ICD-9 CM) diagnostic codes 433.11, 433.31, 433.81, 433.91, 434.01, 434.11, 434.91, and 436). Patients with the diagnosis of intracranial hemorrhage (ICD-9 CM diagnostic codes 430, 431, and 432.x) and patients with the diagnosis of posterior intracranial circulation occlusion (ICD-9 CM diagnostic codes 433.01 & 433.21) were excluded. doi:10.1371/journal.pone.0029193.t002 Table 2. Numbers and prevalence of decompressive craniectomy with and without rtPA among hospitalizations for ischemic stroke by age (n = 4,248,955), Nationwide Inpatient Sample, 1999–2008. Hospitalizations with ischemic stroke listed as the primary diagnosis were identified using the first listed International Classification of Disease 9th Revision clinical modifier (ICD-9 CM) diagnostic codes 433.11, 433.31, 433.81, 433.91, 434.01, 434.11, 434.91, and 436). Patients with the diagnosis of intracranial hemorrhage (ICD-9 CM diagnostic codes 430, 431, and 432.x) and patients with the diagnosis of posterior intracranial circulation occlusion (ICD-9 CM diagnostic codes 433.01 & 433.21) were excluded. doi:10.1371/journal.pone.0029193.t002 among hospitalizations with ischemic stroke in the same dataset.[19] remainder of the study years). No variables for the timing of surgery, dimensions of bony decompression, stroke severity, or procedure laterality exist in the NIS dataset. Two primary end points were examined: in-hospital mortality percentage and discharge to institutions other than home. Results During 199922008 we identified 884,729 adult hospitalizations with ischemic stroke as a primary diagnosis in the NIS, representing 4,320,950 hospitalizations across the United States during this time period. (Figure 1) After excluding 71,995 patients with the diagnosis of intracranial hemorrhage and patients with the diagnosis of posterior intracranial circulation occlusion, our sample size was 4,248,955 hospitalizations. The estimated rates of hospitalizations in DC per 10,000 hospitalizations with ischemic stroke increased from 3.9 in 1999–2000 to 14.46 in 2007–2008 (p for linear trend,0.001) (Table 1). In 2007–2008, 1 out of 7 hospitalizations with DC also had performed intravenous thrombolysis with rtPA (recombinant tissue plasminogen activator). The rates of DC by two age groups are presented in table 2. The 18–59 year old group had significantly higher rates compared to 60+ year old group. Patient age, sex, primary payer (public, private, and others), type of admission (emergency, urgent, elective), admission source (emergency room, transfer from another hospital, transfer from long term care, and routine), hospital region (Northeast, Midwest, South, or West), hospital location and status (urban-teaching, urban-nonteaching, and rural), and bed size (small, medium, large), length of stay (days, continuous) and total hospital charges (US dollars, continuous) were coded in the NIS data. Hospital charges were converted to United States 2008 dollars using all- cities Consumer Price Index (http://data.bls.gov/cgi-bin/cpicalc. pl) Medical comorbidities were defined using markers described by Elixhauser et al[17] and calculated by use of the AHRQ software publically available at http://www.hcup-us.ahrq.gov/toolssoft- ware/comorbidity/comorbidity.jsp. We selected nine co-morbid- ity conditions (congestive heart failure, peripheral vascular disease, hypertension, paralysis, other neurological disorders, chronic pulmonary disease, diabetes with chronic complications, renal failure and coagulopathy) based on the significance of associations with in-hospital mortality reported in the previous studies.[18] We also identified four medical complications by searching the secondary diagnostic codes (ICD-9-CM codes) for pneumonia, deep vein thrombosis, pulmonary embolism, and acute myocardial infarction that were significant predictors of in-hospital mortality Among hospitalizations with ‘‘DC + rtPA’’, in-hospital mortality was higher among 60+ years old group compared to 18–59 years old group (44% vs. 24%), although the difference did not reach statistical significance (p =0.14) (Table 3). However, the difference was not statistically significant after adjustment for admission type, admission source, length of stay, total hospital charges, chronic comorbidities, and medical complications. Among hospitalizations with ‘‘DC only’’, differences in both unadjusted and adjusted in-hospital mortality rates between two age groups were highly non-significant. Methods To facilitate interpretations, adjusted prevalence was calculated from the overall and two age-specific logistic regression models using the PREDMARG statement in SUDAAN.[20] The adjusted percent- age, also known as predictive margin, was generated using the logistic regression model to estimate the probability of in-hospital mortality or probability of home care discharge status, averaging over the distribution of the covariates among the entire weighted sample. The reported percentage estimates were adjusted for survey design as well as the covariates listed above. The unit of analysis was the hospital discharge. The rates of DC per 10,000 hospitalizations with ischemic stroke were assessed across 5 time intervals: 1999–2000, 200122002, 200322004, 200522006, and 200722008. We have chosen this time frame since the procedure code for infusion of thrombolytic agent (ICD- 9-CM 99.10) was first introduced in October 1998. Orthogonal polynomial coefficients were obtained recursively by the method of Fisher and Yates to test linear trends for the two year intervals. The rates of DC per 10,000 hospitalizations with ischemic stroke were also reported between these two age groups: 18–59 and 60+ years. We focused our analysis on these two groups since age is considered a major prognostic factor of functional recovery after brain infarction in general (and malignant infarction in particular) and randomized trials have focused on patient of 60 years and younger.[15–16] Differences in rates of craniectomy by two age groups were compared by using chi-square tests. December 2011 \| Volume 6 \| Issue 12 \| e29193 PLoS ONE \| www.plosone.org Results Among 18–59 years old hospitalizations, no differences existed for in-hospital mortality and disposition status between ‘‘DC + PLoS ONE \| www.plosone.org December 2011 \| Volume 6 \| Issue 12 \| e29193 December 2011 \| Volume 6 \| Issue 12 \| e29193 PLoS ONE \| www.plosone.org 3 Craniectomy in Stroke Table 3. In-hospital mortality and routine disposition among hospitalizations for ischemic stroke by intervention group and age (N weighted = 3,121), Nationwide Inpatient Sample, 1999–2008. rtPA’’ and ‘‘DC only’’ in both unadjusted and adjusted analysis. A th 60+ ld h it li ti ‘‘DC + tPA’’ vs. 26%, p = 0.56). In the ‘‘DC + rtPA’’ group, no hospitalizations ith ti /h di iti t t b d p-value for difference 18– 59 years vs. 60+ Intervention group All 18–59 years 60+ years Hospitalizations (weighted) DC + rtPA 338 237 101 DC only 2,783 1,784 999 DC, overall 3,121 2,021 1,100 In-hospital mortality: Estimated percentage, % Unadjusted DC + rtPA 30.39 (0.06) 24.43 (0.06) 43.82 (0.12) .14 DC only 23.39 (0.02) 22.63 (0.02) 24.73 (0.02) .57 p-value for difference DC + rtPA vs. DC only .23 .77 .11 Adjusted: Model 1 DC + rtPA 30.81 (0.06) 24.55 (0.06) 43.13 (0.12) .16 DC only 23.56 (0.02) 22.97 (0.02) 24.75 (0.02) .68 p-value for difference DC + rtPA vs. DC only .25 .81 .13 Adjusted: Model 2* DC + rtPA 26.49 (0.04) 23.79 (0.06) 29.68 (0.07) .56 DC only 23.79 (0.02) 23.00 (0.02) 25.51 (0.03) .53 p-value for difference DC + rtPA vs. DC only .56 .89 .59 Routine/home care disposition: Estimated percentage, % Unadjusted DC + rtPA 10.31 (0.06) 15.49 0 NA DC only 11.61 (0.04) 11.76 10.16 .32 p-value for difference DC + rtPA vs. DC only .89 .82 NA Adjusted: Model 1* DC + rtPA 17.11 (0.06) 24.53 (0.08) 0 NA DC only 13.11 (0.01) 13.10 (0.02) 13.11 (0.03) .99 p-value for difference DC + rtPA vs. DC only .50 .16 NA Adjusted: Model 2 DC + rtPA 19.18 (0.06) 26.53 (0.08) 0 NA DC only 12.69 (0.02) 12.82 (0.02) 12.43 (0.03) .92 p-value for difference DC + rtPA vs. Results Model 2: Average Marginal Prediction: percentage estimates are adjusted for survey design as well as model covariates (all variables in the model 1 + length of stay (days, continuous) and total charges (US dollars, continuous). doi:10.1371/journal.pone.0029193.t003 vs. 26%, p = 0.56). In the ‘‘DC + rtPA’’ group, no hospitalizations with routine/home care disposition status were observed. rtPA’’ and ‘‘DC only’’ in both unadjusted and adjusted analysis. Among the 60+ year old hospitalization group, ‘‘DC + rtPA’’ group had a higher in-hospital mortalty rate (44% vs. 25%, respectively) compared to ‘‘DC only’’ group (although the difference did not reach statistical significance, p = 0.14). The difference became highly non-significant after adjustment for admission type, admission source, length of stay, total hospital charges, chronic comorbidities, and medical complications (30% Results DC only .31 .10 NA Model 1: Average Marginal Prediction: percentage estimates are adjusted for survey design as well as model covariates (gender, hospital region (Northeast, Midwest, South, or West), hospital location and status (urban-teaching, urban-nonteaching, and rural), type of admission (emergency, urgent, elective), admission status (emergency room, transfer from another hospital, transfer from long term care, and routine), payer (public, private, and others), hospital bed size (small, medium, and large), congestive heart failure, peripheral vascular disease, hypertension, paralysis, other neurological disorders, chronic pulmonary disease, diabetes with chronic complications, renal failure, coagulopathy, pneumonia, pulmonary embolism, acute myocardial infarction, and deep venous thrombosis. Model 2: Average Marginal Prediction: percentage estimates are adjusted for survey design as well as model covariates (all variables in the model 1 + length of stay (days, continuous) and total charges (US dollars, continuous). doi:10.1371/journal.pone.0029193.t003 Model 1: Average Marginal Prediction: percentage estimates are adjusted for survey design as well as model covariates (gender, hospital region (Northeast, Midwest, South, or West), hospital location and status (urban-teaching, urban-nonteaching, and rural), type of admission (emergency, urgent, elective), admission status (emergency room, transfer from another hospital, transfer from long term care, and routine), payer (public, private, and others), hospital bed size (small, medium, and large), congestive heart failure, peripheral vascular disease, hypertension, paralysis, other neurological disorders, chronic pulmonary disease, diabetes with chronic complications, renal failure, coagulopathy, pneumonia, pulmonary embolism, acute myocardial infarction, and deep venous thrombosis. *Model 2: Average Marginal Prediction: percentage estimates are adjusted for survey design as well as model covariates (all variables in the model 1 + length of stay (days, continuous) and total charges (US dollars, continuous). doi:10.1371/journal.pone.0029193.t003 Model 1: Average Marginal Prediction: percentage estimates are adjusted for survey design as well as model covariates (gender, hospital region (Northeast, Midwest, South, or West), hospital location and status (urban-teaching, urban-nonteaching, and rural), type of admission (emergency, urgent, elective), admission status (emergency room, transfer from another hospital, transfer from long term care, and routine), payer (public, private, and others), hospital bed size (small, medium, and large), congestive heart failure, peripheral vascular disease, hypertension, paralysis, other neurological disorders, chronic pulmonary disease, diabetes with chronic complications, renal failure, coagulopathy, pneumonia, pulmonary embolism, acute myocardial infarction, and deep venous thrombosis. Discussion We studied a population of patients undergoing craniectomy after inpatient admission for ischemic stroke by using a nationally representative hospital discharge database. The results of our December 2011 \| Volume 6 \| Issue 12 \| e29193 PLoS ONE \| www.plosone.org December 2011 \| Volume 6 \| Issue 12 \| e29193 4 Craniectomy in Stroke previously reported randomized trials and metaanalysis when considering age group.[9–10] [15] While the strength of this study is its large cohort size, an inherent deficiency is that long term outcomes, quality of life, and neurological status are unable to be determined. analysis showed that the annual number of craniotomies performed for this condition increased significantly and rapidly during the studied years, from 1999–2008. This large, inclusive cohort likely identifies outcomes related to actual practice patterns rather than those found in carefully selected and controlled randomized trials. It should be noted that a manuscript analyzing the same topic of DC for stroke was published during the editing phase of this manuscript. Alshekhlee et al. came to a completely different conclusion, that the prevalence is the same over a similar time period. [36] While the same database was analyzed over a slightly different time period, their analysis only identified 252 patients undergoing DC (whereas our study identified 3121 patients) for ischemic stroke. These small numbers, in combination with only 502,231 patients identified as having an acute ischemic stroke, raises the possibility that the investigators did not perform a valid, accurate weighted analysis to determine true nationwide estimates. Conclusion DC is being increasingly performed for malignant cerebral edema following large territory cerebral ischemia. We ascertain that the increase in the annual incidence of DC for malignant cerebral edema is directly related to the expanding collection of evidence in randomized trials that the operation is efficacious when performed in the correct patient population. In hospital mortality is high for all patients undergoing this procedure, with the highest being in those greater than 60 years of age. While in-hospital mortality tended to be higher in the 60+ age group, adjustment resulted in no identifiable difference between the two groups. We suspect that this finding relates to the higher incidence of brain herniation in this population and is heavily influenced by medical complications & comorbidities across all ages. The in-hospital mortality associated with surgical interven- tion is high in this cohort (range = 23243%) and comparable to Discussion While in-hospital mortality tended to be higher in the 60+ age group, adjustment resulted in no identifiable difference between the two groups. We suspect that this finding relates to the higher incidence of brain herniation in this population and is heavily influenced by medical complications & comorbidities across all ages. The in-hospital mortality associated with surgical interven- tion is high in this cohort (range = 23243%) and comparable to The pathophysiology of malignant cerebral edema is complex, but ultimately can be traced to the metabolic and hemodynamic changes that result in breakdown of the blood brain barrier. [31– 32] Importantly, the use of rtPA in animal models has been shown to promote disruption of the blood-brain barrier that results in edema.[33] It is unclear how much the use of rtPA has to do with the development of malignant cerebral edema in humans. Patients in the subpopulation of our analysis that received rtPA had a comparable mortality following DC.[34] Confounding factors not accounted for when assessing the outcome of DC following rtPA include bias introduced from variability in stroke severity, access to care, and clinical presentation that correspond to the guidelines for safe rtPA administration.[35] In this respect, rtPA administration can be thought of as a general surrogate marker for these factors. 1. Delashaw JB, Broaddus WC, Kassell NF, Haley EC, Pendleton GA, et al. (1990) Treatment of right hemispheric cerebral infarction by hemicraniectomy. Stroke 21: 874–881. Author Contributions Conceived and designed the experiments: BPW EVK. Performed the experiments: BPW EVK WFA. Analyzed the data: BPW EVK BVN MGG JMS JVCEC. Contributed reagents/materials/analysis tools: EVK MGG. Wrote the paper: BPW EVK BVN MGG KTK JMS WFA JVCEC. Discussion In the early modern era of neurosurgery, DC was thoughtfully employed to alleviate elevated intracranial pressure.[21–22] Since the first use of DC as a specific treatment for the sequelae of stroke came in 1956, many studies have demonstrated evidence for its efficacy.[23–26] Although these studies demonstrate increases in overall survival in selected patients, consideration of quality of life measures and functional outcomes have limited its use.[3][27] In recent years, several randomized controlled trials were performed that demonstrated efficacy of DC for large territory cerebral stroke in patients generally less than 60 years of age.[9–11] There has also been evidence to reverse the longstanding dogma that dominant hemispheric stroke is associated with worse out- comes.[28–30] We suspect that the increase in the annual incidence of DC for malignant cerebral edema is directly related to the expanding collection of evidence in randomized trials that the operation is efficacious when performed in the correct patient population. Limitations of our analysis reflect inherent deficiencies of a nationwide, administrative database. Although coding for proce- dures in administrative data have been shown to be reliable, coding for medical complications and chronic co-morbidities may be prone to coding bias resulting in high specificity but low sensitivity selection.[37] Disposition status to home care must be interpreted with caution as there is no modifier available to denote situations such as comfort measures or home hospice care. There is no accounting for the timing of surgery, extent of bony decompression, stroke severity, or operative laterality in this dataset. p p The pathophysiology of malignant cerebral edema is complex, but ultimately can be traced to the metabolic and hemodynamic changes that result in breakdown of the blood brain barrier. [31– 32] Importantly, the use of rtPA in animal models has been shown to promote disruption of the blood-brain barrier that results in edema.[33] It is unclear how much the use of rtPA has to do with the development of malignant cerebral edema in humans. Patients in the subpopulation of our analysis that received rtPA had a comparable mortality following DC.[34] Confounding factors not accounted for when assessing the outcome of DC following rtPA include bias introduced from variability in stroke severity, access to care, and clinical presentation that correspond to the guidelines for safe rtPA administration.[35] In this respect, rtPA administration can be thought of as a general surrogate marker for these factors. 6. Wirtz CR, Steiner T, Aschoff A, Schwab S, Schnippering H, et al. (1997) Hemicraniectomy with dural augmentation in medically uncontrollable hemispheric infarction. Neurosurg Focus 2: E3; discussion 1 p followingE3. 2. Schwab S, Steiner T, Aschoff A, Schwarz S, Steiner HH, et al. (1998) Early hemicraniectomy in patients with complete middle cerebral artery infarction. Stroke 29: 1888–1893. 8. Kondziolka D, Fazl M (1988) Functional recovery after decompressive craniectomy for cerebral infarction. Neurosurgery 23: 143–147. References 9. Hofmeijer J, Kappelle LJ, Algra A, Amelink GJ, van Gijn J, et al. (2009) Surgical decompression for space-occupying cerebral infarction (the Hemicraniectomy After Middle Cerebral Artery infarction with Life-threatening Edema Trial [HAMLET]): a multicentre, open, randomised trial. Lancet Neurol 8: 326–333. 2. Schwab S, Steiner T, Aschoff A, Schwarz S, Steiner HH, et al. (1998) Early hemicraniectomy in patients with complete middle cerebral artery infarction. Stroke 29: 1888–1893. 10. Ju¨ttler E, Schwab S, Schmiedek P, Unterberg A, Hennerici M, et al. (2007) Decompressive Surgery for the Treatment of Malignant Infarction of the Middle Cerebral Artery (DESTINY): a randomized, controlled trial. Stroke 38: 2518–2525. 3. Gupta R, Connolly ES, Mayer S, Elkind MSV (2004) Hemicraniectomy for Massive Middle Cerebral Artery Territory Infarction: A Systematic Review. Stroke 35: 539–543. 11. Vahedi K, Vicaut E, Mateo J, Kurtz A, Orabi M, et al. (2007) Sequential-design, multicenter, randomized, controlled trial of early decompressive craniectomy in malignant middle cerebral artery infarction (DECIMAL Trial). Stroke 38: 2506–2517. 4. Carter BS, Ogilvy CS, Candia GJ, Rosas HD, Buonanno F (1997) One-year outcome after decompressive surgery for massive nondominant hemispheric infarction. Neurosurgery 40: 1168–1175;discussion 1175-1176. 12. Schneck MJ, Origitano TC (2008) Hemicraniectomy and durotomy for malignant middle cerebral artery infarction. Neurosurg. Clin. N. Am 19: 459–468, vi. 5. Uhl E, Kreth FW, Elias B, Goldammer A, Hempelmann RG, et al. (2004) Outcome and prognostic factors of hemicraniectomy for space occupying cerebral infarction. J. Neurol. Neurosurg. Psychiatr 75: 270–274. 13. Schirmer CM, Ackil AA, Jr., Malek AM (2008) Decompressive Craniectomy. Neurocrit Care 8: 456–470. doi:10.1007/s12028-008-9082-y. 6. Wirtz CR, Steiner T, Aschoff A, Schwab S, Schnippering H, et al. (1997) Hemicraniectomy with dural augmentation in medically uncontrollable hemispheric infarction. Neurosurg Focus 2: E3; discussion 1 p followingE3. 14. HCUP Nationwide Inpatient Sample (NIS). Healthcare Cost and Utilization Project (HCUP) (1992) Available: http://www.hcup-us.ahrq.gov/nisoverview. jsp. Accessed 2011 Nov 23. 7. Steiger HJ (1991) Outcome of acute supratentorial cerebral infarction in patients under 60. Development of a prognostic grading system. Acta Neurochir (Wien) 111: 73–79. 15. Arac A, Blanchard V, Lee M, Steinberg GK (2009) Assessment of outcome following decompressive craniectomy for malignant middle cerebral artery infarction in patients older than 60 years of age. Neurosurg Focus 26: E3. 8. Kondziolka D, Fazl M (1988) Functional recovery after decompressive craniectomy for cerebral infarction. Neurosurgery 23: 143–147. 8. Kondziolka D, Fazl M (1988) Functional recovery after decompressive craniectomy for cerebral infarction. References Neurosurgery 23: 143–147. PLoS ONE \| www.plosone.org 5 December 2011 \| Volume 6 \| Issue 12 \| e29193 December 2011 \| Volume 6 \| Issue 12 \| e29193 Craniectomy in Stroke 16. Simard JM, Sahuquillo J, Sheth KN, Kahle KT, Walcott BP (2011) Managing malignant cerebral infarction. Curr Treat Options Neurol 13: 217–229. improved survival but poor functional outcome. J Neurol Neurosurg Psychiatry 70: 226–228. improved survival but poor functional outcome. J Neurol Neurosurg Psychiatry 70: 226–228. g p 17. 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Stroke 36: 1954–1959. 34. PLoS ONE \| www.plosone.org References Kleindorfer D, Lindsell CJ, Brass L, Koroshetz W, Broderick JP (2008) National US estimates of recombinant tissue plasminogen activator use: ICD-9 codes substantially underestimate. Stroke 39: 924–928. 22. Dandy WE (1937) Intracranial Pressure Without Brain Tumor: Diagnosis and Treatment. Ann. Surg 106: 492–513. g 23. Scarcella G (1956) Encephalomalacia simulating the clinical and radiological aspects of brain tumor; a report of 6 cases. J Neurosurg. 278 p. 35. Adams HP, del Zoppo G, Alberts MJ, Bhatt DL, Brass L, et al. (2007) Guidelines for the Early Management of Adults With Ischemic Stroke: A Guideline From the American Heart Association/American Stroke Association Stroke Council, Clinical Cardiology Council, Cardiovascular Radiology and Intervention Council, and the Atherosclerotic Peripheral Vascular Disease and Quality of Care Outcomes in Research Interdisciplinary Working Groups: The American Academy of Neurology affirms the value of this guideline as an educational tool for neurologists. Circulation 115: e478–534. 24. Pillai A, Menon SK, Kumar S, Rajeev K, Kumar A, et al. (2007) Decompressive hemicraniectomy in malignant middle cerebral artery infarction: an analysis of long-term outcome and factors in patient selection. J Neurosurg 106: 59–65. 25. Rabinstein AA, Mueller-Kronast N, Maramattom BV, Zazulia AR, Bamlet WR, et al. (2006) Factors predicting prognosis after decompressive hemicraniectomy for hemispheric infarction. Neurology 67: 891–893. 26. Foerch C, Lang JM, Krause J, Raabe A, Sitzer M, et al. (2004) Functional impairment, disability, and quality of life outcome after decompressive hemicraniectomy in malignant middle cerebral artery infarction. J Neurosurg 101: 248–254. 36. Alshekhlee A, Horn C, Jung R, Alawi AA, Cruz-Flores S (2011) In-hospital mortality in acute ischemic stroke treated with hemicraniectomy in US hospitals. J Stroke Cerebrovasc Dis 20: 196–201. 37. Ingeman A, Andersen G, Hundborg HH, Johnsen SP (2010) Medical complications in patients with stroke: data validity in a stroke registry and a hospital discharge registry. Clin Epidemiol 2: 5–13. 27. Holtkamp M, Buchheim K, Unterberg A, Hoffmann O, Schielke E, et al. (2001) Hemicraniectomy in elderly patients with space occupying media infarction: PLoS ONE \| www.plosone.org December 2011 \| Volume 6 \| Issue 12 \| e29193 6
https://openalex.org/W2615990115	http://www.tara.tcd.ie/bitstream/2262/91753/1/f1000research-6-12627.pdf	English	null	Metabolic reprogramming during the Trypanosoma brucei life cycle	F1000Research	2,017	cc-by	11,216	REVIEW Metabolic reprogramming during the Trypanosoma life cycle brucei [version 2; referees: 4 approved] Equal contributors * Open Peer Review Referee Status: Invited Referees version 2 published 18 May 2017 version 1 published 16 May 2017 1 2 3 4 16 May 2017, (F1000 Faculty Rev):683 (doi: First published: 6 ) 10.12688/f1000research.10342.1 18 May 2017, (F1000 Faculty Rev):683 (doi: Latest published: 6 ) 10.12688/f1000research.10342.2 v2 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 Open Peer Review REVIEW Metabolic reprogramming during the Trypanosoma life cycle brucei [version 2; referees: 4 approved] Terry K. Smith , Frédéric Bringaud , Derek P. Nolan , Luisa M. Figueiredo 4 Biomedical Sciences Research Complex, University of St Andrews, Fife, UK Laboratoire de Microbiologie Fondamentale et Pathogénicité (MFP), UMR 5234 CNRS, Université de Bordeaux, Bordeaux, France School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Equal contributors Abstract Cellular metabolic activity is a highly complex, dynamic, regulated process that is influenced by numerous factors, including extracellular environmental signals, nutrient availability and the physiological and developmental status of the cell. The causative agent of sleeping sickness, , is an Trypanosoma brucei exclusively extracellular protozoan parasite that encounters very different extracellular environments during its life cycle within the mammalian host and tsetse fly insect vector. In order to meet these challenges, there are significant alterations in the major energetic and metabolic pathways of these highly adaptable parasites. This review highlights some of these metabolic changes in this early divergent eukaryotic model organism. 1* 2* 3 4 1 2 3 4 * Referee Status: Invited Referees version 2 published 18 May 2017 version 1 published 16 May 2017 1 2 3 4 16 May 2017, (F1000 Faculty Rev):683 (doi: First published: 6 ) 10.12688/f1000research.10342.1 18 May 2017, (F1000 Faculty Rev):683 (doi: Latest published: 6 ) 10.12688/f1000research.10342.2 v2 Abstract 16 May 2017, (F1000 Faculty Rev):683 (doi: ) First published: 6 10.12688/f1000research.10342.1 Republic Canadian Institute for Advanced Research Canada , Center for Infectious Kenneth Stuart Disease Research USA 2 , National University of Cynthia He Yingxin Singapore Singapore 3 , Yale University USA Christian Tschudi 4 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. TKS is supported by the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, the Engineering and Grant information: Physical Sciences Research Council, the Medical Research Council (MR/M020118/1) and the European Community Seventh Framework Programme under grant agreement 602773 (Project KINDRED). FB is supported by the Centre National de la Recherche Scientifique (CNRS), the Université de Bordeaux, the Agence Nationale de la Recherche (ANR) through the GLYCONOV grant (ANR-15-CE15-0025-01) of the ‘Générique’ 2015 call and the Laboratoire d’Excellence (LabEx) ParaFrap (grant ANR-11-LABX-0024). DPN is supported by the Wellcome Trust and Science Foundation Ireland. LMF is supported by the Howard Hughes Medical Institute (55007419) and the Fundação para a Ciência e a Tecnologia (FCT) (PTDC/BIM-MET/4471/2014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors declare that they have no competing interests. 16 May 2017, (F1000 Faculty Rev):683 (doi: ) First published: 6 10.12688/f1000research.10342.1 Luisa M. Figueiredo ( ) Corresponding author: lmf@medicina.ulisboa.pt Luisa M. Figueiredo ( ) Corresponding author: lmf@medicina.ulisboa.pt Smith TK, Bringaud F, Nolan DP and Figueiredo LM. How to cite this article: Metabolic reprogramming during the life Trypanosoma brucei 2017, (F1000 Faculty Rev):683 (doi: ) cycle [version 2; referees: 4 approved] F1000Research 6 10.12688/f1000research.10342.2 © 2017 Smith TK . This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. TKS is supported by the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, the Engineering and Grant information: Physical Sciences Research Council, the Medical Research Council (MR/M020118/1) and the European Community Seventh Framework Programme under grant agreement 602773 (Project KINDRED). FB is supported by the Centre National de la Recherche Scientifique (CNRS), the Université de Bordeaux, the Agence Nationale de la Recherche (ANR) through the GLYCONOV grant (ANR-15-CE15-0025-01) of the ‘Générique’ 2015 call and the Laboratoire d’Excellence (LabEx) ParaFrap (grant ANR-11-LABX-0024). DPN is supported by the Wellcome Trust and Science Foundation Ireland. F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 Discuss this article (0) Comments Luisa M. Figueiredo ( ) Corresponding author: lmf@medicina.ulisboa.pt Smith TK, Bringaud F, Nolan DP and Figueiredo LM. How to cite this article: Metabolic reprogramming during the life Trypanosoma brucei 2017, (F1000 Faculty Rev):683 (doi: ) cycle [version 2; referees: 4 approved] F1000Research 6 10.12688/f1000research.10342.2 © 2017 Smith TK . This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. TKS is supported by the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, the Engineering and Grant information: Physical Sciences Research Council, the Medical Research Council (MR/M020118/1) and the European Community Seventh Framework Programme under grant agreement 602773 (Project KINDRED). FB is supported by the Centre National de la Recherche Scientifique (CNRS), the Université de Bordeaux, the Agence Nationale de la Recherche (ANR) through the GLYCONOV grant (ANR-15-CE15-0025-01) of the ‘Générique’ 2015 call and the Laboratoire d’Excellence (LabEx) ParaFrap (grant ANR-11-LABX-0024). DPN is supported by the Wellcome Trust and Science Foundation Ireland. LMF is supported by the Howard Hughes Medical Institute (55007419) and the Fundação para a Ciência e a Tecnologia (FCT) (PTDC/BIM-MET/4471/2014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors declare that they have no competing interests. 16 May 2017, (F1000 Faculty Rev):683 (doi: ) First published: 6 10.12688/f1000research.10342.1 Republic Canadian Institute for Advanced Research Canada , Center for Infectious Kenneth Stuart Disease Research USA 2 , National University of Cynthia He Yingxin Singapore Singapore 3 , Yale University USA Christian Tschudi 4 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 Discuss this article (0) Comments Republic Canadian Institute for Advanced Research Canada , Center for Infectious Kenneth Stuart Disease Research USA 2 , National University of Cynthia He Yingxin Singapore Singapore 3 , Yale University USA Christian Tschudi 4 Luisa M. Figueiredo ( ) Corresponding author: lmf@medicina.ulisboa.pt Smith TK, Bringaud F, Nolan DP and Figueiredo LM. How to cite this article: Metabolic reprogramming during the life Trypanosoma brucei 2017, (F1000 Faculty Rev):683 (doi: ) cycle [version 2; referees: 4 approved] F1000Research 6 10.12688/f1000research.10342.2 © 2017 Smith TK . Abstract Cellular metabolic activity is a highly complex, dynamic, regulated process that is influenced by numerous factors, including extracellular environmental signals, nutrient availability and the physiological and developmental status of the cell. The causative agent of sleeping sickness, , is an Trypanosoma brucei exclusively extracellular protozoan parasite that encounters very different extracellular environments during its life cycle within the mammalian host and tsetse fly insect vector. In order to meet these challenges, there are significant alterations in the major energetic and metabolic pathways of these highly adaptable parasites. This review highlights some of these metabolic changes in this early divergent eukaryotic model organism. F1000 Faculty Reviews are commissioned from members of the prestigious F1000 . In order to make these reviews as Faculty comprehensive and accessible as possible, peer review takes place before publication; the referees are listed below, but their reports are not formally published. Page 1 of 12 Discuss this article (0) Comments Luisa M. Figueiredo ( ) Corresponding author: lmf@medicina.ulisboa.pt Smith TK, Bringaud F, Nolan DP and Figueiredo LM. How to cite this article: Metabolic reprogramming during the life Trypanosoma brucei 2017, (F1000 Faculty Rev):683 (doi: ) cycle [version 2; referees: 4 approved] F1000Research 6 10.12688/f1000research.10342.2 © 2017 Smith TK . This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. TKS is supported by the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, the Engineering and Grant information: Physical Sciences Research Council, the Medical Research Council (MR/M020118/1) and the European Community Seventh Framework Programme under grant agreement 602773 (Project KINDRED). FB is supported by the Centre National de la Recherche Scientifique (CNRS), the Université de Bordeaux, the Agence Nationale de la Recherche (ANR) through the GLYCONOV grant (ANR-15-CE15-0025-01) of the ‘Générique’ 2015 call and the Laboratoire d’Excellence (LabEx) ParaFrap (grant ANR-11-LABX-0024). DPN is supported by the Wellcome Trust and Science Foundation Ireland. LMF is supported by the Howard Hughes Medical Institute (55007419) and the Fundação para a Ciência e a Tecnologia (FCT) (PTDC/BIM-MET/4471/2014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors declare that they have no competing interests. 1. Introduction Trypanosoma brucei is a unicellular protozoan parasite, transmit- ted by the bite of tsetse flies (Glossina genus). Different species/ subspecies of trypanosomes infect a variety of different verte- brates, including animals and humans. Human African trypano- somiasis (HAT), also known as sleeping sickness, is caused by two subspecies: Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. In recent years, the number of reported cases of HAT has decreased steadily, falling to just about 6,000 in 20131. Other trypanosome species infect both domestic and wild animals, causing animal African trypanosomiasis. The infection of livestock has a major impact on the African economy, limiting the produc- tion of milk and meat and the development of agriculture in areas otherwise amenable to animal husbandry2. Throughout the life cycle, parasites encounter and adapt to very different environments. In the mammalian host, such adaptations include avoidance of the host immune system (by employing antigenic variation) as well as metabolic adaptations to use avail- able nutrients. For example, the brain glucose levels is normally 10–20% of blood levels8, whereas adipose tissue may be a bet- ter source of lipids. In the tsetse fly vector, the parasites face a Trypanosomatids are also of intrinsic scientific interest as they separated early (>600 million years ago) and have evolved differ- ently from other well-studied eukaryotes3. T. brucei brucei (here called T. brucei), a subspecies non-infectious to human, is by far Figure 1. Changes in metabolism during the life cycle of Trypanosoma brucei. T. brucei life cycle spans two hosts: a mammal (human, cattle, wild animals) and the tsetse fly. As this protozoan parasite is extracellular, it adapts its metabolism to the available extracellular nutrients. The two stages that have been better characterised in terms of metabolism are the bloodstream long slender and procyclic forms, which mainly catabolise glucose and proline, respectively. Fewer studies have studied bloodstream short stumpy forms. In the mammalian host, parasites accumulate in the interstitial spaces of several tissues, mainly the brain, skin and visceral adipose tissue (adipocytes are shown as an example). The metabolism of parasites in these tissues remains mostly unknown, except for the activation of fatty acid β-oxidation in parasites resident of the adipose tissue. Metabolism of metacyclic stage has not been characterised to date. TAO, trypanosome alternative oxidase. Figure 1. Changes in metabolism during the life cycle of Trypanosoma brucei. T. Amendments from Version 1 REVISED This version 2 highlights an additional question of how the parasite metabolism changes during the day to Section 5, as this was missing from the previous version. See referee reports In the mammalian host, parasites exist in two stages: bloodstream long slender form (B-LS), which doubles every 7 hours by binary fission, and short stumpy form (B-SS), which is terminally cell cycle–arrested (Figure 1). The differentiation from B-LS to B-SS is irreversible and is triggered by a quorum-sensing mechanism7. The B-SS form is pre-adapted to life in the tsetse fly midgut7. These pre-adaptions probably help in the efficient differentiation into the replicative procyclic forms (PFs). Eventually, PFs migrate from the midgut to the proventriculus, where they further differen- tiate into epimastigotes and later into metacyclics in the salivary glands (Figure 1). The latter are cell cycle–arrested and are able to re-colonise/re-infect a mammalian host when a tsetse fly takes a blood meal. F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 16 May 2017, (F1000 Faculty Rev):683 (doi: ) First published: 6 10.12688/f1000research.10342.1 Page 2 of 12 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 the best characterised. In the mammalian host, T. brucei parasites colonise the blood and interstitial spaces of several tissues, includ- ing the brain, adipose tissue and skin4–6. The presence of parasites in the brain is associated with the appearance of the sleep disor- der and neurological symptoms characteristic of later stages of the disease1. the best characterised. In the mammalian host, T. brucei parasites colonise the blood and interstitial spaces of several tissues, includ- ing the brain, adipose tissue and skin4–6. The presence of parasites in the brain is associated with the appearance of the sleep disor- der and neurological symptoms characteristic of later stages of the disease1. F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 LMF is supported by the Howard Hughes Medical Institute (55007419) and the Fundação para a Ciência e a Tecnologia (FCT) (PTDC/BIM-MET/4471/2014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors declare that they have no competing interests. 16 May 2017, (F1000 Faculty Rev):683 (doi: ) First published: 6 10.12688/f1000research.10342.1 Luisa M. Figueiredo ( ) Corresponding author: lmf@medicina.ulisboa.pt Smith TK, Bringaud F, Nolan DP and Figueiredo LM. How to cite this article: Metabolic reprogramming during the life Trypanosoma brucei 2017, (F1000 Faculty Rev):683 (doi: ) cycle [version 2; referees: 4 approved] F1000Research 6 10.12688/f1000research.10342.2 g ( ) p g @ p Smith TK, Bringaud F, Nolan DP and Figueiredo LM. How to cite this article: Metabolic reprogramming during the life Trypanosoma brucei 2017, (F1000 Faculty Rev):683 (doi: ) cycle [version 2; referees: 4 approved] F1000Research 6 10.12688/f1000research.10342.2 © 2017 Smith TK . This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. TKS is supported by the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, the Engineering and Grant information: Physical Sciences Research Council, the Medical Research Council (MR/M020118/1) and the European Community Seventh Framework Programme under grant agreement 602773 (Project KINDRED). FB is supported by the Centre National de la Recherche Scientifique (CNRS), the Université de Bordeaux, the Agence Nationale de la Recherche (ANR) through the GLYCONOV grant (ANR-15-CE15-0025-01) of the ‘Générique’ 2015 call and the Laboratoire d’Excellence (LabEx) ParaFrap (grant ANR-11-LABX-0024). DPN is supported by the Wellcome Trust and Science Foundation Ireland. LMF is supported by the Howard Hughes Medical Institute (55007419) and the Fundação para a Ciência e a Tecnologia (FCT) (PTDC/BIM-MET/4471/2014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. mith TK . This is an open access article distributed under the terms of the , whic et al Creative Commons Attribution Licence e, distribution, and reproduction in any medium, provided the original work is properly cited. 2.2. Metabolic adaptations in eukaryotes 2.2. Metabolic adaptations in eukaryotes Textbooks on metabolism explain that in nutrient-rich conditions, model unicellular organisms undergoing exponential growth often use fermentation11. Proliferating cells in a multicellular organism also metabolise glucose primarily through glycolysis, secreting ethanol, lactate, or another organic acid such as acetate. When unicellular organisms are starved of nutrients, they switch and rely primarily on oxidative metabolism, as do terminally differenti- ated cells in a multicellular organism. It is no surprise that there are many exceptions to these generalised concepts, and as we will describe below (Section 4), T. brucei is a quintessential example of these exceptions. Most organisms derive energy from the breakdown of glucose, by a process known as glycolysis, a universal and evolutionarily ancient metabolic pathway, which converts glucose (6-carbon) into pyruvate (3-carbon). Under aerobic conditions, pyruvate can undergo further breakdown to acetyl coenzyme A (acetyl-CoA) (2-carbon) and subsequently to carbon dioxide (CO2) via the tricarboxylic acid (TCA) cycle with the concomitant production of reducing equivalence (NADH and FADH2) and GTP. Transfer of electrons from these reduced cofactors to oxygen via an electron transport chain generates a proton electrochemical gradient across the mitochondrial inner membrane that is used to generate ATP by a membrane-bound ATP synthase, collectively a process termed oxidative phosphorylation (OXPHOS). The complete oxidation of each glucose molecule leads to the production of about 36 ATP molecules, showing how OXPHOS is a very efficient mechanism of producing energy. The metabolism of cells is a highly regulated process that is influ- enced by numerous extracellular factors. For example, yeast uses glucose from the environment as their preferred carbon source. Even in the presence of oxygen, glucose is converted into excreted ethanol, with a low yield of ATP production. Although this process may seem wasteful, it is a highly efficient way to support expo- nential growth. When glucose levels are low, yeast undergoes a diauxic switch to another carbon source, ethanol, which requires an alteration of its mitochondrial metabolism. By using this alternative carbon source, cells are able to continue to grow and divide, but at a significantly reduced rate12. In the absence of oxygen, the glycolysis product (pyruvate or phos- phoenolpyruvate) can be further metabolised by fermentation into excreted end-products, such as lactate (for instance, in humans during running) and ethanol (for instance, in yeast) in the cyto- plasm, leading to the net production of two ATP molecules per molecule of glucose consumed. 1. Introduction brucei life cycle spans two hosts: a mammal (human, cattle, wild animals) and the tsetse fly. As this protozoan parasite is extracellular, it adapts its metabolism to the available extracellular nutrients. The two stages that have been better characterised in terms of metabolism are the bloodstream long slender and procyclic forms, which mainly catabolise glucose and proline, respectively. Fewer studies have studied bloodstream short stumpy forms. In the mammalian host, parasites accumulate in the interstitial spaces of several tissues, mainly the brain, skin and visceral adipose tissue (adipocytes are shown as an example). The metabolism of parasites in these tissues remains mostly unknown, except for the activation of fatty acid β-oxidation in parasites resident of the adipose tissue. Metabolism of metacyclic stage has not been characterised to date. TAO, trypanosome alternative oxidase. Page 3 of 12 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 proteolytic rather than immune challenge and also have to adapt to an environment that is free of glucose but rich in amino acids, particularly proline9. T. brucei re-programmes its metabolism in order to benefit from the nutrients available in the environment. In this review, we will compare the metabolic differences that take place during the T. brucei life cycle, highlighting the questions that remain unanswered. To facilitate the understanding of this review by a non-metabolism expert, we will first summarise the main metabolic pathways present in most eukaryotic cells. Amino acids can also contribute to total energy production by oxidation to urea and CO2. The first reaction is the removal of the amino group by transaminases. While the amino group enters the urea cycle, the ketoacid carbon skeletons typically enter the TCA cycle and fuel OXPHOS. The relative abundances of sugars, amino acids, and fatty acids along with the availability of sufficient oxygen to use OXPHOS influence which metabolic pathways are preferentially used to produce ATP. Thus, the metabolic profile of a cell is a consequence of the regulated expression of pathway-specific proteins and asso- ciated transporters in response to extracellular nutritional and environmental conditions10. 2. Basics of eukaryote metabolism 1. Multiple carbon sources for energy production All living organisms use adenosine triphosphate (ATP) as an intracellular energy source. ATP is generated by the catabolism (breakdown) of nutrients. The most common nutrients or carbon sources are carbohydrates (such as glucose), fatty acids and amino acids. 3.2. Trypanosomes have specific pathways and unique enzymesi Kinetoplastida are of great intrinsic scientific interest as they have diverged very early compared with most studied eukaryotic models (for example, yeast, plants and animals), on which the founda- tions of molecular, biochemical and cellular biology have been built. Cytochrome oxidase (COX) is the terminal oxidase of the mammalian electron transport chain and is responsible for the In contrast, ADP and ATP molecules can be exchanged between the cytosolic and mitochondrial compartments through the Figure 2. Multiple pathways to produce ATP in trypanosomes. Catabolism of the most abundant carbon sources in procyclic form grown in glucose-depleted (A) or in glucose-containing (B) conditions and in bloodstream long slender form (C). Excreted end-products from glucose and proline degradation (pyruvate, acetate, succinate and alanine) are underlined. Arrows with different thicknesses tentatively represent the metabolic flux at each enzymatic step. In (B), the direction of ADP/ATP exchange between the cytosol and the mitochondrion (step 14) is unknown and is represented by double arrows. Key enzymatic steps: 1a, glycosomal phosphoglycerate kinase; 1b, cytosolic phosphoglycerate kinase; 2, pyruvate kinase; 3, phosphoenolpyruvate carboxykinase; 4, glycosomal malate dehydrogenase; 5, cytosolic fumarase (for simplification this reaction is placed in the glycosome); 6, glycosomal NADH-dependent fumarate reductase; 7, pyruvate phosphate dikinase; 8, acetate:succinate coenzyme A-transferase, or ASCT; 9, acetyl-coenzyme A thioesterase; 10, succinyl-coenzyme A synthetase; 11, trypanosome alternative oxidase; 12, respiratory chain; 13, F0F1-ATP synthase; 14, mitochondrial ADP/ATP exchanger. AcCoA, acetyl-coenzyme A; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde 3-phosphate; Gly3P, glycerol 3-phosphate; MAL, malate; PEP, phosphoenolpyruvate; PYR, pyruvate; SUC, succinate. Figure 2. Multiple pathways to produce ATP in trypanosomes. Catabolism of the most abundant carbon sources in procyclic form grown in glucose-depleted (A) or in glucose-containing (B) conditions and in bloodstream long slender form (C). Excreted end-products from glucose and proline degradation (pyruvate, acetate, succinate and alanine) are underlined. Arrows with different thicknesses tentatively represent the metabolic flux at each enzymatic step. In (B), the direction of ADP/ATP exchange between the cytosol and the mitochondrion (step 14) is unknown and is represented by double arrows. 2.2. Metabolic adaptations in eukaryotes Regulatory T (Treg) cells predominantly use OXPHOS for development and survival15,16, while activated B cells show increased glucose uptake and induction of glycolysis17. These examples demonstrate how malleable metabolism is, as cells react to environmental factors and signals to acquire new functions. 3. Trypanosomes have unusual metabolic features 3.1. Trypanosomes have a single mitochondrion and glycosomes Significant differential expression of mitochondrial and glyco- somal proteins occurs during the life cycle21. Indeed, during the differentiation of B-LS into PFs, degradation of glycosomes prob- ably via autophagy is enhanced and new glycosomes with different enzymatic contents are produced, so that parasites become rapidly metabolically adapted to the new host environment22. In fact, the differential expression of glycosomal and mitochondrial proteins is a clear indicator of the difference in metabolic life styles between the two major life-cycle stages of T. brucei. Trypanosomes are characterised by the presence of a dense network of circularised interlocking rings of mitochondrial DNA termed the kinetoplast, located within the large, single mitochondrion of the cell. The single mitochondrion in PFs has a highly defined branched structure with discoid cristae, whereas in the B-LS forms the organelle is a less well-developed narrow tubular structure with an acristate morphology similar to that of the promitochondrion of anaerobic yeast18,19. T. brucei also contains peroxisome-like organelles, named glycosomes, which contain the first six (PF) or seven (B-LS) glycolytic enzymes3. Since the glycosomal membrane is impermeable to ATP, no net ATP production occurs inside these organelles. Thus, net ATP production from glycolysis occurs during the cytoplasmic steps (Figure 2). 2.2. Metabolic adaptations in eukaryotes Although the flux through fermen- tation can be very high, the pathway is energetically inefficient in terms of ATP production. In the absence of oxygen, some micro- organisms use nitrate ions, sulfate ions, and carbon dioxide as final electron acceptors, in a process named anaerobic respiration. For example, the final product of glycolysis could be converted into acetyl-CoA, which enters the TCA cycle or is converted into acetate. The electrons are then donated to the final acceptor through the mitochondrial electron transport chain. In mammals, most non-proliferating differentiated cells use glycolysis and OXPHOS to generate ATP and convert glucose to CO2 and H2O. However, most proliferating cancer cells convert glucose into pyruvate and lactate (3-carbon) even under aerobic conditions, a phenomenon known as the Warburg effect, named after its discoverer11,13. Although this fermentation-like process is intrinsically energetically less efficient, these cells use far higher rates of glycolysis to meet their higher metabolite demand as they divide faster. This metabolic reprogramming allows cancer cells to rapidly produce the building blocks and increase total biomass for a faster propagation time14. In many organisms, fatty acids can be catabolised via β-oxidation in the mitochondria to again generate the 2-carbon unit acetyl- CoA, which feeds into the TCA cycle and OXPHOS. Fatty acid β-oxidation of a palmitate molecule (a fatty acid with 16 carbons that is very abundant in mammalian adipocytes) can generate 106 ATP molecules. The balance between making and breaking down fatty acids is tightly regulated. Major metabolic changes also take place when immune cells are activated and initiate proliferation. When T cells are activated upon infection or inflammation, gene expression is reprogrammed, resulting in rapid growth, proliferation and the acquisition of new effector functions. Effector T cells, like cancer cells, rely upon aerobic glycolysis when proliferating11. In contrast, T cells destined Page 4 of 12 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 TbMCP5 mitochondrial ADP/ATP exchanger20. This exchanger is required because oxidative phosphorylation does not occur in B-LS and thus no ATP is generated inside this organelle. To maintain the mitochondrial proton electrochemical gradient across the mitochondrial membrane, the F1F0-ATPase operates in the reverse direction, hydrolysing ATP into ADP (Figure 2). This unu- sual way of generating a mitochondrial potential also implies that a functional phosphate/H+ exchanger must be present. to become memory cells maintain an oxidative metabolism, which allows them to keep their quiescence and longevity. 3.2. Trypanosomes have specific pathways and unique enzymesi Key enzymatic steps: 1a, glycosomal phosphoglycerate kinase; 1b, cytosolic phosphoglycerate kinase; 2, pyruvate kinase; 3, phosphoenolpyruvate carboxykinase; 4, glycosomal malate dehydrogenase; 5, cytosolic fumarase (for simplification this reaction is placed in the glycosome); 6, glycosomal NADH-dependent fumarate reductase; 7, pyruvate phosphate dikinase; 8, acetate:succinate coenzyme A-transferase, or ASCT; 9, acetyl-coenzyme A thioesterase; 10, succinyl-coenzyme A synthetase; 11, trypanosome alternative oxidase; 12, respiratory chain; 13, F0F1-ATP synthase; 14, mitochondrial ADP/ATP exchanger. AcCoA, acetyl-coenzyme A; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde 3-phosphate; Gly3P, glycerol 3-phosphate; MAL, malate; PEP, phosphoenolpyruvate; PYR, pyruvate; SUC, succinate. Page 5 of 12 Page 5 of 12 Page 5 of 12 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 via glycolysis (Figure 1 and Figure 2C). Unlike proliferative yeast and tumour cells, B-LS does not undergo fermentation per se. In fact, instead of being metabolised and generating ethanol or lactate, most pyruvate in B-LS is immediately excreted, and only about 1% is fermented into succinate30,31. To oxidise the NADH produced during glycolysis back to NAD+, B-LS consume large amounts of oxygen that act as an electron acceptor in a reaction catalysed by the unusual TAO. This type of glucose metabolism is uncommon and does not fit textbook knowledge. Interestingly, B-LS cells also tolerate anaerobic conditions where they convert glucose to equi- molar amounts of glycerol and pyruvate, with a two-fold reduction of the ATP production rate. reduction of oxygen to water. However, T. brucei possesses an additional plant-like, non-energy-conserving terminal oxidase called alternative oxidase (TAO). Indeed, B-LS forms are unique in the sense that they do not use COX but rely on TAO (step 11 in Figure 2C). TAO is 100-fold more expressed in B-LS than PFs and thus is considered a potential drug target23. reduction of oxygen to water. However, T. brucei possesses an additional plant-like, non-energy-conserving terminal oxidase called alternative oxidase (TAO). Indeed, B-LS forms are unique in the sense that they do not use COX but rely on TAO (step 11 in Figure 2C). TAO is 100-fold more expressed in B-LS than PFs and thus is considered a potential drug target23. Sphingolipids are a class of lipids important in cell recognition and signal transmission. To date, T. brucei is the only organism known to make all three types of sphingolipids (sphingomyelin, inositol- phosphoceramide and ethanolamine-phosphoceramide). These lipids are synthesised via four sphingolipid synthases (SLSs) that are encoded by genes organised in a tandem array. 4. Metabolic adaptations during the Trypanosoma brucei life cycle The bloodstream of a mammalian host is a very rich environment, containing 5 mM of glucose, 95% to 99% oxygen saturation levels and 0.6 to 0.8 g/mL proteins, including lipoproteins (low-density lipoprotein and high-density lipoprotein). In contrast, when para- sites are ingested by the tsetse during a blood meal, they end up in a glucose-poor but amino acid-rich environment that is very different from the mammalian bloodstream. Given that we can mimic these growing conditions in vitro, most of our knowledge about meta- bolic changes during the T. brucei life cycle originates from the comparison between B-LS and PFs. 4.1. Amino acids: an abundant carbon source in the midgut of the flyl The midgut of the tsetse fly has a temperature of about 28°C and a variable pH and contains hardly any glucose, but is rich in amino acids, such as proline (about 100 μM)9. It is well accepted that, in a glucose-depleted environment, PFs primarily use proline for their energy production25,26 (Figure 1), but catabolism of other amino acids, such as threonine and leucine, is also used27,28. These latter amino acids feed fatty acid biosynthesis and/or enter into the meval- onate pathway to produce the building blocks to generate essen- tial lipids, including isoprenoids and sterols. Proline is catabolised within the mitochondrion and excreted from the cell as the end- product alanine, with the production of several reduced cofactors, which are reoxidised in the respiratory chain for ATP produc- tion by OXPHOS (Figure 2A). However, if glucose is provided, PFs adjust their metabolism and produce most of their ATP via glucose degradation (glycolysis), even in the presence of proline (Figure 2B)25,29. These findings highlight that these parasites, like most other eukaryotes, are extremely flexible at adapting their central metabolism to their environment. Thirdly and probably the most important reason for a higher rate of glucose degradation in B-LS is that some biological proc- esses require more ATP in B-LS compared with PFs. This is the case of endocytosis, which is at least about 10-fold upregulated in B-LS compared with PFs and other trypanosomatids37,38. The high endocytic activity observed in B-LS is required for rapid recy- cling of cell-surface glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) for internalisation and removal of bound antibodies, facilitating escape from the host immune defences, but also for nutrient scavenging from the mammalian host. 3.2. Trypanosomes have specific pathways and unique enzymesi Sphingolipid synthesis is highly controlled during development: a dedicated inositolphosphoceramide synthase (SLS1) is highly upregulated in B-SS parasites and maintained in PFs24. As a consequence of more ceramide being used for inositolphosphoceramide synthesis, the synthesis of sphingomyelin is reduced, causing an alteration in the levels of phosphatidylinositol species. As mentioned above, although PFs rely upon proline in vivo, they prefer glucose to produce ATP25. Interestingly, the rate of glucose degradation is about 10-fold higher in B-LS than in PFs30,32. This considerable difference is probably due to metabolic adaptations developed by B-LS in response to a much higher ATP demand compared with PFs. Firstly, B-LS replicate faster than PFs (dou- bling times of about 7 and about 12 hours, respectively), which means that theoretically B-LS should show a 1.5-fold higher rate of ATP production. Secondly, the estimated number of ATP molecules produced per glucose consumed is about two-fold lower in B-LS. This difference is explained by the different strat- egies used by B-LS and PFs to degrade glucose into excreted end-products, which are mainly pyruvate in B-LS (Figure 2C) and acetate plus succinate in PFs (Figure 2B)33. Indeed, at the end of glycolysis, PFs convert pyruvate into acetate and ATP by the acetate:succinate CoA-transferase (ASCT)/succinyl-CoA synthetase cycle34,35. This pathway accounting for about 70% of the glycolytic flux in PFs is reduced to 5% in B-LS30. In addi- tion, the glycosomal succinate fermentation pathway (steps 3–6 in Figure 2B), pyruvate phosphate dikinase (step 7), and cytosolic localisation of phosphoglycerate kinase (step 1b in Figure 2B) improve the rate of ATP production within the cytosol of PFs29,36. 4. Metabolic adaptations during the Trypanosoma brucei life cycle Knockdown of actin resulted in a significant decrease (>70%) in endocytic activity and clearance of anti-VSG antibodies by B-LS forms, but did not significantly affect cellular ATP levels38,39. Surprisingly, measurement of the rates of pyruvate production and oxygen consumption, under conditions identical to those employed for the ATP and transferrin uptake assays, revealed a decrease of about four-fold in both rates after a knockdown of 15 hours (D. P. Nolan, unpublished data). Although the consumption of glucose was not measured, these data suggest that membrane traf- ficking in the B-LS may represent a significant additional ATP demand compared with the PFs and even more surprisingly that 4.2. Glucose: differences in consumption rate and efficiency of ATP production So far, the only carbon source for ATP production described for replicative bloodstream parasites is glucose, which is converted 4.2. Glucose: differences in consumption rate and efficiency of ATP production So far, the only carbon source for ATP production described for replicative bloodstream parasites is glucose, which is converted Page 6 of 12 Page 6 of 12 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 undergo several cycles of β-oxidation, releasing acetyl-CoA, which could enter the TCA cycle, leading to the production of NADH and FADH2, which could result in the production of ATP by OXPHOS. This hypothesis, however, implies that complexes III and IV of the respiratory chain are active, and this has not been observed so far in T. brucei mammalian forms. An alternative scenario is that the fatty acid chains (normally, 16 carbon palmitate) released by adipocytes are shortened through β-oxidation to enter an anabolic process in order to produce complex lipids. The resulting acetyl-CoA could be converted into acetate and lead to the production of one ATP molecule by the action of ASCT enzyme. the rate of ATP utilisation may also influence its rate of production via glycolysis. However, knockdown of actin also led to a rapid cessation in cell division and eventual cell death, so the implica- tions of these preliminary metabolic investigations may not be so straightforward. Interestingly, although B-SS live in a glucose-rich environment, they undergo morphological and gene expression alterations that are consistent with a preparation to survive within the tsetse midgut environment7,24 (Figure 1). These adaptions also include increased sensitivity to specific environmental cues that signal entry to the tsetse fly vector, as well as resistance to extracellular acidic and proteolytic stress40,41. Given that B-SS are non- proliferative and existent only in pleomorphic strains, it is more difficult to obtain large and pure quantities of this life-cycle stage in vitro. As a result, its metabolism has been less characterised. Nevertheless, we know that B-SS consume glucose and produce pyruvate and intermediate levels of acetate42, suggesting that metabolism is being pre-adapted to the conditions in which procy- clic forms will live within the tsetse midgut. Transcriptomic studies have confirmed the downregulation of several genes that encode for components of the glycosomes and are involved in glucose uptake and breakdown24. Genes upregulated in B-SS include TAO, fructose-2,6- biphosphatase, specific membrane proteins, and specific lipid biosynthesic genes, including TbSLS1 involved in inositolphosphoceramide synthesis. Further biochemical studies will be necessary to characterise and allow a better understanding of B-SS metabolism. 4.3. Lipids: responding to a great demand for myristate h bl d bl i In the bloodstream, trypanosomes are able to survive extracellu- larly in the mammalian host as they are coated by a dense homog- enous layer of GPI-anchored VSGs. VSG coats are periodically exchanged by a mechanism of antigenic variation, protecting para- sites against the host’s innate and adaptive immune responses43. In B-LS forms, GPI anchors exclusively contain two myristate molecules (14 carbon fatty acid); however, myristate is present at very low levels within the mammalian bloodstream, which could not sustain the B-SL demand44. Thus, it was initially thought that de novo synthesis of myristate occurred via a type II prokaryotic- like synthase45,46, but this synthesis is not sufficient for the GPI requirement47. Hence, it has been discovered that T. brucei uses four microsomal elongases, with different specificities, to synthesise fatty acids in a stepwise manner47. These elongases are responsible for the majority of de novo fatty acid synthesis in T. brucei. In bloodstream forms, downregulation of the elongase 3 in the pathway (which converts C14 to C18) explains the high production of myristate required for GPI anchors. However, much remains to be discovered. Some questions for the future are the following: However, much remains to be discovered. Some questions for the future are the following: • Besides the known glucose and proline transporters, what are the transporters of other essential nutrients? • What is the signalling cascade that coordinates metabolism remodelling? In most eukaryotes, two key kinases are involved in nutrient sensing: target of rapamycin protein (TOR) and AMP-activated kinase (AMPK). T. brucei, the eukaryote with the most complex network of TOR proteins described so far, is composed of four TOR proteins57, which are necessary for cell proliferation. Interestingly, knockdown of one of these proteins, TbTOR4, which appears to be kinetoplastid- specific, caused irreversible differentiation of the B-LS form into a quiescent form with properties very similar to those of the B-SS form, which suggested that TbTOR4 negatively regulates the slender-to-stumpy transition58. Activation of AMPK also triggers differentiation to the quiescent B-SS • What is the signalling cascade that coordinates metabolism remodelling? In most eukaryotes, two key kinases are involved in nutrient sensing: target of rapamycin protein (TOR) and AMP-activated kinase (AMPK). T. brucei, the eukaryote with the most complex network of TOR proteins described so far, is composed of four TOR proteins57, which are necessary for cell proliferation. 4.2. Glucose: differences in consumption rate and efficiency of ATP production Tryponosoma cruzi also infects adipocytes (specialised cells of adipose tissue) and is capable of consuming stored lipids48. It is unclear how T. brucei, an extracellular parasite, accesses lipids that are stored inside adipocytes, which constitute the largest storage of lipids in a mammalian host. Inside adipocytes, the stored triglycerides can be converted via lipolysis into fatty acids and glycerol, which are eventually secreted. During a T. brucei infection, animals typically lose weight and serum shows hyperlipidaemia6,49. We speculate that during a T. brucei infection, lipolysis is increased, leading to the secretion of fatty acids, which could be readily taken up and used by parasites. 5. Concluding remarks and future perspectives 5 Co c ud g e a s a d u u e pe spec es Fields such as immunometabolism emerged because of the devel- opment of highly sensitive metabolomic approaches, including untargeted metabolomic analysis, stable isotope labelling, mass spectrometry, and nuclear magnetic resonance50–52. Researchers discovered that during immune cell activation, the levels of many metabolites undergo alterations and these changes are directly linked to immune cell effector functions. In a way, the life cycle of a pathogen is a series of irreversible differentiation steps, in which the cells adapt to a new environment to perform new functions. In addition, the use of modern metabolomics approaches has revealed that T. brucei uses an incomplete TCA cycle in PFs53 and that pro- line has a different fate in PFs depending upon high- or low-glucose availability in the medium and has allowed the identification of the carbon and nitrogen sources of essential metabolites53–56. 4.3. Lipids: responding to a great demand for myristate h bl d bl i Interestingly, knockdown of one of these proteins, TbTOR4, which appears to be kinetoplastid- specific, caused irreversible differentiation of the B-LS form into a quiescent form with properties very similar to those of the B-SS form, which suggested that TbTOR4 negatively regulates the slender-to-stumpy transition58. Activation of AMPK also triggers differentiation to the quiescent B-SS Trindade et al. have shown that at the RNA level, parasites in adipose tissue and blood are quite different and that many genes associated with metabolism are differentially expressed6. Using pulse-chase experiments with stable isotope-labelled myristate, the authors showed that the first three enzymatic steps of fatty acid β-oxidation were active in parasites occupying the adipose tissue. In this pathway, a fatty acid molecule is converted into acetyl-CoA and a shorter fatty acid. The fate of each of these molecules remains unknown. The most ‘classic’ scenario would be that fatty acids Page 7 of 12 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 forms59. It is likely that some of these kinases are directly involved in remodelling metabolism during the T. brucei life cycle. It is interesting that changes in inositol metabolism lead to perturbations in VSG gene expression, suggesting that inositol metabolites are important for the control of this B-LS-specific process60. encode for metabolic enzymes are circadianly regulated63. This cycling expression pattern leads to two peaks of intracellular ATP concentration, indicating that metabolism is indeed under circadian control. How is this control achieved? Which metabolic pathways are affected? Does this adaptation reflect the circadian variation of nutrients in the host? • What is the metabolism of other stages of the life cycle? With the possibility of generating in vitro multiple stages of tsetse life cycle by overexpressing RBP661, it may be possible in the near future to understand metabolism of epimastigotes and metacyclics. The interplay between the host and pathogen and the influences upon their respective metabolisms is likely to be complex but is probably very significant since adaptation to nutrient availability is a major driving force during evolution. With the help of new and more sensitive biochemical and metabolic methodologies, it should be possible to use systems biology approaches to simultaneously characterise the metabolic changes undergone by parasites and host during an infection and within different tissues. Grant information TKS is supported by the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, the Engineering and Physi- cal Sciences Research Council, the Medical Research Council (MR/M020118/1) and the European Community Seventh Frame- work Programme under grant agreement 602773 (Project KINDRED). FB is supported by the Centre National de la Recher- che Scientifique (CNRS), the Université de Bordeaux, the Agence Nationale de la Recherche (ANR) through the GLYCONOV grant (ANR-15-CE15-0025-01) of the ‘Générique’ 2015 call and the Laboratoire d’Excellence (LabEx) ParaFrap (grant ANR-11- LABX-0024). DPN is supported by the Wellcome Trust and Sci- ence Foundation Ireland. LMF is supported by the Howard Hughes Medical Institute (55007419) and the Fundação para a Ciência e a Tecnologia (FCT) (PTDC/BIM-MET/4471/2014). • What are the consequences of a T. brucei infection in the host? Trypanosomiasis is characterised by decreased levels of aromatic amino acids, especially tryptophan, in the host and these levels are accompanied by the excretion of abnormal amounts of aromatic ketoacids, such as indole-3-pyruvate62. It was recently shown that B-LS generates indole-3-pyruvate by transamination of tryptophan and secretes significant amounts of this aromatic ketoacid. Indole-3-pyruvate appears to be able to modulate the host inflammatory responses, which may prolong host survival and thereby potentiate transmission of the parasite to the tsetse fly vector and ensure completion of the life cycle62. The roles of other secreted parasite metabolites that can possibly modify the host’s metabolism remain to be established. • What are the consequences of a T. brucei infection in the host? Trypanosomiasis is characterised by decreased levels of aromatic amino acids, especially tryptophan, in the host and these levels are accompanied by the excretion of abnormal amounts of aromatic ketoacids, such as indole-3-pyruvate62. It was recently shown that B-LS generates indole-3-pyruvate by transamination of tryptophan and secretes significant amounts of this aromatic ketoacid. Indole-3-pyruvate appears to be able to modulate the host inflammatory responses, which may prolong host survival and thereby potentiate transmission of the parasite to the tsetse fly vector and ensure completion of the life cycle62. The roles of other secreted parasite metabolites that can possibly modify the host’s metabolism remain to be established. • How does the parasite metabolism change during the day? We have recently shown that, in T. brucei, many genes that The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author contributions All authors participated in the writing of the manuscript. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. 4. Caljon G, van Reet N, De Trez C, et al.: The Dermis as a Delivery Site of 4.3. Lipids: responding to a great demand for myristate h bl d bl i • Do other trypanosome species undergo similar or different metabolic adaptations during their life cycle as they encounter different environments? • What is the metabolism of slender and stumpy forms when these occupy other tissues within the mammalian host? Trindade et al. have shown that at the RNA level, parasites in adipose tissue and blood are quite different and that many genes associated with metabolism are differentially expressed6. It will be important in the future, perhaps for drug development, to confirm these observations at protein and metabolite levels not only in visceral adipose tissue but also in the skin and importantly in the brain. • What is the metabolism of slender and stumpy forms when these occupy other tissues within the mammalian host? Trindade et al. have shown that at the RNA level, parasites in adipose tissue and blood are quite different and that many genes associated with metabolism are differentially expressed6. It will be important in the future, perhaps for drug development, to confirm these observations at protein and metabolite levels not only in visceral adipose tissue but also in the skin and importantly in the brain. Author contributions All authors participated in the writing of the manuscript. 1. Franco JR, Simarro PP, Diarra A, et al.: Epidemiology of human African trypanosomiasis. Clin Epidemiol. 2014; 6: 257–75. 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PubMed Abstract \| Publisher Full Text acquiring and understanding global metabolite data. Trends Biotechnol. 2004; 22(5): 245–52. P bM d Ab \| P bli h F ll T 58. Barquilla A, Saldivia M, Diaz R, et al.: Third target of rapamycin complex negatively regulates development of quiescence in Trypanosoma brucei. Proc Natl Acad Sci U S A. 2012; 109(36): 14399–404. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation 58. Barquilla A, Saldivia M, Diaz R, et al.: Third target of rapamycin complex negatively regulates development of quiescence in Trypanosoma brucei. Proc Natl Acad Sci U S A. 2012; 109(36): 14399–404. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation 51. Dettmer K, Aronov PA, Hammock BD: Mass spectrometry-based metabolomics. Mass Spectrom Rev. 2007; 26(1): 51–78. PubMed Abstract \| Publisher Full Text \| Free Full Text 52. Breitling R, Takano E: Synthetic Biology of Natural Products. Cold Spring Harb Perspect Biol. 2016; 8(10): pii: a023994. PubMed Abstract \| Publisher Full Text 59. Saldivia M, Ceballos-Pérez G, Bart JM, et al.: The AMPKα1 Pathway Positively Regulates the Developmental Transition from Proliferation to Quiescence in Trypanosoma brucei. Cell Rep. 2016; 17(3): 660–70. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation 53. van Weelden SW, Fast B, Vogt A, et al.: Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation. J Biol Chem. 2003; 278(15): 12854–63. PubMed Abstract \| Publisher Full Text 60. Cestari I, Stuart K: Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes. Proc Natl Acad Sci U S A. 2015; 112(21): E2803–12. 63. Rijo-Ferreira F, Pinto-Neves D, Barbosa-Morais NL, et al.: Trypanosoma brucei metabolism is under circadian control. Nat Microbiol. 2017; 2: 17032. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation Genomics. 2009; 10: 427. PubMed Abstract \| Publisher Full Text \| Free Full Text PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation Editorial Note on the Review Process are commissioned from members of the prestigious and are edited as a F1000 Faculty Reviews F1000 Faculty service to readers. In order to make these reviews as comprehensive and accessible as possible, the referees provide input before publication and only the final, revised version is published. The referees who approved the final version are listed with their names and affiliations but without their reports on earlier versions (any comments will already have been addressed in the published version). Version 2 , Department of Biological Sciences, Centre for BioImaging Sciences, National University Cynthia He Yingxin of Singapore, Singapore, 117543, Singapore No competing interests were disclosed. Competing Interests: 1 , Department of Biological Sciences, Centre for BioImaging Sciences, National University Cynthia He Yingxin of Singapore, Singapore, 117543, Singapore No competing interests were disclosed. Competing Interests: 1 , Department of Epidemiology of Microbial Diseases, School of Public Health, Yale Christian Tschudi University, New Haven, CT, USA No competing interests were disclosed. Competing Interests: 1 , Center for Infectious Disease Research, Seattle, WA, USA Kenneth Stuart No competing interests were disclosed. Competing Interests: 1 , Biology Centre, Institute of Parasitology, Czech Academy of Sciences, České Julius Lukes Budějovice, Czech Republic 1 1,2,3 1 Faculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic 2 Canadian Institute for Advanced Research, Toronto, Canada 3 No competing interests were disclosed. Competing Interests: PubMed Abstract \| Publisher Full Text 54. Coustou V, Biran M, Breton M, et al.: Glucose-induced remodeling of intermediary and energy metabolism in procyclic Trypanosoma brucei. J Biol Chem. 2008; 283(24): 16342–54. PubMed Abstract \| Publisher Full Text 56. Bringaud F, Biran M, Millerioux Y, et al.: Combining reverse genetics and nuclear magnetic resonance-based metabolomics unravels trypanosome-specific metabolic pathways. Mol Microbiol. 2015; 96(5): 917–26. PubMed Abstract \| Publisher Full Text 63. Rijo-Ferreira F, Pinto-Neves D, Barbosa-Morais NL, et al.: Trypanosoma brucei metabolism is under circadian control. Nat Microbiol. 2017; 2: 17032. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation 57. Saldivia M, Barquilla A, Bart JM, et al.: Target of rapamycin (TOR) kinase in Page 10 of 12 Page 10 of 12 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 Current Referee Status: Editorial Note on the Review Process Open Peer Review Current Referee Status: Version 1 , Department of Epidemiology of Microbial Diseases, School of Public Health, Yale Christian Tschudi University, New Haven, CT, USA No competing interests were disclosed. Competing Interests: 1 , Department of Biological Sciences, Centre for BioImaging Sciences, National University Cynthia He Yingxin of Singapore, Singapore, 117543, Singapore No competing interests were disclosed. Competing Interests: 1 , Center for Infectious Disease Research, Seattle, WA, USA Kenneth Stuart No competing interests were disclosed. Competing Interests: 1 , Center for Infectious Disease Research, Seattle, WA, USA Kenneth Stuart N i i di l d C i I 1 , , Biology Centre, Institute of Parasitology, Czech Academy of Julius Lukes Alena Zikova Sciences, České Budějovice, Czech Republic 1 1,2,3 1,4,3 1 Faculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic 2 Page 11 of 12 Canadian Institute for Advanced Research, Toronto, Canada Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic No competing interests were disclosed. Competing Interests: 3 4 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 F1000Research 2017, 6(F1000 Faculty Rev):683 Last updated: 18 MAY 2017 Canadian Institute for Advanced Research, Toronto, Canada Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic No competing interests were disclosed. Competing Interests: 3 4 Page 12 of 12
https://openalex.org/W4302303761	https://hess.copernicus.org/articles/26/4893/2022/hess-26-4893-2022.pdf	English	null	Socio-hydrological modeling of the tradeoff between flood control and hydropower provided by the Columbia River Treaty	Hydrology and earth system sciences	2,022	cc-by	18,674	Correspondence: Ashish Shrestha (ashres15@asu.edu) Correspondence: Ashish Shrestha (ashres15@asu.edu) Received: 16 November 2021 – Discussion started: 22 November 2021 Revised: 20 August 2022 – Accepted: 25 August 2022 – Published: 6 October 2022 Received: 16 November 2021 – Discussion started: 22 November 2021 Revised: 20 August 2022 – Accepted: 25 August 2022 – Published: 6 October 2022 to cooperate for each country is lowest when they are self- interested but ﬂuctuates in other social preference scenarios. Abstract. The Columbia River Treaty (CRT) signed between the United States and Canada in 1961 is known as one of the most successful transboundary water treaties. Under con- tinued cooperation, both countries equitably share collective responsibilities of reservoir operations and ﬂood control and hydropower beneﬁts from treaty dams. As the balance of beneﬁts is the key factor of cooperation, future cooperation could be challenged by external social and environmental factors which were not originally anticipated or change in the social preferences of the two actors. To understand the ro- bustness of cooperation dynamics, we address two research questions. (i) How does social and environmental change inﬂuence cooperation dynamics? (ii) How do social prefer- ences inﬂuence the probability of cooperation for both ac- tors? We analyzed infrastructural, hydrological, economic, social, and environmental data to inform the development of a socio-hydrological system dynamics model. The model simulates the dynamics of ﬂood control and hydropower ben- eﬁt sharing as a function of the probability to cooperate, which in turn is affected by the share of beneﬁts. The model is used to evaluate scenarios that represent environmental and institutional change and changes in political characteristics based on social preferences. Our ﬁndings show that stronger institutional capacity ensures equitable sharing of beneﬁts over the long term. Under the current CRT, the utility of co- operation is always higher for Canada than non-cooperation, which is in contrast to the United States. The probability Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 https://doi.org/10.5194/hess-26-4893-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction The Columbia River Treaty (CRT) was signed in 1961 to manage shared waters between the United States and Canada. Under the treaty, both countries share collective re- sponsibilities of reservoir operations and beneﬁt from ﬂood control and hydropower production from the treaty dams eq- uitably. The CRT is known as one of the most successful transboundary water treaties in the world, as evidenced by continued cooperation and equitable beneﬁt sharing (Hyde, 2010). However, since the CRT was established, external social and environmental factors not originally anticipated, such as the degradation of valued ﬁsh species, have affected the balance of beneﬁts each country receives (Bowerman et al., 2021; Trebitz and Wulfhorst, 2021). In competition and cooperation, actors’ decisions are guided by their so- cial preferences (also referred to as “other-regarding” prefer- ences). Fehr and Fischbacher (2002) and Kertzer and Rath- bun (2015) suggest that decision makers have social prefer- ences that motivate their decisions, which means that such actors care about gain (here, material payoff) not just for themselves but also for others. The perceived fairness of allocated material resources or balance of beneﬁts, in concert Ashish Shrestha1,⋆, Felipe Augusto Arguello Souza2,⋆, Samuel Park3,⋆, Charlotte Cherry5,⋆, Margaret Garcia1, David J. Yu3,4, and Eduardo Mario Mendiondo2 1School of Sustainable Engineering and the Built Environment, Arizona State University, Tempe, AZ, USA 2Department of Hydraulics and Sanitation, São Carlos School of Engineering, University of São Paulo, São Carlos, Brazil 3Lyles School of Civil Engineering, Purdue University, West Lafayette, IN, USA 4Department of Political Science, Purdue University, West Lafayette, IN, USA 5Department of Civil and Environmental Engineering, University of Illinois at Urbana Champaign, Urbana, IL, USA ⋆These authors contributed equally to this work. Department of Civil and Environmental Engineering, University of Illinois at Urbana Champaign, U These authors contributed equally to this work. Socio-hydrological modeling of the tradeoff between ﬂood control and hydropower provided by the Columbia River Treaty Ashish Shrestha1,⋆, Felipe Augusto Arguello Souza2,⋆, Samuel Park3,⋆, Charlotte Cherry5,⋆, Margaret Garcia1, David J. Yu3,4, and Eduardo Mario Mendiondo2 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs Successful management of transboundary river basins de- pends not only on understanding the hydrology, but also consideration of economic needs and political dynamics of the upstream and downstream riparian states; those politi- cal dynamics are shaped by social comparison in which ac- tors compare their position, beneﬁt, or risks with other actors (Gain et al., 2021; Gober and Wheater, 2014). Research in behavioral economics by Frey and Meier (2004) has shown that actors tends to be cooperative if they know many others are contributing too, which could be key to successful man- agement in transboundary river basins. Transboundary rivers are managed by multiple heterogeneous stakeholders with different sovereignty, governance structures, and economic conditions, while diverse, basin populations may be interde- pendent not just hydrologically but also economically and socially (FAO, 2022; Rawlins, 2019). Social factors that can explain cooperation and conﬂict dynamics include asymmet- ric access to water resources due to upstream–downstream locations and varying levels of dependence on different uses of the river (Warner and Zawahri, 2012). y g Development in transboundary river basins can result in conﬂict or cooperation (Bernauer and Böhmelt, 2020). For example, the construction of dams upstream in the Lancang– Mekong River basin has affected the environmental condi- tions and livelihood opportunities of downstream countries (Lu et al., 2021). Further, the ability to sustain coopera- tion can be critically affected by how beneﬁts (e.g., water supply and hydropower) and risks (e.g., ﬂoods, droughts) are shared under changing conditions (Wolf, 2007; Zeitoun et al., 2013). The Nile River basin is an example of in- equitable beneﬁt sharing where Egypt and Sudan hold abso- lute rights to use, motivating conﬂict and international delib- eration (Kameri-Mbote, 2007; Wiebe, 2001). Understanding the history of such transboundary river basins where con- ﬂicts prevailed more than cooperation showed that there is an inequitable distribution of beneﬁts and risks among ac- tors. In the absence of cooperation, the beneﬁts and risks are usually distributed, with advantage to actors with higher po- litical and economic power or following geographic advan- tages (Dombrowsky, 2009). Prevalence of such imbalance in beneﬁts and risks could further diminish the likelihood of successfully negotiating any agreement to cooperatively manage water resources (Espey and Towﬁque, 2004; Song and Whittington, 2004). A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4894 with the social preferences of each actor, can signiﬁcantly affect the stability of cooperation over time (Abraham and Ramachandran, 2021; Hirshleifer, 1978; Kertzer and Rath- bun, 2015; Rivera-Torres and Gerlak, 2021; Sadoff and Grey, 2002; UNESCO, 2021). Understanding these social prefer- ences between the United States and Canada helps us to un- derstand the interplay of competition, cooperation, or con- ﬂict. The United States and Canada are currently renegotiat- ing the CRT beyond 2024, with the aim of maintaining co- operation in a changing environment. This ongoing renego- tiation motivates and raises two research questions. (1) How does social and environmental change inﬂuence cooperation dynamics? (2) How do social preferences inﬂuence the prob- ability of cooperation for both actors? nerabilities in transboundary water management under cli- mate change. Madani et al. (2014) applied bankruptcy reso- lution methods to the challenge of water allocation in trans- boundary river basins. This quantitative approach is rooted in the economic literature and offers insight into efﬁcient and stable allocation schemes. Pohl and Swain (2017) posit that transboundary waters create economic, social, and environ- mental interdependencies that can be leveraged to either pro- mote cooperation or intensify conﬂict. They highlight that this creates the potential for broader peace dividends when negotiating transboundary water management and present strategies for diplomats to engage constructively. Islam and Susskind (2018) presented the Water Diplomacy Framework, which draws on the concepts of complexity science (e.g., in- terconnectedness, uncertainty, and feedbacks), and negotia- tion theory (e.g., stakeholder identiﬁcation, engagement at multiple levels, and value creation for beneﬁt sharing), to un- derstand and resolve transboundary water issues and cooper- ative decision making. Koebele (2021) takes a policy process approach to understand collaborative governance in trans- boundary water management of Colorado River between the United States and Mexico, where over-allocation of water led to environmental problems and water scarcity downstream. The author applies the Multiple Streams Framework, used to explain decision making in a range of policy contexts, to examine the case of transnational policymaking in the Col- orado River Delta. External factors such as climate change affect the sustainable transboundary water management. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs y p Traditional water resource management assumes values and preferences to be exogenous to the water resource sys- tems, but values and preferences can co-evolve with nat- ural systems (Caldas et al., 2015; Sivapalan and Blöschl, 2015). Socio-hydrology, the study of coupled human–water systems, ﬁlls this need by providing tools to represent dy- namic feedback between the hydrological and social systems (Sivapalan et al., 2012; Troy et al., 2015). Socio-hydrological studies have explored a variety of emergent phenomena that result from such feedback, including the levee effect, the irri- gation efﬁciency paradox, and the pendulum swing between human and environmental water uses (Khan et al., 2017). In the study of transboundary rivers, socio-hydrology allows for the explicit inclusion of changing values or preferences and enables assessment of cooperation and conﬂict as values and preferences shift (Sivapalan and Blöschl, 2015). Thus, we develop a socio-hydrological system dynamics model mo- tivated by the experience of the Columbia River to answer the research questions deﬁned above. This research builds upon the work of Lu et al. (2021), where the authors applied socio-hydrological modeling to the case of the transboundary Lancang–Mekong River, by assessing how preferences and attitudes toward cooperation affect their probability of adher- ing to the agreement. Extending the work by Lu et al. (2021), we apply behavioral economics to incorporate the role of so- cial preferences between actors to quantify the probability of cooperation for each actor. Furthermore, the power dynamics between actors is very different in the Columbia River basin than in the Lancang–Mekong River basin. The objective of this study is to quantify the balance of beneﬁts under coop- erative reservoir operations to assess the impact of changing social and environmental conditions as well as shifts in the social preferences of the United States and Canada. While the study does not aim to provide speciﬁc recommendations for treaty re-negotiations, it explores the role that changes in environmental priorities play in cooperation and presents scenarios to inform future renegotiations of the CRT. g ( ) The fairness consideration behind the CRT is consistent with the now well-established behavioral insight that most human actors are not selﬁsh rational actors that seek to max- imize short-term material beneﬁts with complete information (Henrich et al., 2005). A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs (4) beneﬁts beyond the river (Sadoff and Grey, 2002, 2005). Examples of these beneﬁts include ﬂood and drought mit- igation, improved environmental conditions, and economic beneﬁts from hydropower or agriculture (Qaddumi, 2008). sideration), preference for rational self-interest maximization (homo economicus), and preference for having their own beneﬁts to be higher than those of others (competitiveness) (Charness and Rabin, 2002). Among these four types, par- ticularly relevant to transboundary river management is that human actors have a strong social preference for inequality aversion at both individual and organizational level and that this preference is often a key to why cooperation emerges and is sustained among unrelated parties (Choshen-Hillel and Yaniv, 2011; Kertzer and Rathbun, 2015). Thus, the decisions of organizational actors and their reciprocal interactions over time in the context of the CRT can be described and plausibly explained by inequality aversion. Understanding the social preferences between organizational actors (here the United States and Canada) can capture how their cooperation behav- ior may evolve over time and shape the robustness of CRT. y p g (Q , ) In the case of the Columbia River, the upstream actor (Canada) operates its dams in a way that provides a greater beneﬁt to the downstream actor (the United States) in the form of ﬂood protection because the beneﬁt sharing pro- vision of the CRT ensures that Canada receives a share of those beneﬁts in return. The United States operates its dams to maximize hydropower production and, in exchange, com- pensates Canada for half of the estimated increase in hy- dropower beneﬁt generated by the treaty, which provides an economic incentive to cooperate. This is consistent with the theory that countries tend to cooperate when the net eco- nomic and political beneﬁts of cooperation are greater than the beneﬁts from unilateral action and when the generated beneﬁts are shared in a way that is perceived to be “fair” by both parties (Grey et al., 2016; Jägerskog and Zeitoun, 2009; Qaddumi, 2008). The CRT was established on these grounds, as both actors agreed that the greatest beneﬁt of the Columbia River could be secured through cooperative management (BC Ministry of Energy and Mines, 2013; Yu, 2008). This agreement focuses on the equitable sharing of beneﬁts cre- ated from cooperation, rather than on water allocation itself, which is a key provision of some of the world’s most suc- cessful water agreements (Giordano and Wolf, 2003). A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs In the case of cooperative trans- boundary river management, actors mutually achieve several beneﬁts, including (1) beneﬁts to the river, (2) beneﬁts from the river, (3) the reduction of costs because of the river, and ( ) Globally, 310 international transboundary river basins cover almost 47.1 % of the Earth’s land surface, which in- cludes 52 % of the global population and are the source of 60 % of freshwater supplies (McCracken and Wolf, 2019; UN-Water, 2015; United Nations, 2022). Transboundary wa- ter management compounds the challenges of managing wa- ter between competing users because the river is managed be- tween different jurisdictions and under different policy struc- tures (Bernauer and Böhmelt, 2020). Transboundary water management has been studied through different disciplines. Kliot et al. (2001) reviewed the institutional evolution of wa- ter management in 12 transboundary river basins, identifying legal principles that organize transboundary water manage- ment and discussing their characteristics and shortcomings. The authors discuss that the key challenges in transbound- ary water management arise from water scarcity, maldistribu- tion, over-utilization, and misuse of shared resources. Odom and Wolf (2011) examined the 1994 Israel–Jordan Treaty of Peace where climate extremes and drought created conﬂicts on water sharing and hydropower agreements, but the mod- iﬁed institutional arrangements mitigated conﬂicts and vul- https://doi.org/10.5194/hess-26-4893-2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 4895 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs Rather, there is overwhelming empir- ical evidence that humans are learning and norm-adopting actors, whose decisions are sensitive to contextual condi- tions, including those of how material beneﬁts are relatively distributed between oneself and others (Fehr and Schmidt, 1999; Gintis et al., 2003). Among several social science the- ories that have emerged to explain this empirical regularity about human behavior (note that, as explained by Sanderson et al., 2017, the social sciences are characterized by theo- retical pluralism and that there is no single universal theory about human behavior), perhaps the most rigorous theory is that of social preference which is also referred to as proso- cial preference or other-regarding preference (Fehr and Fis- chbacher, 2002; Kertzer and Rathbun, 2015). This theory as- sumes that humans not only care about their own material beneﬁts, but also about the material beneﬁts received by oth- ers and that this intrinsic nature is consistent with why many people (but not all) exercise social norms such as inequality aversion and conditional cooperation. In line with this the- ory, the utility of individual and organizational actors can be formalized and categorized into four general types of social preferences: preference for having the beneﬁts among all ac- tors to be equal (inequality aversion), preference for maxi- mizing group- or societal-level beneﬁts (social welfare con- This article is organized as follows. Section 2 provides a general background of the Columbia River system and treaty https://doi.org/10.5194/hess-26-4893-2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4896 Figure 1. Map showing (a) the Columbia River basin across Canada and the United States, (b) the Snake River basin and its tributaries within the Columbia River basin, and (c) location of treaty dams along Canada and the United States which are also included in the socio- hydrological system dynamics model. Figure 1. Map showing (a) the Columbia River basin across Canada and the United States, (b) the Snake River basin and its tributaries within the Columbia River basin, and (c) location of treaty dams along Canada and the United States which are also included in the socio- hydrological system dynamics model. dams. Section 3 discusses the conceptualization and formu- lation of the socio-hydrological model. Four scenarios based on environmental and institutional change and four scenarios based on behavioral economics using social preferences are presented here. A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs Section 4 explains the model testing and sce- nario analysis. Section 5 discusses the ﬁndings of this study, draws out major conclusions gained through this study, and identiﬁes remaining questions for future research. (HP), and millions of people in the Paciﬁc Northwest (includ- ing 8 million people in the Columbia Basin; Lower Columbia Estuary Partnership, 2022) rely on the river for hydropower, ﬁshing, irrigation, recreation, navigation, and other environ- mental services (White et al., 2021). Hydropower development started in the Paciﬁc Northwest in 1933 and expanded after the CRT was established. Be- tween 1938 and 1972, 11 dams were built on the US portion of the Columbia River, which generates over 20 000 MW of power (BC Ministry of Energy and Mines, 2013). In total, there are 31 federal dams in the Columbia River basin that are owned and operated by the U.S. Army Corps of Engi- neers (USACE) and the U.S. Bureau of Reclamation, which produce around 40 % of electricity for the Paciﬁc North- west (Bonneville Power Administration, 2001; Northwest Power and Conservation Council, 2020c, d; Stern, 2018). Dams along the Canadian side of the Columbia River pro- duce around half of the province’s hydropower generation (Government of British Columbia, 2019). Figure 1c shows the locations of major CRT dams considered in the sys- tem dynamics model. The reservoir capacity of Canadian treaty dams is 36810 × 106 m3, of which 28387 × 106 m3 is 3 Methodology In this section we present the conceptual model of Columbia River system under CRT, the formulation of a system dy- namics model, model calibration and validation, and sce- nario analysis. To incorporate the transboundary dynamics and feedback between the hydrological and social systems, we simplify the representation of the hydrology and reservoir operations by aggregating the CRT treaty dams for Canada and the United States. To understand the long-term dynam- ics of cooperation and robustness of the cooperation under change, four scenarios based on plausible cases of environ- mental and institutional change and four scenarios based on social preferences were developed and tested as discussed below. g g The original agreement during 1960s prioritized ﬂood con- trol and hydropower, but emerging social and environmental concerns have shifted the way that reservoirs are operated within the Columbia River basin. Dam construction altered the hydrology signiﬁcantly by moderating the strong sea- sonal ﬂow variability, impacting ecosystem health. For ex- ample, changes to salmon spawning habitat elevated smolt and adult migration mortality and led to declines in the salmon population (Kareiva et al., 2000; Karpouzoglou et al., 2019; Natural Resource Council, 1996; Northwest Power Planning Council, 1986; Williams et al., 2005). After the 1970s, mounting social pressure to protect the aquatic envi- ronment resulted in changes in dam operations that shifted the economic beneﬁts that the countries receive from co- operation (Bonneville Power Administration, 2013; Leonard et al., 2015; Northwest Power and Conservation Council, 2020b, a). This increased prioritization of ecosystem health is also seen in other transboundary river basins (Giordano et al., 2014). With changing priorities and operations affect- ing both actors’ share of beneﬁts, incentives to cooperate are shifting. 2 Columbia River system and treaty dams This ﬂood was the impetus for the United States to seek cooperation with Canada because it was not possible to build sufﬁcient storage along the downstream portion of the river to ensure protection from large ﬂoods. The summary of dams along the Columbia River is given is Table 1. allocated for ﬂood protection in the United States, and the capacity of the US treaty dams is 11577 × 106 m3. Grand Coulee is the largest and furthest upstream dam on the US side. Thus, inﬂow to the Grand Coulee includes the outﬂow from the Canadian dams and external tributaries that intersect with the river. Flooding has been the major concern in the downstream portion of the Columbia River. For example, the ﬂood in Vanport, Oregon, in 1948 motivated the construction of additional storage dams along the river (Sopinka and Pitt, 2014). This ﬂood was the impetus for the United States to seek cooperation with Canada because it was not possible to build sufﬁcient storage along the downstream portion of the river to ensure protection from large ﬂoods. The summary of dams along the Columbia River is given is Table 1. 2 Columbia River system and treaty dams The Columbia River, as depicted in Fig. 1, with its headwa- ters located in the mountains of British Columbia, has a basin that extends 670 807 km2 into seven US states – Washington, Oregon, Idaho, Montana, Nevada, Utah, and Wyoming – be- fore reaching the Paciﬁc Ocean in Oregon (Cosens, 2012). Figure 1 also shows the location of the treaty dams along the Columbia River. While only 15 % of the river’s length ﬂows through Canada, 38 % of the average annual ﬂow originates there (Cosens, 2012). By volume, it is the fourth largest river in North America, producing 40 % of all US hydropower https://doi.org/10.5194/hess-26-4893-2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 489 Table 1. List of dams represented by the model. Projects that do not present usable storage capacity are run-of-the-river dams. Treaty storag commitment refers to the room available to accommodate glacier waters under the CRT. Project Reservoir formed Country Total Usable Treaty HP Year of storage storage storage capacity completion capacity capacity commitment (MW) (km3) (km3) (km3) Mica Dam Kinbasket Lake Canada 24.7 14.8 8.6 1736 1973 Duncan Dam Duncan Lake Canada 1.77 1.73 1.73 – 1967 Keenleyside Dam Arrow Lake Canada 10.3 8.76 8.8 185 1968 Grand Coulee Franklin D. Roosevelt Lake USA 11.6 6.4 – 6809 1941 Chief Joseph Rufus Woods Lake USA 0.6 – – 2069 1955 McNary Lake Wallula USA 0.23 – – 980 1994 John Day Lake Umatilla USA 0.54 – – 2160 1971 The Dalles Lake Celilo USA 0.41 – – 2100 1957 Bonneville Lake Bonneville USA 0.66 – – 660 1938 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4897 allocated for ﬂood protection in the United States, and the capacity of the US treaty dams is 11577 × 106 m3. Grand Coulee is the largest and furthest upstream dam on the US side. Thus, inﬂow to the Grand Coulee includes the outﬂow from the Canadian dams and external tributaries that intersect with the river. Flooding has been the major concern in the downstream portion of the Columbia River. For example, the ﬂood in Vanport, Oregon, in 1948 motivated the construction of additional storage dams along the river (Sopinka and Pitt, 2014). A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs The basis of the model is that each country has responsibility over operating its own dams. The modeling framework is illustrated with a causal loop (CL) diagram in Fig. 2. The CL diagram illustrates all the key hydrological, environmental, economic, and social variables, relationships, direction of those relationships, and feedback. The storage capacity of Canada (upstream) and the United States (downstream) are two important state (hydrological) variables which represent the aggregated storage of the treaty dams (Fig. 2), the operation of which is determined by the storage thresholds. The increase in a storage threshold results in an increase in the storage level. Three Canadian dams, namely Mica, Duncan, and Arrow/Keenleyside, are lumped into a single storage as all three dams are multifunctional for ﬂood control and hydropower production. However, it should also be noted that Mica and Arrow dams are the ma- jor dams in Canada contributing to ﬂood control as those are along the primary stream order of Columbia River and Duncan Dam is in the small tributary (Fig. 1). In terms of storage volume Mica, Arrow and Duncan dams are 24.7, 10.3, and 1.77 km3, or 67 %, 28 %, and 5 % of total stor- age, respectively (Table 1). In the United States, the Grand Coulee dam is the only multifunctional dam with useable storage for ﬂood control. Given that the Grand Coulee is the only dam with storage in the US system, we have only lumped the reservoirs for hydropower generation, not ﬂood control. We used the lumped reservoir approach to simplify the system process required to investigate our research ques- tions. The lumped approach is particularly appropriate be- cause all the treaty dams work in coordination to achieve either the hydropower beneﬁts (by US dams) or the ﬂood control (by Canadian dams). The schematic of the lumped system is also shown in Fig. S18 and Sect. S4 in the Supple- ment. In lumping the system, we have considered external input variables such as tributaries and added to the outﬂow from the Canadian reservoir or inﬂow to the US reservoir. These dams along the Columbia River either have signiﬁ- cant ﬂood control capacity or signiﬁcant hydropower pro- duction capacity (Table 1). Thus, the simpliﬁed reservoir operation described below in Sect. 3.2.1 was implemented in the lumped storages on each side of the border, which represent collective operation of all the treaty dams within each country. A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeof 4898 more outﬂow yields higher hydropower beneﬁt. However, the need for ﬂood control is intermittent depending on the sea- sonal high ﬂows. Thus, Canada does not reduce the storage level throughout the year but just before the incoming higher ﬂows. Reservoir levels in the United States (under CRT) are kept as high as feasible to maximize hydropower generation. Each country’s reservoir outﬂow is used to calculate ﬂood control and hydropower production (Fig. 2, economic vari- ables), which is converted into monetary units as shown in the CL diagram. Fish spill is included as an environmental variable as the reduced salmon migration causes depletion of the salmon population in Columbia River. Thus, a coun- termeasure, increase in ﬁsh spill, is in place. However, the increase in ﬁsh spill has a tradeoff in hydropower production as less water ﬂows through the turbine. The United States provides additional beneﬁts to Canada through the Canadian Entitlement, a payment equal to half of the expected addi- tional hydropower generated due to cooperative management of the CRT dams. The collective monetary beneﬁt from ﬂood control and hydropower among countries determines the util- ity of cooperation and non-cooperation (economic variables) for each country as described in Sect. 3.2.2. The social pref- erences in different scenarios determine different values for utility of cooperation and non-cooperation depending on the actor’s social preference. Thus, the directions of these rela- tionships are conditional (Fig. 2). Having higher utility for cooperation under CRT results in a higher probability of co- operation. However, under changing social preferences, if the utility of non-cooperation is higher, the probability of cooperation decreases. In sum, increase in cooperation for Canada results in decrease of dynamic storage threshold, so Canada operates their reservoirs for downstream ﬂood con- trol. Similarly, increase in cooperation for the United States results in increase of the dynamic storage threshold, so the United States operates for maximum hydropower generation, thus creating two similar feedback loops for Canada and the United States (Fig. 2). be maintained at higher levels to achieve the highest hy- draulic head possible. In a non-cooperative regime, Canada would likely switch operations to maximize hydropower pro- duction, while the United States would have to decrease stor- age or water level to provide ﬂood control, at the detriment of US hydropower production. A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs Other hydrological variables in the model (i.e., ﬂows in the CL diagram) are inﬂow into Canadian storage, outﬂow from Canadian storage plus intermediate tributaries, inﬂow into the US storage, and outﬂow from the US stor- age. The higher the outﬂow from the dams, the lower the ﬂood control as ﬂood damages increase. A portion of the reservoir outﬂow passes through hydroelectric turbines; thus 3.1 Socio-hydrological system dynamics model Under the cooperative regime, both Canada and the United States operate their dams to fulﬁll the requirements of the CRT. This means that Canada operates to maximize ﬂood control, while the United States operates to maximize hy- dropower, and the beneﬁts are shared between both coun- tries. As discussed in the literature (BC Ministry of Energy and Mines, 2013; Giordano and Wolf, 2003; Grey et al., 2016; Jägerskog and Zeitoun, 2009; Qaddumi, 2008; Yu, 2008), countries are expected to continue cooperating if they perceive the beneﬁts to be shared equitably. On the other hand, under the non-cooperative regime, the balance of beneﬁts is not perceived to be equitable; thus, the countries would operate their reservoirs for their own beneﬁt. Reser- voir operation to maximize ﬂood control and to maximize hydropower production are in opposition for Canada and the United States. This is because operation for maximizing ﬂood control requires drawdown of reservoir storage to pro- vide space for incoming high ﬂows, while operation for max- imizing hydropower production requires reservoir storage to https://doi.org/10.5194/hess-26-4893-2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 3.2 Equations and parameters Equations describing the links between stocks and ﬂow vari- ables as shown in the CL diagram (Fig. 2) are categorized into reservoir operation, cooperation dynamics, economic beneﬁts, and environmental spills. These equations mathe- matically describe hydrological processes, as well as feed- back from social and economic variables. The following sec- tions describe the formulation of equations for each part of the system in greater detail. The inﬂow, outﬂow, water level, and storage data obtained from the Environment Canada (2022), USACE (2013), USACE (2022) and USGS (2022) are presented in Figs. S2–S10. https://doi.org/10.5194/hess-26-4893-2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4899 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4899 Figure 2. The causal loop diagram presents the hydrological and cooperation feedbacks between the Canada and the United States. Different colors shows the hydrological, environmental, economic, and social variables. Figure 2. The causal loop diagram presents the hydrological and cooperation feedbacks between the Canada and the United States. Different colors shows the hydrological, environmental, economic, and social variables. Figure 2. The causal loop diagram presents the hydrological and cooperation feedbacks between the Canada and the United States. Different colors shows the hydrological, environmental, economic, and social variables. A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs Figure 3. Schematic representation of the dynamic storage thresh- old (Sthreshold), represented by the green line. Sthreshold can range between the blue line, which represents the target storage to opti- mize hydropower production (SHPthreshold), and the red line, which represents the target storage to avoid ﬂood damages downstream of the dam (SFCthreshold). where I1 is the condition when SCA + QiCA · 86400 < SCAthreshold, and nCA parameter maintains the dynamic stor- age threshold required for ﬂood control. QoUS QoUS =       QiUS, for QiUS ≥QUSmax QiUS + max 0,min QUSmax −QiUS, SUS−SUSthreshold 86 400 i , (for I2) QiUS + SUS−SUSthreshold 86 400 , (otherwise) (4) =     QiUS, QiUS ≥QUSmax QiUS + max 0,min QUSmax −QiUS, SUS−SUSthreshold 86 400 i , (for I2) QiUS + SUS−SUSthreshold 86 400 , (otherwise) (4) (4) Figure 3. Schematic representation of the dynamic storage thresh- old (Sthreshold), represented by the green line. Sthreshold can range between the blue line, which represents the target storage to opti- mize hydropower production (SHPthreshold), and the red line, which represents the target storage to avoid ﬂood damages downstream of the dam (SFCthreshold). where I2 is the condition when SUS +QiUS ·86400 < SUSmax. where I2 is the condition when SUS +QiUS ·86400 < SUSmax. US Outﬂow was computed as a dependent variable of the fol- lowing: a. inﬂows (QiCA and QiUS), a. inﬂows (QiCA and QiUS), steps where the United States endures higher ﬂood dam- ages, the United States switches from the hydropower pro- duction regime (SUSHP) to the ﬂood control regime to op- timize its beneﬁts (SUSFC). The target ﬂood control storage in Canada (SCAFC) was determined based on average histori- cal storage in the three treaty reservoirs, while the hypothet- ical hydropower scheme was assumed as the dams operating at 95 % of their full production capacity. The US monthly target storage under the hydropower scheme (SUSHP) was determined based on the historical monthly average, while the hypothetical target storage to provide themselves protec- tion against ﬂoods was calculated as the additional room that Canada would not provide in case of switching to the hy- dropower scheme SCAHP, as presented in Eqs. (5) and (6). Therefore, the storage will be dependent on cooperation. A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs The probability-to-cooperate variables CCA and CUS are de- scribed in Sect. 3.2.2. b. maximum outﬂows observed in the Canadian side (Ar- row and Duncan dams – QCAmax) and in the US side (Grand Coulee – QUSmax), c. the maximum storage capacity of Canadian lumped dam (SCAmax) and the Grand Coulee dam (SUSmax), d. the updated storage stage at each time step in the lumped Canadian reservoir and the Grand Coulee reser- voir (SCA, SUS) and d. the updated storage stage at each time step in the lumped Canadian reservoir and the Grand Coulee reser- voir (SCA, SUS) and e. the dynamic storage threshold for each side (SCAthreshold, SUSthreshold). The dynamic storage threshold (m3) variable, mentioned in Eqs. (3) and (4), was estimated according to the simpliﬁed reservoir operation given by Eqs. (5) and (6) and is schemat- ically represented by Fig. 3. It determines the operational level of the reservoirs based on the probability of coopera- tion (i.e., the higher the cooperation, the higher the coherence with the CRT agreement). 3.2.1 Reservoir operation the Canadian side was calculated as the difference between the streamﬂow at the International Border and outﬂow from the Canadian side was calculated as the difference between the streamﬂow at the International Border and outﬂow from Duncan and Arrow dams, while the tributaries between the International Border and the Grand Coulee reservoir were es- timated by a linear regression (Fig. S12). The change in Canadian and US storage (m3 d−1) as the func- tion of inﬂow and outﬂow is given in Eqs. (1) and (2). Duncan and Arrow dams, while the tributaries between the International Border and the Grand Coulee reservoir were es- timated by a linear regression (Fig. S12). dSCA dt = QiCA −QoCA (1) dSUS dt = QiUS −QoUS (2) (1) The regulated Canadian (QoCA) and US (QoUS) outﬂows were simulated using Eqs. (3) and (4). (2) QoCA QoCA =       QCAmax, for nCA · QiCA ≥QCAmax nCA · QCAmax + max h 0,min QCAmax −nCA · QiCA, SCA−SCAthreshold 86 400 i , (for I1)    QCAmax, for QiCA ≥QCAmax QiCA + max h 0,min QCAmax −QiCA, SCA−SCAthreshold 86 400 i , (otherwise) (3) The Canadian inﬂow (QiCA) corresponds to the streamﬂow observed upstream of Mica and Duncan dams and the dif- ference between Mica outﬂow and Arrow inﬂow (i.e., ﬂow from intermediate tributaries). The data were retrieved from the Bonneville Power Administration (Bonneville Power Ad- ministration, 2020). The US inﬂow (QiUS) is equal to the outﬂow from Canadian storage (QoCA) plus the tributaries between the outlet of Duncan and Arrow dams and inlet of the Grand Coulee reservoir. The ﬂow from tributaries on (3) Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 https://doi.org/10.5194/hess-26-4893-2022 4900 3.2.2 Cooperation dynamics Equations (12) and (13) represent the rate of change in the cooperation probability of the two state actors based on logit dynamics: (8) UUS = wUS −αUS · max(wUS −wCA,0) + βUS · max(wCA −wUS,0) (9) (9) dCCA dt = χ " eγ ·E[UCA_coop] eγ ·E[UCA_coop] + eγ ·E[UCA_NoCoop] −CCA # , (12) dCUS dt = χ " eγ ·E[UUS_coop] eγ ·E[UUS_coop] + eγ ·E[UUS_NoCoop] −CUS # , (13) wCA = ω · (HPCA + FCCA + E) (10) wCA = ω · (HPCA + FCCA + E) (10) dC d wCA = ω · (HPCA + FCCA + E) (10) wUS = ω · (HPUS + FCUS −E), (11) (11) where w of each country is the utility from monetary beneﬁts, HP of each country is the hydropower beneﬁt, FC of each country is the beneﬁt from ﬂood prevention, E is the Canadian Entitlement, and ω is the coefﬁcient that can con- vert the monetary values to utility. The subscripts CA and US refer to Canada and the United States, respectively. Here, α and β values are set to be positive to capture inequality aversion for the behavioral model of Canada and the United States. This is because the balance of beneﬁts (Bankes, 2017; Shurts and Paisley, 2013) between these two countries is be- lieved to be a key factor to explain the level of cooperation. where CCA and CUS represent the probability of each country to cooperate (ranging from 0 for non-cooperation to 1 for full cooperation), and the parameter χ represents the probability that each actor engages in internal comparison of two choices and updates their probability to cooperate per time step. A small value implies the conservativeness of each actor. E[...] stands for an expected value. The parameter γ controls the stochasticity of the choice of strategy. A small value indi- cates that the choice is nearly random, whereas a very large value means a nearly deterministic choice. We assumed γ to be large and constant as both actors aim for higher expected utility. For probability to cooperate, if CCA equals 0.9, that means there is 90 % likelihood that Canada will cooperate with the United States and 10 % likelihood it will not coop- erate. We use logit dynamics functions to capture the rate of change in the cooperation probability of the two state actors (Iwasa et al., 2010). 3.2.2 Cooperation dynamics Cooperation amongst the two actors both impacts and is im- pacted by reservoir operations and beneﬁt sharing. Unequal distribution of beneﬁts alters the sense of fairness and reci- procity, two behavioral traits that are known to be widespread (Fehr and Fischbacher, 2002). To conceptualize and under- stand the cooperation dynamics between two actors in the context of CRT, the theory of social preferences is drawn from the ﬁeld of behavioral economics. Social preferences – which means that actors care not only on their own ma- terial beneﬁts, but also about the material beneﬁts of other actors – have been widely observed in behavioral studies and are consistent with the empirical pattern that many people have aversion to inequality and cooperate only when their initial cooperation is reciprocated by others (Fehr and Fis- chbacher, 2002). Generally, the “actors” could be individuals or groups of individuals occupying positions ranging from household members to decision makers in multiple levels of government. In line with Charness and Rabin (2002), these preferences can be formalized as a general utility function SCAthreshold = SCAFC · CCA + (1 −CCA) · SCAHP (5) SUSthreshold = SUSHP · CUS + (1 −CUS) · SUSFC (6) (5) (6) (5) (6) As explained above, we consider two operation schemes for each country: (1) operate to maximize for ﬂood control or (2) operate to maximize for hydropower production. Depend- ing on the state of cooperation, the choice will change. In most cases, the system will depend on what Canada chooses, and the United States will have to alter its operations in re- sponse. Therefore, when the Canadian probability to coop- erate parameter (CCA) approaches 1, Canada is fully coop- erating. Under cooperation, we assume that Canada operates to maximize ﬂood control, and the United States operates to maximize hydropower. Conversely, when CCA approaches zero, this would indicate lack of cooperation. Under non- cooperation, the Canadian side does not provide ﬂood stor- age to the United States, and, after a few simulation time https://doi.org/10.5194/hess-26-4893-2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4901 ui, given by Eq. (7): ui, given by Eq. (7): Table 2. 3.2.2 Cooperation dynamics The payoff matrix of the mixed-strategy prisoner’s dilemma between Canada and United States showing monetary ben- eﬁt for Canada (wCA_) and the United States (wUS_) in four con- ditions: CC – the US and Canada both cooperate, CN – the United States cooperates and Canada does not, NC – the United States does not cooperate and Canada does, and NN – the United States and Canada both do not cooperate. ui = wi −αi · max wi −wj,0 + βi · max(wj −wi,0), (7) where ui is actor i’s net utility, wi is actor i’s material pay- off, and wj is actor j’s material payoff. Depending on how the signs of α and β are set, the four general types of social preferences described in Sect. 1 can be captured. Note that a positive value of α represents actor i’s disutility from having more than the other actor (the guilt coefﬁcient), and a posi- tive value of β represents actor i’s disutility from having less than the other actor (the jealousy coefﬁcient). Thus, positive α and β values mean that actor i has inequality aversion. United States Canada Coop No coop (CCA) (1 −CCA) Coop (wUSCC, wCACC) (wUSCN, wCACN) (CUS) No coop (wUSNC, wCANC) (wUSNN, wCANN) (1 −CUS) The general utility function of Eq. (7) can be applied to the context of CRT by structuring the utility function U of each country as shown in Eqs. (8)–(11): UCA = wCA −αCA · max(wCA −wUS,0) + βCA · max(wUS −wCA,0) (8) following a particular strategy, compares that strategy’s pay- off to the material payoff of his or her current strategy, and then deterministically chooses the better strategy. Because logit dynamics is more compatible with representation of so- cial preferences, and because of its stochastic best response nature, we chose logit dynamics. Equations (12) and (13) represent the rate of change in the cooperation probability of the two state actors based on logit dynamics: following a particular strategy, compares that strategy’s pay- off to the material payoff of his or her current strategy, and then deterministically chooses the better strategy. Because logit dynamics is more compatible with representation of so- cial preferences, and because of its stochastic best response nature, we chose logit dynamics. 3.2.3 Economic beneﬁt equations The model simulates the beneﬁts that both countries receive from the river. The default operation assumes that the coun- tries cooperate to maximize beneﬁts across the whole system, while in the counter case, beneﬁts are based on operation of each side individually. The economic beneﬁts related to ﬂood control are accounted as the damages prevented by the reser- voir storage operations. Although the U.S. Corps of Engi- neers reports that ﬂood damages in Trail, British Columbia, a city near the International Border, occur when streamﬂow ex- ceeds 6371 m3 s−1 (225 000 cfs) (USACE, 2003), we did not ﬁnd details about the damages related to the seasonal ﬂows in Canada. Therefore, the associated economic beneﬁt due to the damages prevented for the Canadian side due to reservoir operation was assumed to be negligible. HPGrand Coulee = 0.042 · (Qout · h) + 9802.7 (19) (19) In addition, we calculated the daily electricity produced by the other ﬁve dams in Eq. (20): In addition, we calculated the daily electricity produced by the other ﬁve dams in Eq. (20): In the United States, signiﬁcant damages occur when streamﬂow exceeds 12 742 m3 s−1 at Dalles, Oregon, and major damages are caused when ﬂows reach 16 990 m3 s−1 (Bankes, 2012). Therefore, when they are operating jointly, Canada must draw down storage reservoirs before 1 April to accommodate spring runoff and avoid peak ﬂows down- stream. Otherwise, we assume that the United States must switch to a ﬂood control scheme. Flood damages prevented because of reservoir management under CRT were explored by Sopinka and Pitt (2014). They compared the maximum annual daily peak ﬂows at Dalles after the implementation of the CRT and the corresponding monetary damages they could have caused without ﬂood control storage provided. The results of their study were ﬁtted to an exponential curve using Eq. (17), which gives economic beneﬁt in the United HP5 dams = ( 40.3 · (Wﬁsh · Qout) for Wﬁsh · Qout ≤4000m3 s−1 27.8 · (Wﬁsh · Qout) for Wﬁsh · Qout > 4000m3 s−1, (20) (20) where HP5 dams is the hydropower in megawatt hours (MWh) produced by Chief Joseph, McNary, John Day, the Dalles, and Bonneville dams. The variable Qout is Grand Coulee’s daily outﬂow, and Wﬁsh is the weighting factor that considers the operations to meet environmental demands, which is detailed in Sect. 3.2.4. A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs and Canada chooses not to cooperate. The expected mone- tary payoff of Canada is calculated as shown in Eq. (14) (al- though not shown here, an equation with the same structure was used for the expected utility of the United States). The expected net utility of Canada that reﬂects its inequality aver- sion is derived using Eqs. (15) and (16) (although not shown, equations with the same structure were used for the United States). FCUS = 4.007 · exp(2×10−4·QDalles), (17) (17) which presented a R-squared value equal to 0.76. This func- tion was used to estimate the value of ﬂood protection. More details on ﬂood control beneﬁt are presented in Fig. S11– S13. The economic beneﬁt in the United States due to ﬂood damages avoided (FCUS) is based on inﬂow (m3 s−1) into the Dalles dam (QDalles). Thereafter, we found the corre- lation between the Dalles’ inﬂow and the combined out- ﬂow of Grand Coulee (QGrand Coulee) and the Snake River (QSnake River) (Eq. 18). E[wCA] = E[wCACoop] · CCA + E[wCANoCoop] · (1 −CCA) (14) (14) E[UCAcoop] = E[wCACoop] −αCA · max(E[wCACoop] −E[wUS],0) + βCA · max(E[wUS] −E[wCA_Coop],0) (15) QDalles = 1.3329·(QGrand Coulee+QSnake River)−122.91 (18) (15) The Snake River discharge was included in this analysis because its basin is the major tributary to the Columbia River, contributing to ﬂow at the Dalles. The other economic beneﬁt resulting from management of the Columbia River is the electricity produced by the hy- dropower facilities installed in the dams listed in Table 1. Although other dams on the Canadian side of the Columbia Basin have capacity to generate hydropower, the model only considers those three that are part of the CRT. Similarly, we only consider the six federal dams on the US side whose surplus production contributes to the determination of the Canadian Entitlement. Since all six dams produce energy, but only the Grand Coulee operations were modeled, we split the economic beneﬁt from hydropower generation into two parts. Equation (19) resulted from the regression performed between the product of the forebay level (h) times Grand Coulee’s daily average outﬂow (Qout) versus the daily histor- ical hydropower produced by Grand Coulee (HPGrand Coulee) (MWh), which resulted in an R-squared value equal to 0.84. E[UCAnocoop] = E[wCANoCoop] −αCA · max(E[wCANoCoop] −E[wUS],0) + βCA · max(E[wUS] −[wCA_NoCoop],0) (16) (16) 3.2.2 Cooperation dynamics We chose to use logit dynamics (Hof- bauer and Sigmund, 2003) over replicator dynamics (Taylon and Jonker, 1978) because the former enables us to incorpo- rate actors’ innate social preferences, i.e., each actor inter- nally compares two choices (e.g., cooperation vs. defection) in terms of net utilities that reﬂect their social preferences and then makes a probabilistic choice. In comparison, replicator dynamics are based on social comparisons of externally ob- servable material payoffs and social imitation; i.e., each ac- tor sees externally observable material payoffs of other actors It is commonly observed that actors cooperate if they ex- pect others will do the same (Fehr and Fischbacher, 2002). In line with this notion, a mixed-strategy prisoner’s dilemma is used to calculate the expected monetary payoffs, E[w], according to the combination of strategic decisions across countries (Table 2). For example, wCACN is the monetary ben- eﬁt of Canada when the United States chooses to cooperate, https://doi.org/10.5194/hess-26-4893-2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs h d S d ﬂ d l 4902 3.3 Model setup and testing HPCA = µ · γ · Qturb · H 103 · HPCAD (22) (22) The equations described above are formulated into the sys- tem dynamics model and implemented in R, a statistical pro- gramming environment. In this study we used the library package deSolve, version 1.28 (Soetaert et al., 2010, 2020), to solve the initial value problem of ordinary differential equations (ODEs), differential algebraic equations, and par- tial differential equations. The ordinary differential equations wrapper (i.e., lsoda) that uses variable-step, variable-order backward differentiation formulae to solve stiff problems or Adams methods to solve non-stiff problems (Soetaert et al., 2010) was used to compute dynamic behavior of the lumped reservoir system and to assess how the reservoir level and operation rules change as a function of time and different variables. The model was simulated using daily time steps, and the outputs are extracted and presented at monthly scale. Sensitivity analysis was conducted to test the sensitivity of the parameters and identify the parameters that are most im- portant. However, all unknown parameters were used in cal- ibration due to the limited computational cost. The details of the sensitivity analysis are presented in Sect. S3. Since this equation is based on the Mica dam, and, in the model, the three Canadian dams are modeled together, the Qturb and H were interpolated according to the actual and maximum recorded Canadian outﬂow and Canadian storage, respectively. The last economic beneﬁt modeled in this study is the en- titlement that United States returns to Canada as a payment for increased hydropower generation due to the collabora- tion between both countries. The Canadian Entitlement (E), simulated in USD, is a function of the actual entitlement in MWh provided by the United States, the κ parameter, which corresponds to a dimensionless correction factor of the total energy produced by the United States, and the average energy price HPUSD of Oregon and Washington states (Eq. 23). E = Entitlement · κ · HPUSD (23) (23) E = Entitlement · κ · HPUSD 3.2.3 Economic beneﬁt equations The correlation for the ﬁrst and second conditions in Eq. (20) presented R-squared val- ues equal to 0.99 and 0.94, respectively. Correlation to pre- dict hydropower generation from outﬂows and forebay levels is presented in Figs. S14 and S15. In Eq. (21) we calculate the Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 https://doi.org/10.5194/hess-26-4893-2022 A Shrestha et al : Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4903 4903 r production (HPUS) detailed in Eq. (24). Wﬁsh = P5 i=1 Qﬁshi Qoutﬂowi · MaxHPi P5 i=1MaxHPi (24) (24) (21) where HPUSD is the average energy price of Oregon and Washington states according to the U.S. Energy Information Administration (2022). This weighting factor (Wﬁsh) accounts for the fraction of ﬂow ( Qﬁshi Qoutﬂowi ) that no longer goes through the hydropower turbines between April and August because it is released through a spillway or a regulating outlet to meet the biologi- cal demands. We calculated the average monthly fraction for each of the i dams downstream of Grand Coulee and multi- plied it by the maximum hydropower produced by each dam (MaxHPi) to address individual contributions and the partic- ular effect of FOPs at treaty dams. For the Canadian dams, historical data on hydropower production are not available. Therefore, Eq. (22) estimates the economic beneﬁt due to electricity produced in Canada (HPCA) in USD based on the generation ﬂow capacity (Qturb), the maximum hydraulic head (H), the hydropower facility efﬁciency (µ), the speciﬁc water weight (γ ), and the electricity price in British Columbia according to BC Hydro (2020) converted to USD equivalent. 3.4 Scenario analysis Scenario analysis explores dynamics within cooperation and beneﬁt sharing as a result of external environmental factors, institutional capacity, and social and behavioral preferences. 3.4.1 Scenarios based on environmental and institutional change The CRT’s success has been based on beneﬁt sharing be- tween the two countries (Hyde, 2010). However, due to in- creased environmental ﬂows in the United States, some par- ties feel beneﬁts are no longer equitable. Based on these is- sues, four scenarios were developed to represent the changes in institutional capacity and environmental factors that could affect the probability of cooperation. The model was used to simulate the probability of cooperation under these scenar- ios for 28 years between 1990 to 2017, which was compared with the baseline scenario that represents the existing system obtained from the calibrated model. These scenarios are as follows: Figure 4. Overview of calibration process to optimize parameter values using the genetic algorithm. The stopping criteria include either the maximum iteration for the algorithm to run, which is set at 200 generations, or the number of iterations before the algorithm stops, in case no further optimal ﬁtness value can be found, which is set at 70 generations. i. Chi (χ) decreases. The calibrated value of 0.5 decreases to 0.05. χ represents the institutional capacity which de- termines the growth potential of the probability of co- operation. This type of condition could occur due to a more tense relationship between the United States and Canada that could arise due to lack of cooperation in other areas or weaker institutions. A maximum of 200 iterations and a population size of 200 were used to run the algorithm with a stopping criterion of 70 iterations before the algorithm stops when no further im- provement can be found. The selected larger population size and iterations, for eight parameters, ensure that search space is not restricted. The range of parameter values assigned was 0.01 to 0.8 for χ, 0.95 to 1.05 for Wﬁsh, 0.1 to 0.5 for nCA, 0.95 to 1.05 for κ, 0 to 1.3 for αUS and αCA, and −4 to −0.01 for βUS and βCA. The model was calibrated using daily time series data from 1990 to 2005, and ﬁtted parameters were used to validate the model using data from 2006 to 2017. ii. Chi (χ) increases. The calibrated value of 0.5 increases to 0.7. This scenario represents the strengthening of in- stitutions. A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4904 Figure 4. Overview of calibration process to optimize parameter values using the genetic algorithm. The stopping criteria include either the maximum iteration for the algorithm to run, which is set at 200 generations, or the number of iterations before the algorithm stops, in case no further optimal ﬁtness value can be found, which is set at 70 generations. simulated and observed data, with its values ranging from 0 to 1 and 1 indicating better ﬁt. For mathematical expressions of these metrics, readers are referred to Zambrano-Bigiarini (2020). 3.4.1 Scenarios based on environmental and institutional change Note that the selection of χ values for sce- narios “Chi (χ) increases” and “Chi (χ) decreases” was done based on experimentation, where drastic change in CCA and CUS is observed at both ends of increasing and decreasing χ from the calibrated value. iii. High ﬁsh spills. Environmental concerns result in prior- itization of spills for ﬁsh passage. Water for ﬁsh spills increases by 40 % from April through August. g The model assessment for the goodness of ﬁt between modeled and observed values was done using four goodness- of-ﬁt metrics, including root mean square error (RMSE), per- cent bias (PBIAS), volumetric efﬁciency (VE), and relative index of agreement (rd). RMSE gives the standard deviation of the model prediction error, with lower RMSE indicating better ﬁtness. PBIAS measures average tendency of the sim- ulated values to be higher or lower than the observed data, which range from −∞to +∞, and its optimal value is 0. VE is a modiﬁed form of mean absolute error in which ab- solute deviation is normalized by the total sum of observed data, which could range from 0 to 1, with 1 indicating bet- ter agreement. Lastly, rd measures the agreement between iv. Chi (χ) decreases and high ﬁsh spills. Chi (χ) decreases to 0.05, and ﬁsh spills increase by 40 %. It represents the scenario when environmental pressure is high, and institutions are weaker. Calibration and validation The Fish Operation Plan (FOP) details the spills dams must release to meet biological requirements. Fish passage facili- ties have decreased hydropower generation. The Bonneville Power Administration, which operates the US treaty dams, estimates that losses due to forgone revenue and power pur- chases are about USD 27 million to USD 595 million per year (Northwest Power and Conservation Council, 2019). Although the historical data between 1985 and 2018 of hy- dropower generated by the six US dams listed in Table 1 re- veal hydropower production increased after the FOP imple- mentation, when normalized as the ratio of hydropower pro- duction to inﬂows, there is in fact a decrease in production after FOP is implemented. The calibration and selection of appropriate parameter val- ues are essential to accurately reproduce the system’s behav- ior. The calibration parameters can be found in Fig. 4. These parameters are related to both the hydrological and socio- economic components of the system. A genetic algorithm (GA) (Scrucca, 2021) was used to optimize the system dy- namics model, using observation for the period from 1 Jan- uary 1990 to 31 December 2005. The methodological frame- work for model calibration is presented in Fig. 4. A single ob- jective function was deﬁned as minimizing the average root mean square error of reservoir water levels in Canada and the United States (Z), which is given by Eq. (25). In order to address the impact of biological spills on hy- dropower production, we created a weighting factor in the hydropower beneﬁt equation for the United States, which is Z = RMSESca + RMSESus 2 (25) RMSESca + RMSESus (25) 2 https://doi.org/10.5194/hess-26-4893-2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4.1 System dynamics model parameterization and testing During the calibration period from 1990 to 2005 (and to the present) Canada and the United States have conformed to the treaty, irrespective of changes in beneﬁt sharing and proba- bility to cooperate. The selection of these social, economic, and behavioral parameters therefore represents conditions of cooperation regime. Based on the objective function, the goal was to calibrate the model to simulate reservoir levels that match past observations. Figure 5a–d show the simulated and observed time series, during 1990 to 2005, of the stock (storages) and ﬂow (outﬂow) variables, along with the eco- nomic variable of hydropower beneﬁts for the United States. The model performance metrics for the calibration period are shown in Table 4. The metrics show good calibration results with respect to all four metrics. The root mean square error and percent bias are minimal, and volumetric efﬁciency is higher, for both stock and ﬂow variables. Although the mag- nitude of the RMSE is large, it is considered a good ﬁt when compared proportionally with reservoir volumes, streamﬂow, and beneﬁts. i. Scenario 0. We posit that both Canada and the United States have the same inequality aversion (αCA = αUS = 0.9, βCA = βUS = −1). The same inequality aversion means that the actors prefer the beneﬁts to be equally distributed; i.e., each actor wants to increase/de- crease their beneﬁts up to the equitable benchmark when there is imbalance in beneﬁts. This scenario is not the same as the “baseline” scenario discussed above in Sect. 3.4.1, where four scenarios based on environmen- tal and institutional change are compared. As seen in Fig. 5a and b, the total reservoir capacity in the Canadian treaty dams far exceeds the capacity of the US treaty dams, and it is to be noted that the treaty ﬂood control (FC) level in the Canadian dams is 28387 × 106 m3 (equivalent to the 8.95 × 106 AF (acre-feet) ﬂood storage re- quested by the United States). Grand Coulee inﬂow is the primary input to the US storage. Thus, the observed and com- puted inﬂows are compared to ensure accurate model behav- ior (Fig. 5c). The hydropower beneﬁt for Canada depends on US hydropower production due to the Canadian Entitlement; thus, only the beneﬁt of the United States was selected for assessing the calibration results, as estimating hydropower beneﬁt of the United States correctly is an important process in the model (Fig. 5d). 4 Results This section presents results of model parameterization us- ing the genetic algorithm, including results from the scenario analysis. effect of material or beneﬁt payoffs. The key assumption in economic science that economic reasoning is mostly based on self-interest or that all actors are exclusively motivated by their material self-interest is invalid as this assumption rules out the heterogeneity arising from social preferences which substantial fraction of people exhibit (Fehr and Fis- chbacher, 2002). To explore the effect of inequality aversion of each country on the cooperation dynamics, we develop four scenarios with different conﬁguration of α and β val- ues for Canada and the United States (shown in Table 3). Theoretically, the value of the two coefﬁcients should range from β < 0 < α ≤1, and jealousy is more likely than guilt (\|β\| > \|α\|) (Fehr and Schmidt, 1999). The four scenarios are the following: A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoff We did not include the change of the jealousy of the United States or the guilt of Canada in the scenario analysis. This choice is justiﬁed because the net monetary beneﬁt of the United States is always higher than that of Canada, so the United States never feels jealousy, nor does Canada feel guilt. In each scenario, we impose a small amount of white noise to each country’s α and β values, which introduces an element of stochasticity. Table 3. The conﬁguration of different other-regarding preferences of Canada and the United States for scenario analysis. In Scenario 0, both countries have the same level of inequality aversion, while in Scenario 1, the United States has less guilt than Scenario 0, in Sce- nario 2, Canada is more jealous than in Scenario 0, and in Sce- nario 3, both countries are only concerned with their own utility. αCA αUS βCA βUS Scenario 0 0.9 0.9 −1 −1 Scenario 1 0.9 0.3 −1 −1 Scenario 2 0.9 0.9 −3 −1 Scenario 3 0 0 0 0 3.4.2 Scenarios based on social preferences As discussed by Fehr and Fischbacher (2002) and Kertzer and Rathbun (2015), consideration of social preferences is required to understand mechanisms of cooperation and the Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 https://doi.org/10.5194/hess-26-4893-2022 4905 4.1 System dynamics model parameterization and testing Here, the Canadian Entitlement pro- vided in terms of energy supply is converted into monetary units to compare hydropower with other beneﬁts. The sim- ulated hydropower production for the United States is com- ii. Scenario 1. The United States has less guilt than Canada (αCA = 0.9, αUS = 0.3, βCA = βUS = −1). That means the United States is willing to have more beneﬁts than Canada. ii. Scenario 1. The United States has less guilt than Canada (αCA = 0.9, αUS = 0.3, βCA = βUS = −1). That means the United States is willing to have more beneﬁts than Canada. iii. Scenario 2. Canada has more jealousy than the United States (αCA = αUS = 0.9, βCA = −3, βUS = −1). This means Canada is unwilling to have fewer beneﬁts than the United States. iv. Scenario 3. We assume that the both countries have no social preferences (αCA = αUS = βCA = βUS = 0), which signiﬁes self-interest or selﬁshness. In this sce- nario, each country is only concerned with its own util- ity and indifferent to the utility of the other. https://doi.org/10.5194/hess-26-4893-2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4906 4906 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs Figure 5. Calibration result from 1990–2005 showing (a) Canadian storage, (b) US storage, (c) Grand Coulee inﬂow, and (d) hydropower beneﬁt for the United States. Note that sim. – simulated, obs. – observed, full capacity – maximum capacity, CRT FC level – CRT ﬂood protection target level, and min. level – minimum capacity for the US dams. Figure 5. Calibration result from 1990–2005 showing (a) Canadian storage, (b) US storage, (c) Grand Coulee inﬂow, and (d) hydropower beneﬁt for the United States. Note that sim. – simulated, obs. – observed, full capacity – maximum capacity, CRT FC level – CRT ﬂood protection target level, and min. level – minimum capacity for the US dams. Table 4. Calibration (1990–2005) and validation (2006–2017) result. https://doi.org/10.5194/hess-26-4893-2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 https://doi.org/10.5194/hess-26-4893-2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 4.2.1 Scenarios based on environmental and institutional change The four scenarios tested here are based on changes in envi- ronmental and institutional conditions. The results are com- pared with the baseline scenario, which represents cooper- ation between both countries. In the quantile-quantile plot (Fig. 8a–f), the baseline scenario is shown on the horizontal axis and four scenarios on the vertical axis, where each point represents a time step. The scenario “χ decreases” signiﬁ- cantly reduces the probabilities to cooperate for both coun- tries as the maximum CCA reduced from 0.9 to 0.8 and max- imum CUS reduced from 0.7 to 0.6. Reducing χ showed that the maximum as well as minimum probability to cooperate or CCA reduces. The probability to cooperate for Canada under the “χ decreases” scenario is similar to the “χ decreases and high ﬁsh spills” scenario (Fig. 8a); thus blue and cyan points mostly overlap. Similar results were seen for the US prob- ability to cooperate (Fig. 8b). Lowering the χ resulted in lower CCA, and, therefore, Canada would be expected to in- crease the level of storage in its dams to produce more hy- dropower as compared to the baseline (Fig. 8c). This could mean the Canada maintains its reservoir at ∼1300 × 106 m3 higher than in baseline. Lowering the χ impacted CUS too, along with CCA, because, if Canada increased its hydropower production, the United States would have to provide its own ﬂood control. Therefore, reservoir levels in the United States would decrease as compared to the baseline when χ de- creases (Fig. 8d). Since Canada would produce its own hy- dropower in this scenario, the monetary beneﬁt slightly in- creases or remains similar compared to the baseline at the daily timescale, and the result is similar to the “χ decreases and high ﬁsh spills” scenario for Canada (Fig. 8e). PBIAS for both calibration and validation showed that the result is close to optimal, and Grand Coulee inﬂow showed the best ﬁt with the PBIAS value that is closest to 0. VE is only applied to the reservoir volumes and streamﬂow, as per the suitability of the metric. VE values are greater than 0.72, suggesting a good ﬁt. Similarly, agreement index or rd values indicated better performance for all the comparisons, except for Canadian storage. The result of these metrics show that the model is able to replicate and predict the desired behavior. 4.2 Scenario analysis pared to the observed cumulative energy production data re- trieved from the U.S. Army Corps of Engineers database. The beneﬁt in terms of the monetary value is obtained by multiplying the average unit cost (USDMWh−1) of energy by the hydropower quantity (MWh). The scenario analysis results presented below are based on environmental and institutional change and social pref- erences. The scenario analysis covers the same time pe- riod from 1990 to 2017, utilizing observed inﬂow, tributary streamﬂow, and storages, and the same initial conditions as these simulations are not for projection but rather to gain a deeper understanding of dynamics in the socio-hydrological system. y y p q y ( ) The model validation period was 12 years from 2006– 2017 (Fig. 6a–d). Compared to calibration results, model val- idation presented slightly better results in terms of RMSE and PBIAS (Table 4). The simulated behavior of the reser- voir level in Canada and the United States during calibra- tion and validation is quite similar (Fig. 6a and b). In Cana- dian reservoirs, the model accurately simulates the maximum peaks, but the simulated low reservoir level is higher than the observed (Figs. 5a and 6a). Meanwhile, for the US reser- voirs, the simulated lower reservoir level is lower than ob- served (Figs. 5b and 6b). It is to be noted that the actual op- erating rules for these dams are dynamic, based on seasonal changes and weather forecasts. In practice, they may change suddenly from the predetermined plan given unforeseen cir- cumstances. Therefore, it is impossible to capture the exact behavior in a lumped model of this kind. The validation re- sult for Grand Coulee inﬂow (Fig. 6c) and hydropower bene- ﬁt for the United States (Fig. 6d) showed similar performance as the calibration period with the ability to simulate accurate model outputs. A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4.1 System dynamics model parameterization and testing Stock and ﬂow variables Metric Calibration Validation Storage Canada RMSE 5317.07 × 106 m3 4069.82 × 106 m3 PBIAS (%) 14.30 6.00 VE 0.82 0.87 rd 0.68 0.81 Storage United States RMSE 1407.39 × 106 m3 1153.32 × 106 m3 PBIAS (%) −7.3 −5.60 VE 0.90 0.91 rd 0.78 0.84 Grand Coulee dam (GCL) inﬂow RMSE 874.73 m3 s−1 839.71 m3 s−1 PBIAS (%) −7.50 −8.50 VE 0.76 0.77 rd 0.80 0.85 HP beneﬁt RMSE USD 5.77 million USD 5.65 million PBIAS (%) 4.5 8.8 VE – – rd 0.71 0.74 Table 4. Calibration (1990–2005) and validation (2006–2017) result. 4907 https://doi.org/10.5194/hess-26-4893-2022 4.2.1 Scenarios based on environmental and institutional change Figure 7a and b show the utility of monetary beneﬁt and dynamics of the probability to cooperate for the United States and Canada during the calibration and validation peri- ods. This model simulation with calibrated parameters over 1990 to 2017 is also referred to as baseline in the next sec- tion. The share of beneﬁts that the United States receives is higher than the beneﬁt in Canada, relatively, despite the Canadian Entitlement (Fig. 7a). The minimum probabilities to cooperate for the Canada converge at 0.5 and for the United States at 0.4, while peak amplitude for cooperation dynamics is higher for Canada compared to the United States (Fig. 7b). During each time step, the probability to coopera- tion changes, as shown in Eqs. (12) and (13). The periodicity in the probability to cooperation is due to the seasonality in the streamﬂow pattern. It is to be noted that for the key deci- sions regarding the reservoir operations, the peak amplitude is the deciding criterion. The change in χ represents the higher or lower rate of change in probability to cooperate. The “χ increases” sce- nario indicates better institutional capacity that favors coop- eration to either maintain its highest level or increase in the magnitude for cooperation. Maintaining the highest level of the probability to cooperate is most important, which deter- mines the storage thresholds. Increasing χ helped maintain the maximum probabilities to cooperate (i.e., CCA and CUS) and also slightly increase its magnitude (Fig. 8a and b). With increasing χ, Canada would continuously provide ﬂood con- trol to the United States as agreed upon in the CRT; hence https://doi.org/10.5194/hess-26-4893-2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4908 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs Figure 6. Validation result 2006–2017 showing (a) Canadian storage, (b) US storage, (c) Grand Coulee inﬂow, and (d) hydropower beneﬁt for the United States. Note that sim. – simulated, obs. – observed, full capacity – maximum capacity, CRT FC level – CRT ﬂood protection target level, and min. level – minimum capacity for the US dams. 4908 Figure 6. 4.2.1 Scenarios based on environmental and institutional change Validation result 2006–2017 showing (a) Canadian storage, (b) US storage, (c) Grand Coulee inﬂow, and (d) hydropower beneﬁt for the United States. Note that sim. – simulated, obs. – observed, full capacity – maximum capacity, CRT FC level – CRT ﬂood protection target level, and min. level – minimum capacity for the US dams. beneﬁts. It is to be noted that this loss of hydropower pro- duction affects the United States but has no effect on Cana- dian beneﬁt because the United States remains obligated to pay the Canadian Entitlement, even if hydropower produc- tion is lower. The combined scenario of “χ decreases and high ﬁsh spills” has similar results to the “χ decreases” sce- nario (Fig. 8a–e), but reduction in monetary beneﬁt is slightly higher compared to the “χ decreases” and “high ﬁsh spills” scenarios. storage level remains similar to the baseline (Fig. 8c). In addition, the United States continues its existing operations to produce maximum hydropower; hence the storage level in the United States remains the same as in the baseline (Fig. 8d). With increasing χ, Canada’s and the United States’ beneﬁt continues to be the same as the baseline (Fig. 8e). When χ increases or decreases, the utility beneﬁt that the United States receives does not change signiﬁcantly. This is due to the United States balancing the increased ﬂood dam- age control, while hydropower production is compromised. The “high ﬁsh spills” scenario refers to strict regulation to protect ﬁsh passage along the Columbia River, which has negative implications for hydropower production. Increasing ﬁsh spills in US dams has no effect on the Canadian prob- ability to cooperate (CCA) as it does not affect Canadian dam operation (Fig. 8a). Increasing the ﬁsh spills decreases peak CUS slightly, but the average remained similar to the baseline (Fig. 8b). This also does not affect the reservoir op- eration and storage level in the US dams (Fig. 8d), but mon- etary beneﬁt for the US decreases due to regulation as water is diverted from the hydropower turbines (Fig. 8f). It could mean the loss of ∼6000–26 000 MWh worth of hydropower 4.2.2 Scenario analysis in terms of social preferences In addition to the scenarios above, four different scenarios of social preferences were tested and compared to each other. Figure 9 shows the differences between the expected utility of cooperation and non-cooperation from each country ac- cording to different scenarios. Figure 10a–c show the changes in the probability to coop- eration (CCA and CUS) according to the different conﬁgura- tions of social preferences. As shown in Fig. 10a–c, Canada’s probability of cooperation is always higher than 0.5 in all sce- narios because Canada can get higher expected utility when Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 https://doi.org/10.5194/hess-26-4893-2022 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4909 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4909 Figure 7. Change in (a) the utility of monetary beneﬁt and (b) probability to cooperate during calibration and validation period for Canada and the United States. Note that the lower initial probability to cooperate during 1990 is only due to the warmup period of model simulations. Figure 7. Change in (a) the utility of monetary beneﬁt and (b) probability to cooperate during calibration and validation period for Canada and the United States. Note that the lower initial probability to cooperate during 1990 is only due to the warmup period of model simulations. it chooses to cooperate, no matter which behavioral types the two countries possess. This explains why the probabil- ity to cooperate in Canada is always higher than the United States in Fig. 10a–c. Conversely, since the expected utility of cooperation in the United States is always smaller than the expected utility of non-cooperation in Fig. 9b, the probabil- ity of cooperation of the United States is always less than Canada (Fig. 10a–c). cooperate due to the Canadian Entitlement (Fig. 10b). In Scenario 3, the differences between the expected utility of cooperation and non-cooperation decrease compared to Sce- nario 0 if Canada does not care about the counterpart’s pay- offs and focuses on its own payoffs (Fig. 9a). Cooperation will decline as Canada is narrowly self-interested in the fair distribution of material payoffs (Fig. 10c). In terms of coop- eration, selﬁshness is worse than jealousy. Comparing Scenario 0 and Scenario 1 from the standpoint of Canada, we found that there was no difference in the out- puts between Scenario 0 and Scenario 1 (Fig. 10a). 4.2.2 Scenario analysis in terms of social preferences This means that a decrease in the guilt coefﬁcient of the United States does not affect Canadian decision-making on whether to cooperate or not. However, in Scenario 2, the gap between the expected utilities with cooperation and without cooper- ation widens, and Canada is more likely to continue coop- erating when Canada feels more jealousy (more sensitive to disadvantageous inequity) (Fig. 9a). From the standpoint of Canada, it is always economically beneﬁcial to cooper- ate with the United States because Canada can receive the entitlement from the United States under the CRT. In other words, the more unfair the distribution of material beneﬁts between Canada and the United States, and the greater the jealousy of Canada, the more Canada will be motivated to From the standpoint of United States, there was no dif- ference between Scenario 0 and Scenario 2 in terms of out- puts (Fig. 10b). This implies that a rise in Canada’s jealousy coefﬁcient has no effect on the decision of United States of whether to cooperate. Comparing Scenario 0 and Scenario 1, the difference between expected utilities with and without cooperation is expanded, but the expected utilities of non- cooperation are larger than those of cooperation (Fig. 9b). As a result, the United States is less inclined to cooperate in the future when it feels less guilty (less sensitive to advanta- geous inequity) (Fig. 10a). In other words, the more material beneﬁts Canada receives, and the less guilt the United States has, the more driven the United States will be motivated to break the treaty. Like Scenario 3, if the United States does not care about the counterpart’s payoffs and focuses on its own payoffs, the relative magnitude of expected utility of co- https://doi.org/10.5194/hess-26-4893-2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4910 ure 8. Quantile–quantile plot of the baseline versus other scenarios (χ decrease, χ increase, high ﬁsh spills, and combined χ decrease d high ﬁsh spills) comparing probabilities to cooperate, reservoir storage volumes, and utility of monetary beneﬁts. Figure 8. Quantile–quantile plot of the baseline versus other scenarios (χ decrease, χ increase, high ﬁsh spills, and combined χ decrease and high ﬁsh spills) comparing probabilities to cooperate, reservoir storage volumes, and utility of monetary beneﬁts. 4.2.2 Scenario analysis in terms of social preferences United States has zero or negative beneﬁt from the CRT. The United States has some beneﬁts, but it would not continue to cooperate because the beneﬁts of not cooperating are greater than the beneﬁts of cooperating. As environmental concerns increase, the net beneﬁt of the United States is expected to decline further because of lower hydropower beneﬁt, so the United States is less likely to agree with continuation of the treaty until it is changed to create greater beneﬁts for the United States from cooperation. operation will decrease. As the guilt of the United States de- creases, the United States becomes less concerned about a “fair deal” with Canada and loses the motivation to continue cooperation. Therefore, the United States can maximize its proﬁts by halting cooperation (not paying the Canadian En- titlement) and operating unilaterally. Since Canada gets the entitlement due to the CRT, Canada is likely to continue cooperating. If the US preference for a fair distribution of beneﬁts declines during future CRT ne- gotiations, such as in Scenario 1 and Scenario 3, the United States is more likely to break the treaty or change its stance on the Canadian Entitlement. That does not mean that the https://doi.org/10.5194/hess-26-4893-2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4911 Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4911 ure 9. The differences between the expected utility of cooperation and no cooperation from each country according to different scenarios a) Canada and (b) the United States. Figure 9. The differences between the expected utility of cooperation and no cooperation from each country according to different scenarios for (a) Canada and (b) the United States. 5 Discussion and conclusion derlying drivers of a successful cooperative regime and the factors that inﬂuence each country’s choice about whether to cooperate or not. The provisions of the CRT expire in 2024, and negotiations for the next phase of the treaty are ongoing. There have been many prominent discussions about what the future of the treaty should look like, including is- sues related to hydropower generation versus ﬁsh and how to account for spills (Blumm and Deroy, 2019; Harman and Stewardson, 2005; Leonard et al., 2015; Muckleston, 1990; Northwest Power and Conservation Council, 2019; United States Government Accountability Ofﬁce, 2018). Addition- ally, both countries perceive imbalances in the beneﬁts that are received from the CRT relative to what each deserves or compared to what they perceive the other side’s beneﬁts to be (Holm, 2017; Stern, 2018). As discussed in Gain et al. (2021) and Gober and Wheater (2014), the success in treaties or in- stitutions managing river basins depends not only on the con- trol of hydrology, but also on consideration of socio-political dynamics. This study shows that addressing emerging social and environmental issues are critical to continued coopera- The CRT is regarded as one of the most successful trans- boundary river agreements. As the upstream and downstream actors, Canada and the United States have asymmetric ac- cess to water resources and different positions with regard to the risk of ﬂoods and potential for hydropower produc- tion. Within the Columbia River basin, Canada is less sus- ceptible to ﬂood risk relative to the United States, and the United States has capacity for higher hydropower produc- tion relative to Canada. The unique feature of the CRT is that the two countries developed a plan to manage the river as a uniﬁed system and to share the costs and beneﬁts equitably (Bankes and Cosens, 2012; Shurts and Paisley, 2013). This collective sharing of risks from ﬂooding and beneﬁt from hydropower as indicated by Wolf (2007) and Zeitoun et al. (2013) makes the CRT successful among other transbound- ary river treaties. This study examines the dynamics of coop- eration and how it is affected by feedback between humans and natural systems. It is important to understand the un- https://doi.org/10.5194/hess-26-4893-2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4912 A. https://doi.org/10.5194/hess-26-4893-2022 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs waterway is. These scenarios represent current and plausible future socio-political and environmental changes. We found that institutional capacity (χ) plays an important role in long- term cooperation (Figs. 8a, b and S17). Stronger environ- mental regulation for increased ﬁsh spills affects the ben- eﬁt for the United States but not as substantially as when χ (institutional capacity) decreases. Canada continues to re- ceive payment through the Canadian Entitlement, even when the United States is producing less hydropower, something that is interesting to explore further for future negotiations of the CRT. Different conﬁgurations of social preferences for the behavioral model of Canada and United States were used to demonstrate how the probability to cooperate changes. The expected utility of cooperation as compared to expected util- ity of non-cooperation is higher for Canada and lower for the United States (Fig. 9). Thus, the probability to cooperate was simulated to be higher for Canada. The results show that both the guilt coefﬁcient of the United States and the jealousy co- efﬁcient of Canada affect the level of cooperation. For future CRT negotiations, the ideas considered in this study could help provide insight into the long-term dynamics of coopera- tion and the impacts of beneﬁt sharing. For other transbound- ary rivers, e.g., along Nepal and India, Bangladesh and India, or India and Pakistan (Ho, 2016; Mirumachi, 2013; Saklani et al., 2020; Thomas, 2017; Uprety and Salman, 2011), the jealousy and guilty coefﬁcient between actors and their so- cial preferences will not be the same as in Columbia River basin. Similarly, the tipping points for the balance of coop- eration arising from environmental and social change could be different, and this warrants future research in other trans- boundary river basins. Our approach of integrating concept of behavioral science such as social preferences is suitable, particularly (and extendable) in cases when reciprocity be- tween actors is the main driver for cooperation and where system operates to share beneﬁts equitably while ensuring the resources are sustainable. A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs As mentioned previously, the results of this study can help inform the renegotiation of the CRT in two ways: (1) the methods of modeling the hydrological and social systems in tandem, and using behavioral economics, could be used to help formulate policies or management priorities, and (2) un- derstanding of the connection between the share of beneﬁts received by each side and cooperation can support negoti- ation discussions to ﬁnd solutions that would satisfy both sides. More generally, the model demonstrates that under- standing the motivations of each country in terms of guilt and jealousy might provide insight into the factors driving each country and the thresholds that might inﬂuence their decision about whether to cooperate. We also ﬁnd that it is of great importance to maintain institutional strength in support of cooperation. Unlike the United States and Canada where a non- cooperative regime or resort to direct conﬂict is unantici- pated, even if the beneﬁts are perceived to be severely im- balanced, there are many other river basins where different environmental challenges are evolving (UNEP, 2016), and political tensions are high. Globally, conﬂicts do arise be- tween countries that share a water source, with root causes that extend far beyond the water system (Sadoff and Grey, 2002). However, transboundary rivers support the livelihoods of millions of people, preserve ecosystems, and provide a vi- tal resource that needs to be managed sustainably. Using the methodologies presented in this study and the insights gained could be applied to other river basins around the world to help us understand what behaviors and beneﬁts are driving choices about cooperation. Code and data availability. Data supporting the conclusions in this paper are publicly available at Environment Canada (2022; https: //waterofﬁce.ec.gc.ca/mainmenu/historical_data_index_e.html, last access: 1 July 2022), the Bonneville Power Admin- istration (2020; https://www.bpa.gov/p/Power-Products/ Historical-Streamﬂow-Data/Pages/Monthly-Data.aspx, last access: 1 July 2020), the U.S. Army Corps of Engineers (https: //www.nwd-wc.usace.army.mil/dd/common/dataquery/www/, last access: 1 July 2022; USACE, 2022; https://usace.contentdm.oclc. org/digital/collection/p266001coll1/id/3193/, last access: 10 Au- gust 2022; USACE, 2013), and the United States Geological Survey (USGS, 2022; https://waterdata.usgs.gov/nwis/uv?14105700, last access: 1 July 2022). Other processed data are available on request from the corresponding author (ashres15@asu.edu). This socio-hydrological system dynamics model can be further improved by considering additional variables related to climate change, land use change, and water use regime changes. 5 Discussion and conclusion Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs e probability to cooperate of each country according to different scenarios: (a) Scenario 1, (b) Scenario 2, and (c) Scenario 3. ng valuable insights for the current renegotiation common interests and multi layered economic ties The A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tra e 10. The probability to cooperate of each country according to different scenarios: (a) Scenario 1, (b) Scenario 2, and (c) Scen ure 10. The probability to cooperate of each country according to different scenarios: (a) Scenario 1, (b) Scenario 2, and ( to cooperate of each country according to different scenarios: (a) Scenario 1, (b) Scenario 2, and (c) Scenario 3 Figure 10. The probability to cooperate of each country according to different scenarios: (a) Scenario 1, (b) tion, providing valuable insights for the current renegotiation process, as well as future treaty negotiations on transbound- ary waterways similar to the Columbia River. common interests, and multi-layered economic ties. The socio-hydrological system dynamics model developed for this study captures the dynamics of cooperation to reﬂect ex- ternal perturbations. Explicitly incorporating the probability to cooperate CCA and CUS (Eqs. 5 and 6) into the model en- ables exploration of the factors inﬂuencing cooperation. This study further illustrates the utility of simpliﬁed lumped mod- els in understanding complex systems. Natural and social systems evolve over time. Under un- foreseen and uncertain changes, the balance of these sys- tems could shift. A subtle social change can be induced by environmental and hydrological changes, which in turn lead to further unforeseen changes in hydrologic or physi- cal systems. For the Columbia River basin, sudden change in cooperation and deviation from cooperation to conﬂict is not anticipated because both countries that have simi- lar economy and political power and have shared values, This socio-hydrological model presented here further al- lowed for the exploration of scenarios under environmen- tal and institutional changes and social preferences, to un- derstand how robust the cooperation on this transboundary https://doi.org/10.5194/hess-26-4893-2022 Hydrol. Earth Syst. Sci., 26, 4893–4917, 2022 4913 A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs The key limitation of this study is the explicit con- sideration of water use for hydropower production and ﬂood control only. The study does not consider future projections of these variables, which would be a possible direction for future research. Another limitation is the method of estima- tion of ﬂood damages. We estimated the economic beneﬁts involving ﬂood damage prevention, which does not include the monetary beneﬁt of ﬂood control in Canada due to treaty dams because little information is available in the scientiﬁc literature and ofﬁcial reports, and existing resources indi- cate signiﬁcantly less ﬂood damage in Canada relative to the United States (BC Ministry of Energy and Mines, 2013; Northwest Power and Conservation Council, 2022). How- ever, future studies should investigate the magnitude of this beneﬁt since there are certainly ﬂood risks averted by Cana- dian storage. Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/hess-26-4893-2022-supplement. Author contributions. AS, FAAS, SP, and CC planned this work as participants of “Socio-Hydrology Summer Institute on Transbound- ary Rivers”. AS focused on model development and analysis, FAAS and AS focused on data collection and data analysis, SP focused on A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs 4914 formation Processing, vol. 420, edited by: Morais, D. C., Fang, formation Processing, vol. 420, edited by: Morais, D. C., Fang, L., and Horita, M., Springer, Cham, https://doi.org/10.1007/978- 3-030-77208-6_8, 2021. formation Processing, vol. 420, edited by: Morais, D. C., Fang, behavior economics, and CC focused on review and synthesizing the Columbia River Treaty. AS, FAAS, SP, and CC conceptualized the system dynamics framework, FAAS and AS formulated stock and ﬂow equations, SP formulated cooperation dynamics equations, and AS, FAAS, and SP formulated hydropower and ﬂood control beneﬁt equations. CC conducted assessment of past and current is- sues affecting treaty renegotiation, AS wrote the model script and performed model testing, scenario analysis, and data visualization, and SP performed social preference scenario analysis and assess- ment. AS, FAAS, SP, and CC wrote the manuscript draft, AS re- vised the manuscript, and MG, DJY, and EMM provided guidance and funding and reviewed and edited the manuscript. L., and Horita, M., Springer, Cham, https://doi.org/10.1007/978- 3-030-77208-6_8, 2021. L., and Horita, M., Springer, Cham, https://doi.org/10.1007/978- 3-030-77208-6_8, 2021. Bankes, N.: Flood Control Regime of the Columbia River Treaty: Before and after 2024, Washington Journal of Environmental Law & Policy, 2, 1–73, 2012. Bankes, N.: The Columbia River Treaty between Canada and the United States of America–time for change?, in: Water Resource Management and the Law, edited by: Hollo, E. J., Edward El- gar Publishing, https://doi.org/10.4337/9781785369834.00019, 2017. Bankes, N. and Cosens, B.: The Future of the Columbia River Treaty, Program on Water Issues, Munk School of Global Affairs at the University of Toronto in collaboration with the University of British Columbia, the University of Idaho and the University of Calgary, Toronto, Ontario Canada, 2012. Competing interests. The contact author has declared that none of the authors has any competing interests. BC Hydro: Electricity rates & energy use, https://app.bchydro. com/accounts-billing/rates-energy-use.html (last access: 1 June 2021), 2020. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. BC Ministry of Energy and Mines: US Beneﬁts from the Columbia River Treaty – Past, Present and Future: A Province of British Columbia Perspective, https://engage.gov.bc.ca/app/uploads/ sites/6/2012/07/US-Beneﬁts-from-CRT-June-20-13-2.pdf (last access: 1 June 2021), 2013. Special issue statement. This article is part of the special issue “Socio-hydrology and transboundary rivers”. It is not associated with a conference. Bernauer, T. and Böhmelt, T.: International conﬂict and coop- eration over freshwater resources, Nat. Sustain., 3, 350–356, https://doi.org/10.1038/s41893-020-0479-8, 2020. Blumm, M. A. Shrestha et al.: Socio-hydrological modeling of ﬂood control and hydropower tradeoffs C. and Deroy, D.: The Fight over Columbia Basin Salmon Spills and the Future of the Lower Snake River Dams, Washington Journal of Environmental Law & Policy, 9, 1–26, 2019. Acknowledgements. We acknowledge “Summer Institute on Socio- hydrology and Transboundary Rivers” held in Yunnan University, China, in 2019, and Jing Wei for support and feedback. We also acknowledge our professors – Giuliano Di Baldassarre, Günter Blöschl, Megan Konar, Amin Elshorbagy, Fuqiang Tian, and Mu- rugesu Sivapalan – for their feedback we received during and after the institute. 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https://openalex.org/W2792221206	https://research.vu.nl/ws/files/72064392/2.484.pdf	English	null	Determinants of selective reporting: A taxonomy based on content analysis of a random selection of the literature	PloS one	2,018	cc-by	8,343	Determinants of selective reporting Determinants of selective reporting van der Steen, Jenny T; van den Bogert, Cornelis A; van Soest-Poortvliet, Mirjam C; Fazeli Farsani, Soulmaz; Otten, René H J; Ter Riet, Gerben; Bouter, Lex M Link to publication in VU Research Portal Link to publication in VU Research Portal citation for published version (APA) van der Steen, J. T., van den Bogert, C. A., van Soest-Poortvliet, M. C., Fazeli Farsani, S., Otten, R. H. J., Ter Riet, G., & Bouter, L. M. (2018). Determinants of selective reporting: A taxonomy based on content analysis of a random selection of the literature. PLoS ONE, 13(2), e0188247. 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E-mail address: vuresearchportal.ub@vu.nl E-mail address: vuresearchportal.ub@vu.nl Download date: 24. Oct. 2024 RESEARCH ARTICLE Design Inductive qualitative content analysis of a random selection of the pertinent literature includ- ing empirical research and theoretical reflections. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Jenny T. van der Steen1,2, Cornelis A. van den Bogert3, Mirjam C. van Soest-Poortvliet4, Soulmaz Fazeli Farsani5¤, Rene´ H. J. Otten6, Gerben ter Riet7, Lex M. Bouter8,9 Jenny T. van der Steen1,2, Cornelis A. van den Bogert3, Mirjam C. van Soest-Poortvliet4, Soulmaz Fazeli Farsani5¤, Rene´ H. J. Otten6, Gerben ter Riet7, Lex M. Bouter8,9 1 Department of Public Health and Primary Care (PHEG), Leiden University Medical Center (LUMC), Leiden, the Netherlands, 2 Department of Primary and Community Care, Radboud university medical center, Nijmegen, the Netherlands, 3 Division of Pharmacoepidemiology & Clinical Pharmacology, Utrecht University, Utrecht, the Netherlands, 4 Program on Aging, Netherlands Institute of Mental Health and Addiction (Trimbos-instituut), Utrecht, the Netherlands, 5 Division of Pharmacoepidemiology & Clinical Pharmacology, Utrecht University, Utrecht, the Netherlands, 6 Medical Library, Vrije Universiteit, Amsterdam, the Netherlands, 7 Department of General Practice, University of Amsterdam, Academic Medical Center, Amsterdam, the Netherlands, 8 Department of Epidemiology & Biostatistics, VU University Medical Center, Amsterdam, the Netherlands, 9 Vrije Universiteit, Faculty of Humanities, Department of Philosophy, Amsterdam, the Netherlands a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ¤ Current address: Boehringer Ingelheim GmbH, Ingelheim am Rhein, Germany. ¤ Current address: Boehringer Ingelheim GmbH, Ingelheim am Rhein, Germany. * jtvandersteen@lumc.nl urrent address: Boehringer Ingelheim GmbH, Ingelheim am * jtvandersteen@lumc.nl OPEN ACCESS Citation: van der Steen JT, van den Bogert CA, van Soest-Poortvliet MC, Fazeli Farsani S, Otten RHJ, ter Riet G, et al. (2018) Determinants of selective reporting: A taxonomy based on content analysis of a random selection of the literature. PLoS ONE 13(2): e0188247. https://doi.org/10.1371/journal. pone.0188247 To develop a taxonomy of determinants of selective reporting in science. Copyright: © 2018 van der Steen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Selective reporting is wasteful, leads to bias in the published record and harms the credibility of science. Studies on potential determinants of selective reporting currently lack a shared taxonomy and a causal framework. Editor: Martin Chalumeau, Universite Paris Descartes, FRANCE Objective To develop a taxonomy of determinants of selective reporting in science. Determinants of selective reporting: A taxonomy based on content analysis of a random selection of the literature Jenny T. van der Steen1,2, Cornelis A. van den Bogert3, Mirjam C. van Soest-Poortvliet4, Soulmaz Fazeli Farsani5¤, Rene´ H. J. Otten6, Gerben ter Riet7, Lex M. Bouter8,9 ¤ Current address: Boehringer Ingelheim GmbH, Ingelheim am Rhein, Germany. jtvandersteen@lumc.nl Conclusions We designed a taxonomy of putative determinants of selective reporting consisting of 12 categories. The taxonomy may help develop theory about causes of selection bias and guide policies to prevent selective reporting. Results Saturation was reached after analyzing 64 articles. We identified 497 putative determinants, of which 145 (29%) were supported by empirical findings. The determinants represented 12 categories (leaving 3% unspecified): focus on preferred findings (36%), poor or overly flexi- ble research design (22%), high-risk area and its development (8%), dependence upon sponsors (8%), prejudice (7%), lack of resources including time (3%), doubts about report- ing being worth the effort (3%), limitations in reporting and editorial practices (3%), academic publication system hurdles (3%), unfavorable geographical and regulatory environment (2%), relationship and collaboration issues (2%), and potential harm (0.4%). Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Determinants of selective reporting: A taxonomy from the Dutch Ministry of Health, Welfare and Sports. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Methods Using search terms for bias and selection combined with terms for reporting and publication, we systematically searched the PubMed, Embase, PsycINFO and Web of Science data- bases up to January 8, 2015. Of the 918 articles identified, we screened a 25 percent ran- dom selection. From eligible articles, we extracted phrases that mentioned putative or possible determinants of selective reporting, which we used to create meaningful catego- ries. We stopped when no new categories emerged in the most recently analyzed articles (saturation). Funding: The design of the study and writing of the article was funded by a career award for JTS from the Netherlands Organisation for Scientific Research (NWO; Innovational Research Incentives Scheme: Vidi grant number 917.11.339), and Leiden University Medical Center, Leiden, The Netherlands. CAvdB was supported by a PhD grant Funding: The design of the study and writing of the article was funded by a career award for JTS from the Netherlands Organisation for Scientific Research (NWO; Innovational Research Incentives Scheme: Vidi grant number 917.11.339), and Leiden University Medical Center, Leiden, The Netherlands. CAvdB was supported by a PhD grant 1 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 Introduction Complete, accurate and timely reporting of all (study protocol-stipulated) outcomes is essential for syntheses of research to be valid and as precise as possible [1, 2]. Complete or unselective reporting refers to both unselective publication of all results of a study as well as unselective or complete reporting within publications on all planned outcomes [3]. In other words, all planned outcomes should be reported on within a reasonable time frame (and the exploratory nature of analyses with any unplanned outcomes should be disclosed). Selective reporting leads to bias if specific results remain unpublished because the decision to report depends on the nature of the results (e.g., direction or magnitude of the target associ- ation). Reporting bias is an important threat to the validity of systematic reviews which clini- cians, researchers, policy makers and citizens rely on. Reporting bias is wasteful, distorts the aggregate body of scientific evidence, threatens the credibility of science, but it may also result in suboptimal treatment or even in avoidable harm to, e.g., patients’ health. Therefore, in addi- tion to validity and efficiency reasons, there is an ethical imperative to report all results includ- ing those of clinical trials [4, 5]. Journals increasingly require that protocols have been registered before commencing or completing a study to facilitate the detection of selective reporting and increase transparency, and there is some evidence that registration of trials is effective [6, 7]. However, around half of planned outcomes of clinical trials are not reported, and a third to half of registered clinical tri- als remain unpublished [8–11]. There are numerous reports that suggest that selective publica- tion is a major problem in the clinical domain, but it is also pervasive in basic and translational research [12, 13] and in the social sciences [14]. Selective reporting occurs in various other types of studies and across various designs, e.g., trials with psycho-educational interventions, and quantitative observational and qualitative studies [11, 15, 16]. So-called “protocol-to-publication” and similar studies point to selective reporting of statisti- cally significant results [11, 17, 18]. More generally, “preferred findings” are more often reported. Preferred findings are often statistically significant findings. However, there are exceptions such as in case of studies into adverse effects, or equivalence trials where no difference is preferred. Design, protocol and reporting To develop a taxonomy of putative determinants of selective reporting, we combined princi- ples of systematic reviews [27] with those of inductive qualitative content analysis [28, 29]. Before analyzing full-texts, we piloted search strategies, we piloted abstract and full-text eligi- bility criteria and reviewed procedures as detailed in the protocol (S1 File). We developed the study protocol based on the preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) guidelines [30,31] as far as applicable. In this article, we report applica- ble items from the PRISMA [32] guidelines for systematic review and applicable items from guidelines for the reporting of qualitative studies [33, 34]. Eligibility criteria We included articles that examined or suggested determinants of selective reporting. We searched for articles from any academic discipline reporting on studies employing any type of design based on empirical data, such as intervention studies (with any type of comparator), and observational studies. Additionally, to cover hypotheses on what drives selective reporting, we included non-empirical articles such as editorials presenting opinion, theoretical considerations and anecdotal evidence. To minimize duplication of determinants extracted from articles that were also selected for inclusion in reviews, we excluded reviews. The outcome, selective report- ing, comprised non-publication and selective reporting within publications. Our review also covered the possible consequences of selective reporting, including publication bias and other types of reporting bias. Introduction As PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 2 / 15 Determinants of selective reporting: A taxonomy a consequence, findings preferred by key stakeholders in the research project at issue are likely overrepresented in the scientific literature [19]. Research has suggested that financial conflicts of interest may cause selective reporting (e.g., when studies are sponsored by industry [9, 11, 20, 21]), but non-financial conflicts of interest probably play an important role too. Causes of publication bias, and of reporting bias more generally, may relate to decisions taken by researchers and sponsors, and also decisions by editors [8, 9, 22, 23]. Some have argued that it is human nature to search for positive mes- sages, which suggests that basically, all scientists are at risk [2, 24]. However, certain persons or environments may be at increased risk of selective reporting, such as junior researchers [25] and scientists in more competitive academic environments [26]. Despite numerous studies on selective reporting, there is no accepted taxonomy of its deter- minants and no explicit causal framework. A recently developed framework of non-publica- tion [22] attempted to answer the questions “what?” (defining dissemination), “who?”(is to blame-actor/stakeholder) and “why?” (stakeholders’ motivations). However, it does not pro- vide a single taxonomy of determinants and its scope was limited to clinical trials. We aimed to develop a taxonomy of putative determinants of selective reporting. We there- fore addressed the questions of “what are possible determinants of selective reporting?,” and “how are putative determinants of selective reporting best grouped based on its content?“. Search strategy The search strategy focused on selective reporting to avoid limitation to any preconceived determinants. We used terms for bias and selection combined with terms for reporting and publication (S1 File, Box 1), and we pilot tested the search strategy. For reasons of feasibility of identifying determinants with qualitative content analysis (S1 File, Box 1), we retained a 25% random sample of the total of 918 hits after deduplication, from all databases searched from inception to January 8th 2015 (Fig 1). We used SPSS version 22’s random allocator function “random sample of cases” to randomly select a quarter com- prising 230 hits. Data collection process Full-text data extractions were performed by pairs of researchers (JTvdS, CAvdB, MCvS-P, SFF, and GtR). JTvdS, CAvdB and MCvS-P piloted a standardized full-text data extraction spreadsheet using three empirical and non-empirical articles not included in the random sample. Initially, the reviewers extracted data independently, but after, for each pair of analysists, the analysis of a few articles made it clear that determinants were identified consistently, one reviewer extracted data with verification by another (S1 File, paragraph Data collection process). Data items At the level of the article, we abstracted year of publication, academic discipline and study design (S1 File, box 2). More than one determinant per article was possible. We assessed whether evidence of association of a determinant with the outcome was empirical, indicated an actor (stakeholder), referred to a cause or could possibly be interpreted as a cause (we thus explicitly assessed the degree of interpretation [35]). We assessed the type and scope of selec- tive reporting (whether limited to a single medium, e.g., a specific journal) and the nature of the association between a putative determinant and selective reporting (if there was any associ- ation, the direction, and strength of association, if applicable). Information sources We searched PubMed, Embase, PsycINFO and Web of Science to cover a wide range of aca- demic disciplines from inception to January 8th 2015. We limited the search to the English, French, German and Dutch languages. 3 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 Determinants of selective reporting: A taxonomy Selection of articles Titles and abstracts were screened independently against the inclusion criteria by pairs of two reviewers (JTvdS, CAvdB and MCvS-P). If there was no abstract, we reviewed keywords. We used the first ten percent of abstracts (23) to test a shared understanding of the inclusion crite- ria and we discussed discrepant interpretations. Of the other abstracts, we calculated inter- rater agreement (percentage) of decisions on retrieval as full-text. We evaluated for eligibility all full-text articles and we calculated inter-rater agreement also for this step. PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 Analyses The sample of articles and determinants was summarized using percentages. We used qualitative iterative and inductive content analysis to group determinants by content and form categories of determinants [29]. In particular, the reviewers (pair) extracted putative determinants in the context of the article, and the team subsequently discussed interpreta- tions and categorization. In the content analysis, we coded putative determinants and subsumed them under meaningful categories without imposing any prior model. We considered determinants which were tested even when authors found them unrelated to selective reporting in the particular study, because different studies often vary in finding associations with outcomes. A single dataset with putative determinants was created after discussions on differences of interpretation. 4 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 Determinants of selective reporting: A taxonomy Next, two researchers (JTvdS and CAvdB) independently categorized all determinants into higher-level groupings. This was a non-linear, iterative process as we classified batches of Records identified through database searching (Pubmed n = 721; Embase n=380; PsycINFO n=108; Web of Science n=450) Screening Included Eligibility Idenﬁcaon Additional records identified through other sources (n = 0) Records after duplicates removed (n = 918) Random sample of records (n = 918) Records excluded (n=688) Full-text articles assessed for eligibility (n = 85) Full-text articles excluded, with reasons: no determinant, outcome not selective reporting Articles eligible for qualitative synthesis (n = 127) Articles included in qualitative analysis of categories of selective reporting, until saturation Records screened (n = 230) Records excluded (n=87) Eligible articles based on title / abstract (n=58) Articles not analysed after having ensured data saturation (n = 63) Fig 1. PRISMA flow diagram of identified and analyzed articles. https://doi.org/10.1371/journal.pone.0188247.g001 Additional records identified through other sources (n = 0) Records after duplicates removed (n = 918) Random sample of records (n = 918) Records excluded (n=688) Records excluded (n=87) Records screened (n = 230) Full-text articles excluded, with reasons: no determinant, outcome not selective reporting Full-text articles assessed for eligibility (n = 85) Eligible articles based on title / abstract (n=58) Articles eligible for qualitative synthesis (n = 127) Articles not analysed after having ensured data saturation (n = 63) Articles included in qualitative analysis of categories of selective reporting, until saturation 1. PRISMA flow diagram of identified and analyzed articles. https://doi.org/10.1371/journal.pone.0188247.g001 Next, two researchers (JTvdS and CAvdB) independently categorized all determinants into higher-level groupings. Analyses This was a non-linear, iterative process as we classified batches of Next, two researchers (JTvdS and CAvdB) independently categorized all determinants into higher-level groupings. This was a non-linear, iterative process as we classified batches of 5 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 Determinants of selective reporting: A taxonomy about 50 determinants followed by discussing all classifications of each batch before moving on to the next batch. We avoided overlap in categories [35] by adding descriptions (to serve as definitions) that we developed from the iterative classification of content. In reaching consen- sus about the categorization we often went back to previous classification work to adapt cate- gories, or to the full-texts to ensure we understood the context. Thus, we constructed a structured list of categories of more specific determinants. An initial classification and any unresolved issues about further classifications were discussed with a third researcher (GtR) to achieve full consensus. Following the principles of content analyses [9], we counted the num- ber of determinants per category for descriptive purposes of which categories were more and which were less prominent in the literature. Saturation. We analyzed content of articles randomly selected from different decades (< 1980, 1980s, 1990s, 2000–2009, 2010–2015). We concluded analyses when saturation was reached. Saturated data ensure replicability in categories derived from content analyses, which in turn verifies and ensures comprehensiveness [35]. Saturation of categories was determined in two ways. First, during the process of analyzing the batches of determinants we assessed satura- tion prospectively, during the content analyses, in the usual way for qualitative analyses (i.e., no new categories emerged in the last analyzed articles). After having analyzed about 50 articles, we deemed the newly classified determinants not to be essentially different from those already classi- fied (we could fit them in the categories we had developed), and we assumed saturation. This was confirmed after having analyzed one more batch of determinants representing 10% of articles (13 of 127). Second, retrospectively, after having analyzed the articles, we verified quantitatively how many articles had been analyzed when the first determinant of each category emerged. Subgroup analyses. We assessed the extent to which various determinants were based on empirical studies or (solely) on opinion (as planned and described in the protocol, S1 File). Selection of articles for analyses We identified 918 unique records, and we included 64 records (articles) in the final analysis (Fig 1). The inter-rater agreement between independent assessors on the need to retrieve the full-text as assessed for 90% of the records was 72% (150/207; agreement about 143 records, doubt about 7, no agreement about 57). The initial agreement about eligibility of full-text assessment was 78% (66/85). All initial disagreements were resolved through discussion. Analyses Further, to avoid inclusion of categories exclusively comprising determinants that were consis- tently unrelated to outcome, we calculated the proportion of articles that (quantitatively or qualitatively) reported a non-significant or no association, or an unexpected direction of an association between a determinant and selective reporting. PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 Description of the dataset: Articles and determinants Characteristic % n Year of publication < 1980 1980s 1990s 2000–2009 2010–2015 2 3 16 44 36 1 2 10 28 23 Academic discipline Clinical medicine Biomedicine / Life sciences Humanities No specific discipline 75 9 11 5 48 6 7 3 Type of study / study design Non-empirical (reflective / theoretical) Observational quantitative, longitudinal Observational quantitative, cross-sectional Case description / anecdotal Simulation / modelling† Randomized trial‡ Qualitative Mixed methods (integrated) Review of reviews 48 27 9 5 5 2 2 2 2 31 17 6 3 3 1 1 1 1 Observational quantitative studies included: comparisons of publications (n = 5), comparison of registry records with publications (4), of protocols with publications (4), of submitted with accepted papers (4), of abstracts with publications (4), of public funder database with publications (1) and of industry database with Medline records (1) † Mathematical simulations of reporting bias, subjective decision-making in peer-review, and the selection process in publication bias, whether purely hypothetical or with use of empirical data ‡ The RCT assessed the effect of blinded peer-review on reviewers’ and editors’ decisions about manuscript acceptance [36]. The determinant was prejudice in the peer-review process, and the outcome was non-publication, considering that the editor’s decision dictates whether the manuscript is published Categories of determinants Table 3 lists the 12 categories that emerged from coding the extracted determinants, along with descriptions and examples. Focus on preferred findings was the largest category (180 deter- minants, 36%), which included, for example, significance chasing. This concerned empirical data in 17% (30/180) of cases, and in 93% (168/180) of cases the original authors postulated that a focus on preferred findings was positively associated with selective reporting. By con- trast, the second largest category (109 determinants, 22%), poor or flexible research design, was based on empirical findings in half of the cases, and the original authors mentioned a positive association in 57% of cases. The other 10 categories occurred less frequently (8% or less) yet represented distinct concepts. References to the 64 analyzed articles are provided per category, as a supplement (S3 File). Description of the dataset: Articles and determinants Of the 64 articles analyzed, 48 (75%) concerned clinical medicine, and 51 (80%) were pub- lished in 2000 or later (Table 1). Almost half of the articles (31) were non-empirical. The empirical studies were mostly observational and quantitative; we found one RCT [36]. Of the 64 articles analyzed, 48 (75%) concerned clinical medicine, and 51 (80%) were pub- lished in 2000 or later (Table 1). Almost half of the articles (31) were non-empirical. The empirical studies were mostly observational and quantitative; we found one RCT [36]. We extracted 497 determinants from the 64 articles (median 6; range 1 (9 articles) to 22 (3 articles; Table 2; S2 File). Twenty-nine percent (145 determinants) concerned empirical evi- dence of associations with selective reporting. If an actor (stakeholder) was mentioned (41%, 204 determinants), it was the investigator in about half of cases (110 determinants). In 79% of cases, an association with selective reporting was found or postulated (all in the hypothesized or expected direction). We extracted 497 determinants from the 64 articles (median 6; range 1 (9 articles) to 22 (3 articles; Table 2; S2 File). Twenty-nine percent (145 determinants) concerned empirical evi- dence of associations with selective reporting. If an actor (stakeholder) was mentioned (41%, 204 determinants), it was the investigator in about half of cases (110 determinants). In 79% of cases, an association with selective reporting was found or postulated (all in the hypothesized or expected direction). 6 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 Determinants of selective reporting: A taxonomy Table 1. Characteristics of the 64 analyzed articles. https://doi.org/10.1371/journal.pone.0188247.t002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 Saturation The first determinant in each category usually emerged after analysis of only a few articles (Fig 2). After having extracted 120 determinants (of 497, 24%) from 14 articles, all categories com- prised one or more determinants. Assuming that a category emerges upon identification of at least one or more determinants that are dissimilar from other categories, this indicates that sat- uration could have been reached earlier, with fewer than the set analyzed articles. 7 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 Determinants of selective reporting: A taxonomy Table 2. Characteristics of the determinants (n = 497), outcomes and their associations. Characteristic % (n) Evidence of association of determinant with outcome Empirical Non-empirical (e.g., from viewpoint, or opinion in discussion section, or inference from the literature or theoretical study) 29 (145) 71 (352) Actor (stakeholder) involved investigators or authors editors or journals reviewers sponsors or industry government analyst no actor mentioned 41 (204) 22 (110) 11 (57) 4 (18) 3 (17) 0.2 (1) 0.2 (1) 59 (293) Interpretation of association (hypothesized, whether confirmed or not) in terms of possible causal pathways† Describes a cause Allows for a single and clear interpretation of cause Unclear cause or multiple causal interpretations are possible 22 (111) 20 (98) 58 (288) Type of selective reporting outcome Non-publication Selective publication in general Selective reporting within publication Reporting bias Other (delayed publication–which risks e.g., no uptake in reviews) 59 (292) 16 (78) 14 (70) 11 (53) 1 (4) Scope of selective reporting outcome Within a single medium (journal or conference) General 13 (66) 87 (431) Reported association between determinant and outcome† Present (confirmed) No association 79 (393) 21 (104) Note: the table is based on the pre-planned items for description of 497 determinants abstracted from the 64 articles Examples: “The company often owns the study database and controls decisions about publication and release of data” (describes a cause); authors reported “lack of time” as a reason (allows for a single and clear interpretation of cause– a cause is implicated (lack of time), but that cause itself begs a more detailed explanation (how does the reporting compete with other duties and why?)); sample size (unclear cause or multiple causal interpretations are possible– such as with larger sample size more power, more collaborators, more rigorous design, more quality checks etc.) †Note that a possible causal interpretation of a determinant under study (a hypothesis) does not necessarily mean that in each case a causal association with the outcome was actually confirmed in the particular study or in the Empirical Actor (stakeholder) involved investigators or authors editors or journals reviewers sponsors or industry government analyst no actor mentioned Note: the table is based on the pre-planned items for description of 497 determinants abstracted from the 64 articles Examples: “The company often owns the study database and controls decisions about publication and release of data” (describes a cause); authors reported “lack of time” as a reason (allows for a single and clear interpretation of cause– a cause is implicated (lack of time), but that cause itself begs a more detailed explanation (how does the reporting compete with other duties and why?)); sample size (unclear cause or multiple causal interpretations are possible– such as with larger sample size more power, more collaborators, more rigorous design, more quality checks etc.) †Note that a possible causal interpretation of a determinant under study (a hypothesis) does not necessarily mean that in each case a causal association with the outcome was actually confirmed in the particular study or in the narrative (finding no association was still possible as an empirical result, or a possible causal association could be denied in a comment) 8 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 Determinants of selective reporting: A taxonomy Discussion We developed a taxonomy of putative determinants of selective reporting in science based on saturated qualitative analyses of a representative sample of the relevant literature. The taxon- omy clusters determinants in a meaningful way. It consists of 12 mutually exclusive categories along with descriptions and examples to clarify boundaries and differences between the cate- gories. The taxonomy should give structure and depth to commonly used expressions such as significance chasing (placed in the category focus on preferred findings) and conflict of interest (financial: dependence upon sponsors and relationship or collaboration issues; intellectual: rela- tionship or collaboration issues and academic publication system hurdles). Two categories, focus on preferred findings and poor or flexible research design, covered over half of the determinants we found. These related mostly to choices of individual researchers or teams. The individual or team level was also referred to in six of the 10 other categories (preju- dice, dependence upon sponsors, lack of resources including time, doubts about reporting being worth the effort, limitations in reporting and editorial practices, which refers to individual edi- tors and authors, and relationship and collaboration issues). Four categories referred to the wider environment. These were the categories of academic publication system hurdles and potential harm; the other two included determinants that often lack clear direction or hypothe- sis (e.g., when a range of disciplines or countries are compared based on distributions): high- risk area and its development, and unfavorable geographic or regulatory environment. Relationships and similarities between categories in view of possible causality The descriptions clarified boundaries between categories that were conceptually close, such as high-risk area and its development, and unfavorable geographical and regulatory environment. Both categories represented a wider environment than the research team or institution. The categories of unfavorable geographical and regulatory environment and academic publica- tion system hurdles were distinct as these included determinants that did not clearly refer to a pos- sible hypothesis regarding a mechanism or cause of selective reporting (an example is provided as a Footnote to Table 3). By including these categories, we were able to classify all determinants, except for 15 that only mentioned a stakeholder (actor) as the source of selective reporting (or the denied source, such as when an actor is believed not to cause selective reporting, in which case we recorded no association between determinant and the outcome of selective reporting). We found six described instances of interaction between determinants (effect modifica- tion). We counted these as classified with the main determinant only. The interactions all clearly described causes. For example, “Outcomes could be deemed post hoc to have little clini- cal relevance if they fail to show significant findings and may thus be omitted when accommo- dating space limitations” [37]. In this case, the interaction between focus on preferred findings and limitations in reporting and editorial practices was classified under the first. By contrast, we classified another interaction, between a focus on preferred findings and high-risk area and its development under the latter as the primary category: “Early in the history of a research domain results in either direction are important news but [that] later, when the preponderance of evidence has supported one direction, significant reversals are often more important news than further replications.” [38]. Determinants of selective reporting: A taxonomy Table 3. Taxonomy of determinants (n = 497) resulting from the inductive qualitative content analyses. Determinant classification, category Description Examples % (n in full sample) % empirical result (n in category /-per row) % any relationship† (n in category /per row) 1. Focus on preferred findings A focus on finding results that match preferences, mostly statistically significant or otherwise positive findings, wishful thinking and acting Significance chasing, finding significant results, larger effect size, suppressing publication of unfavorable results, not being intrigued by null findings 36 (180/497) 17 (30/180) 93 (168/180) 2. Poor or flexible research design Attributes of study design relating to power and level of evidence provide much leeway in how studies are performed and in interpretation of their results Not a controlled or blinded study, study protocol unavailable, small sample size 22 (109/497) 50 (54/109) 57 (62/109) 3. High-risk area and its development Area of research or discipline or specialty including its historical development and competitiveness, the currently dominant paradigms and designs, and career opportunities Ideological biases in a research field, area with much epidemiological research versus clinical or laboratory research (“hard sciences”), humanities, experimental analytic methods, “hot” fields, publication pressure in the specific field 8 (39/497) 31 (12/39) 72 (28/39) 4. Dependence upon sponsors Financial conflict of interest resulting in lack of academic freedom Requirements and influence of funding source with financial interests in study results 8 (38/497) 34 (13/38) 82 (31/38) 5. Prejudice A conscious or unconscious belief that may be unfounded, and of which one may or may not be aware Prior belief about efficacy of treatment, author reputation or gender bias in the phase of review 7 (33/497) 24 (8/33) 82 (27/33) 6. Lack of resources, including time Insufficient manpower or finances Lack of time resulting from excessive workload, or lack of personnel due to life events 3 (17/497) 18 (3/17) 100 (17/17) 7. Doubts about reporting being worth the effort Weighing investment of time and means versus likelihood of gain through publication Anticipating disappointment of yet another rejection or low chances of acceptance of a manuscript, belief that findings are not worth the trouble 3 (16/497) 6 (1/16) 100 (16/16) 8. Limitations in reporting and editorial practices Constraints and barriers to the practice of reporting relevant detail Journal space restrictions, author writing skills 3 (14/497) 71 (10/14) 50 (7/14) 9. Related conceptual work on selective reporting The basis of the framework of Bassler et al. [22] comprised 50 highly cited articles published until 2012 identified in Web of Science, and consensus among 10 experts. Our work represents PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 9 / 15 Academic publication system hurdles Various hurdles to full reporting related to submission and processing of manuscripts (other than reporting) including those that represent an intellectual conflict of interest Solicited manuscripts, authors indicating non-preferred reviewers, editor’s rejection rate 3 (14/497) 36 (5/14) 57 (8/14) 10. Unfavorable geographical or regulatory environment‡ Geographical or regulatory environment that affects how research is being performed Continents under study included North America, Europe and Asia; few international collaborations; no governmental regulation of commercially sponsored research 2 (12/497) 67 (8/12) 75 (9/12) 11. Relationship and collaboration issues Intellectual conflict of interest between reporting and maintaining good relationships Disagreements among co-authors and between authors and sponsors, sponsors prefer to work with investigators who share the sponsor’s position 2 (8/497) 13 (1/8) 100 (8/8) 12. Potential harm Publishing data can harm individuals Risk of bioterrorism, or confidentiality restriction 0.4 (2/497) 0 (0/2) 100 (2/2) (13) Not specified Referring to a stakeholder only Selective publication not caused by editors 3 (15/497) 0 (0/15) 67 (10/15) Empirical result as described in Table 2, first row †A l i hi d i h d di i if l i hi b i h h i d l i hi N f h h h i d l i hi i i i l Empirical result as described in Table 2, first row †Any relationship, and in the expected direction if any relationship was being hypothesized, versus no relationship. None of the hypothesized relationships in empirical result were found to be in the opposite direction ‡We aimed to consistently include a direction in all category names. However, the work from which we abstracted the determinants for this category was probably less theory driven (often not providing background or a hypothesis of direction), and more data driven (combining countries in order to attain sufficiently large groups). For example, manuscripts from the US versus all other countries was tested and there were very few manuscripts from other countries. This made it difficult to find a term that clearly describes direction. We therefore used “unfavorable” without further specification https://doi.org/10.1371/journal.pone.0188247.t003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 10 / 15 Empirical result as described in Table 2, first row PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 10 / 15 Determinants of selective reporting: A taxonomy Fig 2. Saturation graph. The horizontal axis displays the articles (records) in chronological order of analysis. The vertical axis displays the percentage of the 12 (plus 1 unspecified) determinant categories containing at least one determinant. The labels describe which determinant category appeared for the first time in which record. Fig 2. Saturation graph. The horizontal axis displays the articles (records) in chronological order of analysis. The vertical axis displays the percentage of the 12 (plus 1 unspecified) determinant categories containing at least one determinant. The labels describe which determinant category appeared for the first time in which record. https://doi.org/10.1371/journal.pone.0188247.g002 a wider scope of literature. For example, potential harm through bioterrorism was identified through veterinary medicine literature [39]. Also, Web of Science identified less than half of the articles in our sample. We included expert views and aggregate understanding, and deter- minants that may not have been studied well, yet in a different manner: through analyzing edi- torials, comments, and the full articles including introduction and discussion sections. We used explicit and transparent inductive qualitative research methods to cover the broad range of putative determinants in the literature. In contrast, Bassler et al. [22] focused on actors and motivations, which complements our work to help understand the multi-causality and multi- ple system pressures on and rewards for individuals and teams. Limitations and strengths The combined quantitative and qualitative approaches including two different ways –prospec- tively and retrospectively– to check saturation increased the likelihood of having captured all relevant categories of determinants and served as an internal validation of our approach. How- ever, the data did not suffice to discern patterns of determinants by academic field or strength 11 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 Determinants of selective reporting: A taxonomy of evidence. Our work does not cover all possible single determinants. We do believe that the framework with categories likely captures the categories that fit with the large majority of determinants. For example, we did not find lack of a statistician on the team being a putative determinant, but depending on the hypothesis of how it may relate to selective reporting, it may fit, for example, with poor or flexible research design or focus on preferred findings, or lack of resources including time. Further, in our work, discussion to reach consensus was essential because abstracting possible determinants without a prior framework was not a straightfor- ward endeavor, as evidenced by modest initial agreement (72% and 78%) about inclusion of articles in reviewing abstracts and full-text. Not publishing research outcomes is unethical. Our findings, however, raise questions about possible rare but legitimate reasons to report selectively or to not publish research. Obvi- ously, potential harm can be considered a legitimate reason, when publication may involve misuse by e.g., terrorists, or involves breaking confidentiality restrictions. Fatally flawed research probably should also not be published. However, poor design is preferably prevented in the first place, the academic reviewing system is in place to improve quality and to prevent fatally flawed work to be published or to be included in reviews and meta-analyses [40]. Future guidance may clarify what should be published in such cases. Future studies New research, using various methods, should verify the categories we created. The categories and their interrelations may clarify causal pathways and inform theory. (Complex) interven- tions should probably and most fruitfully address several determinant categories. So far, most empirical work has been performed on poor or flexible research design, but not all findings refer to clear causes and therefore cannot be used to formulate interventions (such as studies examining the association of sample size with selective reporting of positive findings). Future research should also employ qualitative methods to address researchers’ daily decision making and balancing of interests to better understand causal mechanisms and the multiple factors involved. The taxonomy may also help plan studies on risk profiling (e.g., research domains in which flexible designs are commonly used, or where a particular mission prevails) which in turn may inform efficient policy development on responsible conduct of research. We suggest our work to promote a constructive debate on causes of reporting bias. It is hoped that it will contribute to decrease the mostly deleterious phenomenon of selective reporting in modern science. S1 PRISMA Checklist. (DOC) S1 File. Study protocol dx.doi.org/10.17504/protocols.io.jz3cp8n. (DOCX) S1 File. Study protocol dx.doi.org/10.17504/protocols.io.jz3cp8n. (DOCX) S2 File. Dataset with determinants. S2 File. Dataset with determinants. S2 File. Dataset with determinants. (XLS) Author Contributions Conceptualization: Jenny T. van der Steen, Lex M. Bouter. 12 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0188247 February 5, 2018 Determinants of selective reporting: A taxonomy Formal analysis: Jenny T. van der Steen, Cornelis A. van den Bogert, Gerben ter Riet. Funding acquisition: Jenny T. van der Steen. Investigation: Jenny T. van der Steen, Cornelis A. van den Bogert, Mirjam C. van Soest- Poortvliet, Soulmaz Fazeli Farsani. Methodology: Jenny T. van der Steen, Cornelis A. van den Bogert, Rene´ H. J. Otten, Gerben ter Riet, Lex M. Bouter. Project administration: Cornelis A. van den Bogert, Mirjam C. van Soest-Poortvliet, Rene´ H. J. Otten. Supervision: Gerben ter Riet, Lex M. Bouter. Supervision: Gerben ter Riet, Lex M. Bouter. Validation: Jenny T. van der Steen, Cornelis A. van den Bogert, Gerben ter Riet. Visualization: Jenny T. van der Steen, Cornelis A. van den Bogert. Writing – original draft: Jenny T. van der Steen. 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https://openalex.org/W1835808004	https://europepmc.org/articles/pmc4570799?pdf=render	English	null	Quantification of the Iodine Content of Perigastric Adipose Tissue by Dual-Energy CT: A Novel Method for Preoperative Diagnosis of T4-Stage Gastric Cancer	PloS one	2,015	cc-by	7,106	RESEARCH ARTICLE Quantification of the Iodine Content of Perigastric Adipose Tissue by Dual-Energy CT: A Novel Method for Preoperative Diagnosis of T4-Stage Gastric Cancer Li Yang1☯, Gaofeng Shi1, Tao Zhou2☯, Yang Li1, Yong Li2 1 Department of CT, The fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei Province, China, 2 Department of surgery, The fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei Province, China ☯These authors contributed equally to this work. yangli8008@hotmail.com Editor: Qing-Yi Wei, Duke Cancer Institute, UNITED STATES Editor: Qing-Yi Wei, Duke Cancer Institute, UNITED STATES Received: February 9, 2015 Accepted: August 10, 2015 Published: September 15, 2015 Published: September 15, 2015 Copyright: © 2015 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Abstract This study investigated the utility of quantifying iodine concentration (IC) in perigastric adi- pose tissue, using dual-energy computed tomography (DECT), for the detection of T4a- stage gastric cancer. Fifty-four patients with gastric cancer were enrolled at the Fourth Hos- pital of Hebei Medical University between January and June 2013. Patients were imaged preoperatively with conventional computed tomography (CT) scans and DECT, and the IC in perigastric fat adjacent to the tumor calculated from arterial phase (AP) and portal venous phase (PVP) images. The patients subsequently received surgical treatment (gastrectomy), and histologic analysis of resected specimens was used as a ‘gold standard’ reference for cancer staging. Receiver operating characteristic (ROC) curve analysis was employed to assess the utility of DECT for identifying T4a-stage gastric cancer, with optimal IC thresh- olds determined from the area under the ROC curve (AUC). Postoperative histology revealed that 32 patients had serosal invasion (group A), and 22 did not (group B). The accuracy of conventional CT for distinguishing stage T4 from non-T4 stages was 68.5% (37/54). IC was significantly higher in group A than in group B (AP: 0.60±0.34 vs. 0.09±0.19 mg/mL, p<0.001; PVP: 0.83±0.41 vs. 0.27±0.21 mg/mL, p<0.001). The sensitivity, specific- ity and AUC for detecting serosal invasion were 77.1%, 79.2% and 0.89 at an IC threshold of 0.25 mg/mL for AP images; and 80.0%, 79.2% and 0.90 at an IC threshold of 0.45 mg/mL for PVP images. These results indicated that Iodine quantification in perigastric fat using DECT is an accurate method for detecting serosal invasion by gastric cancer. OPEN ACCESS Citation: Yang L, Shi G, Zhou T, Li Y, Li Y (2015) Quantification of the Iodine Content of Perigastric Adipose Tissue by Dual-Energy CT: A Novel Method for Preoperative Diagnosis of T4-Stage Gastric Cancer. PLoS ONE 10(9): e0136871. doi:10.1371/ journal.pone.0136871 Introduction Gastric cancer is one of the most frequently diagnosed cancers, and is a leading cause of cancer-related deaths worldwide [1–3]. The preoperative staging of gastric cancer is widely rec- ognized as an invaluable aid for determining the optimal therapy and evaluating tumor resect- ability and patient prognosis [4–6]. The TNM system is commonly used to stage gastric cancer, Funding: These authors have no support or funding to report. Funding: These authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 1 / 12 Dual-Energy CT: Iodine Concentration in Gastric Cancer with T4 defined as a tumor that invades the serosa [3]. Accurately differentiating T4a-stage gastric cancer from T3 or earlier stages is particularly important with regard to preoperative selection of appropriate treatment strategies, including the requirement for multi-organ sur- gery [7–9]. Neoadjuvant chemotherapy is strongly recommended for patients with T4 staging and lymph node metastasis, and may be beneficial to those with T4a stage in down grading the tumor prior to resection allowing in some cases curative resection [10]. Multi-detector com- puted tomography (MDCT) is often chosen as the modality for preoperative staging, and has been shown to have an overall accuracy that approaches 90% [5,7,9]. Nonetheless, preoperative staging with MDCT can be difficult because the serosal surface is very rough and the adjacent adipose tissues are generally turbid, so increased density could reflect several different phenom- enon including tumor invasion and reactive fibrous connective tissue hyperplasia; therefore, the specificity of MDCT is relatively low. As MDCT does not show complete agreement with postoperative staging by histologic analysis of surgically resected specimens new approaches are needed to improve the sensitivity, specificity and accuracy of imaging modalities for the preoperative staging of gastric cancer. It has been demonstrated that dual-energy CT, including dual-source dual-energy CT (DECT) is capable of quantifying the iodine concentration (IC) in tissues in vivo. [4] Therefore, DECT could potentially be used to measure the iodine content of tumor-invaded perigastric adipose tissue of the lesser and greater omentum, and this may represent a novel approach to more accurately detect T4a-stage gastric cancer. We hypothesized that quantification of the iodine concentration in perigastric adipose tissue using DECT could help to distinguish T4a- stage gastric cancer from earlier stage tumors. Introduction Therefore, the aim of the present study was to investigate the relationship between the iodine concentration in perigastric fat, measured using DECT, and T4a-stage gastric cancer, and to determine the sensitivity, specificity and accuracy of DECT for identifying T4a-stage gastric cancer, using histologic assessment of surgically resected specimens as the ‘gold standard’ reference for tumor staging. PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 Image acquisition All CT images were acquired with a dual-source dual-energy CT scanner (SOMATOM Defini- tion Flash; Siemens Healthcare, Germany). Each patient was instructed to fast for at least 6 hours before the CT examination. Ten minutes before scanning, each patient was administered 10 mg anisodamine intramuscularly (to reduce the tension of the gastrointestinal tract), and drank 800–1000 mL water (to fully expand the stomach). The pre-contrast images were acquired with a tube voltage of 120 kVp, a tube current of 190 mAs, a collimation of 32 × 1.2 mm, and a pitch of 0.9. Arterial and portal venous phase images were acquired 25 and 70 sec- onds after the start of the injection of contrast medium. A fixed scan delay was used for the arterial phase. The dual-energy mode was used for both arterial and portal venous phase imag- ing, with tube voltages of 100 kVp and 140 kVp with a tin filter, tube currents of 230 and 178 mAs, a collimation of 32 × 0.6 mm for both tubes, a pitch of 0.55, and a gantry rotation time of 0.5s. Non-ionic contrast medium (Iohexol, 300 mg/dL; GE Healthcare, USA) was injected intravenously at a flow rate of 3 mL/s. The amount of contrast medium injected was calculated according to the patient weight (2 mL/kg). Patients This was a cross-sectional diagnostic study that enrolled consecutive patients with gastric can- cer confirmed by endoscopic biopsy, who were referred between January 2013 and June 2013 to the Department of CT, The Fourth Hospital of Hebei Medical University, Shijiazhuang, China, for preoperative CT scanning to stage the disease and assist with treatment planning. Patients were excluded from the study if: surgical resection of the gastric tumor (gastrectomy) was carried out more than 1 week after the CT scan; the patient was allergic to contrast medium; or the patient had T4b stage cancer that was easily diagnosed by CT to have invaded other organs. All the included patients underwent a three-phase CT scan: pre-contrast single- energy CT imaging, and contrast-enhanced DECT imaging at arterial and venous phases. Some patients had too thin fat layers for CT imaging and were considered to have technique failure. Histologic examination of resected specimens was performed in a blinded manner after surgery, and served as the ‘gold standard’ reference for tumor staging. Two senior radiologists, who did not know the endoscopic findings or pathological results, were assigned to reconstruct the merge images during the venous phase (thickness of 1.5 mm, using the B30 algorithm), analyze their axial views and multi-planar reformation (MPR) images and discuss the tumor stages. Criteria for tumor staging were based on the TNM staging system for gastric carcinoma (7th edition) by the American Joint Committee on Cancer [3]. According to the postoperative pathology results, the included patients were assigned to one of two groups: group A, serosal invasion (stage T4a); or group B, intact serosa (stage T1–T3). PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 2 / 12 Dual-Energy CT: Iodine Concentration in Gastric Cancer The study was approved by Institutional Ethics Committee of The Fourth Hospital of Hebei Medical University, and written informed consent was obtained from each patient prior to inclusion. The study was approved by Institutional Ethics Committee of The Fourth Hospital of Hebei Medical University, and written informed consent was obtained from each patient prior to inclusion. PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 Image evaluation To determine the tumor T stage using conventional CT signs, and to compare the sensitivity and specificity between un-enhanced CT and dual-energy CT two experienced abdominal radi- ologists evaluated the three-phase images by consensus in a joint session. The definition of stages T1–T4 followed the 7th edition of the staging manual published by the American Joint Committee on Cancer in 2010 [11]. The pre-contrast, arterial and venous phase images were reconstructed with a 1.5-mm slice thickness and a B30 kernel. The arterial and venous phase images were obtained by mixing high- and low-energy images in a 1:1 ratio, which was the default mixing ratio. These mixed images were considered as simulated single energy 120 kVp images. Image reading was per- formed on a commercial workstation (MMWP; Siemens Healthcare, Germany) using trans- verse, MPR or maximum intensity projection views. Both readers were blinded to the results of the DECT iodine measurements and histologic investigations. To prepare the image for iodine quantification, high- and low-energy arterial and venous phase images were reconstructed with a 5-mm slice thickness and a D30 kernel. The iodine concentration was determined by one radiologist using a commercial dual-energy software package (Liver VNC; Siemens Healthcare, Germany). The iodine concentration was measured by selecting a region of interest (ROI) in the perigastric fat adjacent to the tumor (Fig 1). A strip ROI of 25–50 mm2 and a width 5 mm (considering the range of cancer invasions, we limited the width of the ROI) of the cancer tissue was selected close to and along the gastric wall (we kept a 1 mm gap between the cancer tissue and gastric wall, so the latter was not involved) to measure the adipose iodine concentration in the involved gastric serosa. To obtain a control value for the iodine concentration in fat, an additional ROI was placed in an area dis- tant to the tumor, for example at the greater curvature (Fig 1C and 1D). The ROI was 25–50 mm2 circular and positioned so that it was over a homogenous area, and did not overlap with regions containing tumor or other tissues such as blood vessels. Each measurement was repeated 3 times, and the average iodine concentration recorded for further analysis. Image evaluation In each patient, the iodine concentration was measured from both arterial and venous phase images, PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 3 / 12 Dual-Energy CT: Iodine Concentration in Gastric Cancer Fig 1. Representative CT images obtained from a 68 year-old female patient. (A) The mixed venous phase image shows thickening of the wall of the lesser curvature with transmural enhancement (arrow). Based on the mixed image, the tumor was classified as stage cT3. (B) The histological image, stained with hematoxylin and eosin (HE, ×100), revealed a grade II adenocarcinoma with invasion of surrounding soft tissue. The pathologic stage was pT4a. (C) The iodine map image at the arterial phase shows that the iodine concentration was 0.5 mg/mL in the fat near the tumor (ROI 1), but 0 mg/mL in the fat in a normal region distant from the tumor (ROI 2). (D) The iodine map image at the venous phase shows that the iodine concentration was 0.5 mg/mL in the fat near the tumor (ROI 1), but 0 mg/mL in the fat in a normal region distant from the tumor (ROI 2). This indicates that the serosa was invaded by the tumor. Fig 1. Representative CT images obtained from a 68 year-old female patient. (A) The mixed venous phase image shows thickening of the wall of the lesser curvature with transmural enhancement (arrow). Based on the mixed image, the tumor was classified as stage cT3. (B) The histological image, stained with hematoxylin and eosin (HE, ×100), revealed a grade II adenocarcinoma with invasion of surrounding soft tissue. The pathologic stage was pT4a. (C) The iodine map image at the arterial phase shows that the iodine concentration was 0.5 mg/mL in the fat near the tumor (ROI 1), but 0 mg/mL in the fat in a normal region distant from the tumor (ROI 2). (D) The iodine map image at the venous phase shows that the iodine concentration was 0.5 mg/mL in the fat near the tumor (ROI 1), but 0 mg/mL in the fat in a normal region distant from the tumor (ROI 2). This indicates that the serosa was invaded by the tumor. doi:10.1371/journal.pone.0136871.g001 doi:10.1371/journal.pone.0136871.g001 using a ROI of the same size, shape and positioned at the same anatomic location. Only the means were used for statistical analyses. Image evaluation Since gastric peristalses lasted throughout the whole process and the ROIs were selected manually, we could not guarantee that the ROI selections during the arterial and venous phases were exactly the same. Therefore, we tried our best to ensure a similar choice in shape, size and site. Radiation dose The volume CT dose index (CTDIvol) and dose length product (DLP) were recorded from the CT console for the pre-contrast, arterial and venous phase scans. The effective dose was calcu- lated by multiplying the DLP by a conversion coefficient for the abdomen (k = 0.015 mSvmGy-1cm-1). Histologic examination of resected tumor All specimens obtained by surgery were embedded in paraffin, stained with hematoxylin and eosin (HE) using standard techniques, and then sectioned into slices 4 μm thick. Light micros- copy was used to determine the pathologic type, histologic grade and invasion depth of the tumor, and the presence/absence of lymph node metastasis. Statistical analysis Statistical analysis was performed using SPSS version 11.5 (SPSS Inc., USA). Comparisons of the mean iodine concentration between groups A and B were made using the Wilcoxon signed rank test (the data were not normally distributed). A p-value < 0.05 was considered to be 4 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 Dual-Energy CT: Iodine Concentration in Gastric Cancer statistically significant. Receiver operating characteristic (ROC) curve analysis was used to determine the utility of perigastric fat iodine concentration for diagnosing T4-stage gastric can- cer. The area under the ROC curve (AUC) was used to determine the optimal threshold iodine concentration for tumor classification. Sensitivity was calculated as the true positive rate (num- ber of true positives divided by the sum of the number of true positives and number of false negatives); specificity as the true negative rate (number of true negatives divided by the sum of the number of true negatives and number of false positives); and accuracy as the sum of the number of true positives and true negatives, divided by the total number of positives and negatives. Patient demographic and clinical characteristics Of the 80 patients initially screened for inclusion in the study, 21 were excluded because sur- gery was not performed within one week of imaging and pathology results were not available as a reference. Of these, 5 were unresectable and received chemotherapy, 2 refused any therapy and 14 accepted neoadjuvant chemotherapy. Neoadjuvant chemotherapy is recommended for stage T4 tumors in our hospital so those patients who were included with confirmed T4 stage tumors had opted according to their own judgement to receive early surgery. 5 patients with fat layers that were too thin for measurement by CT, considered to have technique failure, were included in the calculation for sensitivity and specificity but not in the ROC curve analysis. The patients included 1 with T1, 1 with T3 and 3 with T4 stage tumors. Hence, a total of 54 patients (41 males, 13 females; mean age, 61.6 ± 10.5 years; age range, 31–78 years) were included in all of the analysis (Table 1) and 59 were included in the sensitivity and specificity analysis. There Table 1. Clinical and demographic characteristics of the 54 patients. Characteristic Total Group A Group B p value (Group A Vs. Group B) Gender (no. of patients) Male 41 23 18 0.401 Female 13 9 4 Age (years) Mean ± standard deviation 61.6 ± 10.5 62.5±11.2 60.3±10.1 0.071 Anatomic location of gastric carcinoma (no. of patients) Antrum 17 10 7 0.993 Corpus 16 9 7 Cardia-fundus 11 6 5 Cardia 10 6 4 Surgical treatment (no. of patients) Radical subtotal gastrectomy 39 19 20 0.011 Radical total gastrectomy 15 13 2 Pathologic type (no. of patients) Adenocarcinoma 46 24 22 0.010 well differentiated 3 0 3 moderately differentiated 26 11 15 poorly differentiated 17 13 4 Signet ring cell carcinoma 4 4 0 Mucinous adenocarcinoma 3 3 0 Adenocarcinoma combined with mucinous adenocarcinoma 1 1 0 doi:10.1371/journal.pone.0136871.t001 Table 1. Clinical and demographic characteristics of the 54 patients. PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 5 / 12 Dual-Energy CT: Iodine Concentration in Gastric Cancer were no significant differences between groups A and B in terms of age, gender and location of the gastric carcinoma, but there were significant differences in the surgical treatment (p = 0.011) and the pathologic type of carcinoma (p = 0.010). The gastric carcinoma was located in the gastric cardia in 10 patients, cardia-fundus in 11, corpus in 16 and antrum in 17. Tumor staging based on pathology of resected specimens TNM staging, based on post-surgical pathology results, was as follows: 8 patients were classi- fied as T1, 7 as T2, 7 as T3 and 32 as T4; 24, as N0, 16 as N1, 6 as N2 and 8 as N3; no patients had distant metastasis. Seven patients were classified as stage IA, 7 as IB, 6 as IIA, 8 as IIB, 14 as IIIA, 4 as IIIB 4, and 8 as IIIC. According to the T-stages, 32 patients were assigned to Group A, and 22 to group B. Patient demographic and clinical characteristics All patients were treated surgically by radical total gastrectomy (15 patients) or radical subtotal gastrectomy (39 patients). All patients received D2 lymph node dissection. The pathologic types of gastric cancer identified in these patients included adenocarcinoma in 46 patients (G1, well differentiated in 3; G2, moderately differentiated in 26; and G3, poorly differentiated in 17), mucinous adenocarcinoma in 3 patients, signet ring cell carcinoma (SRCC) in 4 patients and adenocarcinoma combined with mucinous adenocarcinoma in 1 patient. doi:10.1371/journal.pone.0136871.t002 Tumor staging based on conventional CT imaging Based on images acquired using conventional CT scanning, 3 patients were classified as having cancer of stage T1, 10 as T2, 18 as T3 and 23 as T4 (Table 2, Figs 1 and 2, S1 and S2 Figs). Com- pared with histologic staging, 22 patients were incorrectly classified into other T stages by con- ventional CT, including 4 cases of pathologic T3 misclassified by CT as T4, and 13 cases of pathologic T4 misclassified by CT as T3 or T2. Using the histologic results as the reference, the T stage was correctly identified in 57.6% (34/59) by evaluation of conventional CT images. Fur- thermore, the accuracy of conventional CT for distinguishing stage T4 from non-T4 stages was 67.8% (40/59). pathologic T4 misclassified by CT as T3 or T2. Using the histologic results as the reference, the T stage was correctly identified in 57.6% (34/59) by evaluation of conventional CT images. Fur- thermore, the accuracy of conventional CT for distinguishing stage T4 from non-T4 stages was 67.8% (40/59). Table 2. T-staging of the gastric cancers using preoperative conventional pre-contrast single-energy CT and dual-energy CT (DECT) compared to postoperative histology staging. CT stage DECT stage Total Non T4 T4 Non T4 T4 Arterial phase Histologic stage Non T4 18 4a 19 3a 22 T4 13b 19 5b 27 32 Total 31 23 24 30 Venous phase Histologic stage Non T4 18 4c 19 3c 22 T4 13d 19 4d 28 32 Total 31 23 23 31 The data represents patient numbers a In the arterial phase 3 patients with non T4 histologic stage was wrongly diagnosed as T4 by conventional CT staging that was corrected by DECT. sing preoperative conventional pre-contrast single-energy CT and dual-energy CT (DECT) compared to Table 2. T-staging of the gastric cancers using preoperative conventional pre-contrast single-energy CT an postoperative histology staging. Table 2. T-staging of the gastric cancers using preoperative conventional pre-contrast single-energy CT and dual-energy CT (DECT) compared to postoperative histology staging. b In the arterial phase 10 patients were correctly diagnosed by DECT after a wrong diagnosis by conventional CT. c In the venous phase 2 patients with non T4 histologic stage was wrongly diagnosed as T4 by conventional CT staging that was corrected by DECT. d In the venous phase there were 12 patients correctly diagnosed by DECT after a wrong diagnosis by conventional CT. e correctly diagnosed by DECT after a wrong diagnosis by conventional CT. c In the venous phase 2 patients with non T4 histologic stage was wrongly diagnosed as T4 by conventional CT staging that was corrected by DECT. d In the venous phase there were 12 patients correctly diagnosed by DECT after a wrong diagnosis by conventional CT. non T4 histologic stage was wrongly diagnosed as T4 by conventional CT staging that was corrected by DECT. patients correctly diagnosed by DECT after a wrong diagnosis by conventional CT. g g y b In the arterial phase 10 patients were correctly diagnosed by DECT after a wrong diagnosis by conventional CT. erial phase 10 patients were correctly diagnosed by DECT after a wrong diagnosis by conventional CT. Tumor staging based on conventional CT imaging PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 6 / 12 Dual-Energy CT: Iodine Concentration in Gastric Cancer Fig 2. Representative CT images obtained from a 46 year-old male patient. (A) The mixed venous phase image depicts thickening of the wall of the antrum with transmural enhancement. The density of the perigastric fat was elevated, with a stripe-like shadow. The clinical stage was determined to be cT4. (B) The histological image, stained with hematoxylin and eosin (HE, ×100), revealed a grade II adenocarcinoma with muscularis invasion. The pathologic stage was pT3. (C) The iodine map at the arterial phase shows that the iodine concentration was 0.1 mg/mL in the perigastric fat (ROI 1). (D) The iodine map at the venous phase shows that the iodine concentration was 0.1 mg/mL in the perigastric fat (ROI 1). doi:10.1371/journal.pone.0136871.g002 Fig 2. Representative CT images obtained from a 46 year-old male patient. (A) The mixed venous phase image depicts thickening of the wall of the antrum with transmural enhancement. The density of the perigastric fat was elevated, with a stripe-like shadow. The clinical stage was determined to be cT4. (B) The histological image, stained with hematoxylin and eosin (HE, ×100), revealed a grade II adenocarcinoma with muscularis invasion. The pathologic stage was pT3. (C) The iodine map at the arterial phase shows that the iodine concentration was 0.1 mg/mL in the perigastric fat (ROI 1). (D) The iodine map at the venous phase shows that the iodine concentration was 0.1 mg/mL in the perigastric fat (ROI 1). doi:10.1371/journal.pone.0136871.g002 doi:10.1371/journal.pone.0136871.g002 doi:10.1371/journal.pone.0136871.g002 Radiation dose The CTDIvol, DLP and effective dose were (respectively) 14.00 ± 0.41 mGy, 338.60 ± 80.14 mGy- cm and 5.08 ± 1.20 mSv for the pre-contrast phase; 12.66 ± 2.90 mGy, 292.87 ± 97.10 mGy-cm and 4.39 ± 1.46 mSv for the arterial phase; and 12.58 ± 2.70 mGy, 305.10 ± 161.60 mGy-cm and 4.58 ± 2.42 mSv for the venous phase. ROC curve analysis ROC curve analysis of using DECT-derived measurements of perigastric fat iodine concentra- tion to stage gastric cancer showed that the AUC was 0.89 for arterial phase images and 0.90 for portal venous phase images. For arterial phase images, the optimal threshold iodine concen- tration (in a ROI near the tumor) for distinguishing between group A and B was 0.25 mg/mL, and this yielded a sensitivity of 77.1%, a specificity of 79.2%, and an accuracy of 78.0%. For venous phase images, the optimal threshold value was 0.45 mg/mL, and its use resulted in a sensitivity of 80.0%, a specificity of 79.2%, and an accuracy of 79.7%. By not considering the 5 (8.5%) patients with technique failure, the sensitivity and specificity can be improved to 84.4%, 86.4% for arterial phase images, and 87.5%, 86.4% for venous phase images, as the cutoff value was actually calculated without these cases. Even with technique failure the method is still via- ble for over 90% of patients. Tumor staging based on DECT measurements of iodine concentration As shown in Table 3, the iodine concentration of the fat adjacent to the tumor was significantly higher in group A than in group B for both arterial phase images (0.60 ± 0.34 mg/mL [range, 0.00–1.30 mg/mL] vs. 0.09 ± 0.19 mg/mL [range, 0.00–0.80 mg/mL]; p < 0.001) and venous phase images (0.83 ± 0.41 mg/mL [range, 0.00–1.80 mg/mL] vs. 0.27 ± 0.21 mg/mL [range, 0.00–0.90 mg/mL]; p < 0.001). In contrast, there were no significant differences between groups A and B in the iodine concentration of fat at sites distant from the tumor for both the arterial phase (0.02 ± 0.07 vs. 0.02 ± 0.04) and venous phase (0.12 ± 0.20 vs. 0.04 ± 0.09). In group A, there was a significant difference in iodine concentration between fat adjacent to the tumor and that at distant sites, both for the arterial and venous phases (p < 0.001). In group B, there was also a significant difference in iodine concentration between fat adjacent to and that Table 3. Iodine concentrations in the perigastric adipose tissue of patients in groups A and B, measured using DECT. Group A (n = 32) Group B (n = 22) Near tumor Distant to tumor Near tumor Distant to tumor Arterial phase 0.60 ± 0.34 0.02 ± 0.07§ 0.09 ± 0.19* 0.02 ± 0.04 Venous phase 0.83 ± 0.41 0.12 ± 0.20§ 0.27 ± 0.21* 0.04 ± 0.09§ Data are presented as the mean ± SD (mg/mL). Near tumor: iodine concentration measured in a ROI near the tumor; Distant to tumor: iodine concentration measured in a ROI distant to the tumor. *p < 0.001 compared with the corresponding value in group A §p < 0.001 compared with the ‘Near tumor’ value for the same phase within the same group. doi:10.1371/journal.pone.0136871.t003 dine concentrations in the perigastric adipose tissue of patients in groups A and B, measured using DECT. erigastric adipose tissue of patients in groups A and B, measured using DECT. Table 3. Iodine concentrations in the perigastric adipose tissue of patients in groups A and B, measu PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 7 / 12 Dual-Energy CT: Iodine Concentration in Gastric Cancer distant from the tumor for the venous phase (p < 0.001), but not for the arterial phase (p = 0.12). PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 Discussion The present study was designed to investigate the feasibility of using the iodine concentration in perigastric adipose tissue adjacent to the tumor, measured with DECT, to detect serosal invasion of gastric cancer. The main findings of the study were that the iodine concentration in perigastric adipose tissue adjacent to the tumor was significantly higher in the presence of sero- sal invasion than when the serosa was intact. Using post-surgery histologic findings as a ‘gold standard’ for staging, ROC curve analysis revealed that the AUC for detecting serosal invasion was 0.89 and 0.90 for arterial and portal venous phases, respectively. When 0.25 and 0.45 mg/ mL were taken as threshold iodine concentration value for the arterial and portal venous phases, respectively, the accuracy of DECT for differentiating between T4a-stage and earlier T- stage gastric cancer was 78.0% and 79.7%, respectively. Taken together, these observations demonstrate that quantification of iodine in perigastric adipose tissue by DECT represents a novel and accurate clinical method for distinguishing T4a-stage gastric cancer from earlier T- stages. To the best of our knowledge, this is the first report demonstrating the utility of this approach in the staging of gastric cancer. DECT iodine measurements provide a quantitative imaging method for detecting advanced local gastric cancer. DECT has been used to characterize various tumors, such as lung cancer nodules [12], insulinoma [13] and adrenal nodules [14]. The present study extended the scope of DECT to advanced gastric cancer. It was found that invasion of the serosa by gastric cancer significantly elevated the iodine concentration in the perigastric adipose tissue adjacent to the tumor. In contrast, adipose tissues without tumor invasion and a normal blood supply showed undetectable or low iodine levels in the arterial and portal venous phases. The high iodine con- centration observed in the perigastric adipose tissue of patients with T4a-stage cancer is likely associated with increased perfusion, possibly caused by tumor invasion or leakage from malig- nant cell membranes as a result of a breakdown in serosal integrity. 8 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 Dual-Energy CT: Iodine Concentration in Gastric Cancer As evidenced by the findings in the present study, DECT provides additional value to con- ventional single-energy CT in the diagnosis of T4a-stage gastric cancer. Discussion According to the national comprehensive cancer network (NCCN) clinical practice guidelines for gastric carci- noma 2010 (Chinese version), preoperative chemotherapy or chemoradiotherapy can be con- sidered in resectable advanced tumors or those with node metastasis. As it is difficult to make a confirmed diagnosis in some patients with conventional CT techniques, iodine concentration imaging may assist with the decision on whether to undertake preoperative adjuvant therapy. When equipped with sub-millimeter thin slicing, single-energy CT can readily depict the serosa adjacent to the epigastric fat [5,14], and the accuracy of diagnosing T4 gastric cancer is signifi- cantly improved with the assistance of the MPR technique [5,7,15–17] or virtual gastroscopy [18]. As the gastric serosa is very thin, we could not observe serosal invasion directly. Conven- tional MDCT determines whether the gastric serosa is invaded mainly by estimating the den- sity of adipose tissues at the serosal surface by direct observation; however, the serosal surface is very rough and the adjacent adipose tissues are generally turbid, thus increased density could reflect several different phenomenon including tumor invasion and reactive fibrous connective tissue hyperplasia. Therefore, the specificity of determining serosal invasion by MDCT is rela- tively low. In addition, for T4a lesions with perigastric microinvasion, even increasing the win- dow width and window level in conventional MDCT could not clearly determine serosal invasion. In the present study, all the patients were treated surgically, and preoperative staging of the lesions was mainly non-T4; however, according to the postoperative pathological results, 12 patients had T4a lesions but were underestimated as T3, and 1 patient had a T4a lesion but was underestimated as T2. These findings demonstrated the limitations of using MDCT in evaluating serosal invasion. The results of the present study demonstrate that DECT iodine quantification represents an accurate method with which to identify T4a-stage gastric cancer, which is comparable in accuracy, sensitivity and specificity to previous studies using MPR images [5,7,8,15]. However, identification of all T4a patients by DECT alone was still not possi- ble. We tested higher sensitivity values of 95% and 100% for the arterial and venous phase, but the resulting specificities were too low to suggest a viable clinical use for the DECT technique in isolation. Therefore, this technique may be useful in addition to other methods or further research may identify methods by which the sensitivity and specificity of the DECT technique can be improved. PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 Discussion The iodine maps provided by DECT are color maps, which are better for the color distinguishing ability of humans compared with grayscale images provided by conven- tional MDCT. In addition, iodine concentration provided by DECT is quantitative data, which could provide better objective evidence for the diagnosis. Abnormal tumor angiogenesis and local microcirculation (compared with normal tissues) exist at the regions with cancerous cell invasion at early stages. DECT could evaluate the microcirculation of the region of interest by measuring the iodine concentration at the serosal adipose tissues, and thus help determine serosal invasion. Therefore, the findings of the present study demonstrated that using DECT to measure iodine concentration could provide more objective and accurate evidence for deter- mining serosal invasion. In addition to enabling iodine quantification, DECT can improve the visualization of invading tumor, adjacent structures and neighboring blood vessels through the use of monochromatic MPR images [13,19], such as those shown in Figs 1C and 2C, without a radiation dose penalty [20]. Therefore, an advantage of DECT is that it allows the evaluation of linear or reticular fat stranding signs as well as iodine concentration, which may be useful in cases of inflammatory reactions. The accuracy of DECT in this study was slightly lower than those studies that used MDCT, which were estimated to be nearly 90% [5 7 9] However there are many points to consider The accuracy of DECT in this study was slightly lower than those studies that used MDCT, which were estimated to be nearly 90% [5,7,9]. However, there are many points to consider when comparing the two methods. The first is the slight alteration in the staging criteria. The 7th TNM staging criteria used here [3], in 2010 increased the requirement of the display of PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 9 / 12 Dual-Energy CT: Iodine Concentration in Gastric Cancer different layers of the gastric wall, and thus increased the difficulty of accurate staging by pre- operative CT scanning. For instance, tumors with muscular and subserosal invasions were clas- sified as stage T2 in the 6th criteria, while in the 7th criteria, tumors with muscular invasion were classified as stage T2, while tumors with subserosal invasion were classified as stage T3; tumors with serosal invasion were classified as stage T3 or T4 in the 6th criteria but as stage T4 in the 7th criteria. Discussion The use of different staging criteria could decrease the comparability of the studies. In addition, the different data of these studies could be associated with the differences in the scanning equipment, examination method (e.g. gastrointestinal preparation before the examination), clinicians’ experience, and different patient subgroups included. In the present study, the density of serosal adipose tissues was evaluated by the naked eye (similar to MDCT), and the accuracy of determining serosal invasion was 68.5%; while for preoperative examina- tions, iodine concentration was measured by DECT to evaluate serosal invasion, and the accu- racy was 78.0% (arterial phase) and 79.7% (venous phase), respectively, compared with the gold standard (pathological examinations). The present study has some limitations. First, only patients with confirmed gastric cancer were enrolled in our study, which may overestimate the capability of DECT for staging gastric cancer. Second, the number of patients included was relatively small, so it should be considered to be a pilot study that merits larger-scale studies to confirm the results. Third, direct compari- sons with other imaging modalities were not made. Fourth, the utility of iodine quantification with DECT was examined only for the diagnosis of T4-stage gastric cancer; the ability of DECT to diagnose earlier stages was not examined and there may be some other factors that will affect the iodine concentration measurement such as inflammation, metastatic lymph nodes, or peri- gastric tumor deposits close to the tumor. So the value of this method will have to be evaluated in further studies involving all of these factors. Finally, some patients were excluded from the study due to insufficient perigastric fat for measurement of iodine concentration using DECT– thus, this methodology may not be appropriate for all patients, and this may have introduced some bias into the study as these patients were not included in the sensitivity and specificity calculations. Conclusions Quantification of iodine content in perigastric adipose tissue with DECT provides an accurate, sensitive and specific method for distinguishing gastric cancer with serosal invasion from that without serosal invasion. Thus, DECT represents a useful clinical tool for preoperatively diag- nosing T4a-stage gastric cancer. References 1. Catalano V, Labianca R, Beretta GD, Gatta G, de Braud F, Van Cutsem E. Gastric cancer. Crit Rev Oncol Hematol. 2009; 71: 127–164. doi: 10.1016/j.critrevonc.2009.01.004 PMID: 19230702 2. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011; 61: 69–90. doi: 10.3322/caac.20107 PMID: 21296855 3. Washington K. 7th edition of the AJCC cancer staging manual: stomach. Ann Surg Oncol. 2010; 17: 3077–3079. doi: 10.1245/s10434-010-1362-z PMID: 20882416 4. Allum WH, Blazeby JM, Griffin SM, Cunningham D, Jankowski JA, Wong R. Guidelines for the manage- ment of oesophageal and gastric cancer. 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(XLSX) S1 Fig. Representative CT images obtained from a 63 year-old male patient. A: Venous phase: cardia wall thickening, with a nontransmural enhanced wall (arrow), preoperative imag- ing staging: T1. B: Postoperative pathological images, (HE, X40), showed a low differentiated adenocarcinoma that had infiltrated the mucous layer. Postoperative pathologic staging: pT1. C: arterial phase IC = 0.0 mg/ml. D: venous phase IC = 0.0 mg/ml, indicated no serous inva- sion. (PDF) S2 Fig. Representative CT images obtained from a 50 year-old female patient. A: Venous phase: cardia wall thickening, with a nontransmural enhanced wall (arrow), preoperative 10 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 Dual-Energy CT: Iodine Concentration in Gastric Cancer imaging staging: T2. B: Postoperative pathological images, (HE, X200), showed a low differen- tiated adenocarcinoma that had infiltrated the muscle layer. Postoperative pathologic staging: pT2. C: arterial phase IC = 0.0 mg/ml. D: venous phase IC = 0.0 mg/ml, indicated no serous invasion. (PDF) Acknowledgments Thanks for Dr. Runze Wu's selfless help of the study design and paper modification. Author Contributions Conceived and designed the experiments: GFS. Performed the experiments: LY GFS TZ Yang Li Yong Li. Analyzed the data: LY GFS TZ Yang Li Yong Li. Contributed reagents/materials/ analysis tools: GFS. Wrote the paper: LY TZ. Provided the critical revision: GFS LY. All authors read and approved the final manuscript. PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 References Functional imaging of lung cancer using dual energy CT: how does iodine related attenuation correlate with standardized uptake value of 18FDG-PET-CT? Eur Radiol. 2012; 22: 93–103. doi: 10.1007/s00330-011-2230-3 PMID: 21822784 13. Lin XZ, Wu ZY, Tao R, Guo Y, Li JY, Zhang J, et al. Dual energy spectral CT imaging of insulinoma- Value in preoperative diagnosis compared with conventional multi-detector CT. Eur J Radiol. 2012; 81: 2487–2494. doi: 10.1016/j.ejrad.2011.10.028 PMID: 22153746 11 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0136871 September 15, 2015 Dual-Energy CT: Iodine Concentration in Gastric Cancer 14. Ho LM, Marin D, Neville AM, Barnhart HX, Gupta RT, Paulson EK, et al. Characterization of adrenal nodules with dual-energy CT: can virtual unenhanced attenuation values replace true unenhanced attenuation values? AJR Am J Roentgenol. 2012; 198: 840–845. doi: 10.2214/AJR.11.7316 PMID: 22451549 14. Ho LM, Marin D, Neville AM, Barnhart HX, Gupta RT, Paulson EK, et al. Characterization of adrenal nodules with dual-energy CT: can virtual unenhanced attenuation values replace true unenhanced attenuation values? AJR Am J Roentgenol. 2012; 198: 840–845. doi: 10.2214/AJR.11.7316 PMID: 22451549 15. Kumano S, Murakami T, Kim T, Hori M, Iannaccone R, Nakata S, et al. T staging of gastric cancer: role of multi-detector row CT. Radiology. 2005; 237: 961–966. PMID: 16251394 16. Hur J, Park MS, Lee JH, Lim JS, Yu JS, Hong YJ, et al. Diagnostic accuracy of multidetector row com- puted tomography in T- and N staging of gastric cancer with histopathologic correlation. J Comput Assist Tomogr. 2006; 30: 372–377. PMID: 16778609 17. Kim AY, Kim HJ, Ha HK. Gastric cancer by multidetector row CT: preoperative staging. Abdom Imaging. 2005; 30: 465–472. PMID: 15785907 18. Kim JH, Eun HW, Hong SS, Kim YJ, Han JK, Choi BI. Gastric cancer detection using MDCT compared with 2D axial CT: diagnostic accuracy of three different reconstruction techniques. Abdom Imaging. 2012; 37: 541–548. doi: 10.1007/s00261-011-9823-9 PMID: 22080389 19. Lv P, Lin XZ, Chen K, Gao J. Spectral CT in patients with small HCC: investigation of image quality and diagnostic accuracy. Eur Radiol. 2012; 22: 2117–2124. doi: 10.1007/s00330-012-2485-3 PMID: 22618521 20. Bauer RW, Kramer S, Renker M, Schell B, Larson MC, Beeres M, et al. Dose and image quality at CT pulmonary angiography-comparison of first and second generation dual-energy CT and 64-slice CT. Eur Radiol. 2011; 21: 2139–2147. doi: 10.1007/s00330-011-2162-y PMID: 21614614 12 / 12
https://openalex.org/W3009709883	https://europepmc.org/articles/pmc7154865?pdf=render	English	null	High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1	Plants	2,020	cc-by	15,981	Received: 14 January 2020; Accepted: 24 February 2020; Published: 1 March 2020 Abstract: Plants employ a number of phosphorylation cascades in response to a wide range of environmental stimuli. Previous studies in Arabidopsis and yeast indicate that histidine kinase AHK1 is a positive regulator of drought and osmotic stress responses. Based on these studies AHK1 was proposed a plant osmosensor, although the molecular basis of plant osmosensing still remains unknown. To understand the molecular role and signaling mechanism of AHK1 in osmotic stress, we have expressed and puriﬁed full-length AHK1 from Arabidopsis in a bacterial host to allow for studies on the isolated transmembrane receptor. Puriﬁcation of the recombinant protein solubilized from the host membranes was achieved in a single step by metal-aﬃnity chromatography. Analysis of the puriﬁed AHK1 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting show a single band indicating that the preparation is highly pure and devoid of contaminants or degradation products. In addition, gel ﬁltration experiments indicate that the preparation is homogenous and monodisperse. Finally, CD-spectroscopy, phosphorylation activity, dimerization studies, and protein–protein interaction with plant phosphorylation targeting AHP2 demonstrate that the puriﬁed protein is functionally folded and acts as phospho-His or phospho-Asp phosphatase. Hence, the expression and puriﬁcation of recombinant AHK1 reported here provide a basis for further detailed functional and structural studies of the receptor, which might help to understand plant osmosensing and osmosignaling on the molecular level. Keywords: histidine kinase; osmosensing; membrane protein puriﬁcation; phosphorelay; protein-protein interaction plants plants High-Level Expression, Puriﬁcation and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1 Alexander Hofmann 1,† , Sophia Müller 1,†, Thomas Drechsler 2,† , Mareike Berleth 1, Katharina Caesar 2, Leander Rohr 2 , Klaus Harter 2 and Georg Groth 1,* Alexander Hofmann 1,† , Sophia Müller 1,†, Thomas Drechsler 2,† , Mareike Berleth 1, Katharina Caesar 2, Leander Rohr 2 , Klaus Harter 2 and Georg Groth 1,* 1 Institute of Biochemical Plant Physiology, Heinrich Heine University, Düsseldorf 40225, Germany; a.hofmann@hhu.de (A.H.); somue106@uni-duesseldorf.de (S.M.); mareike.berleth@hhu.de (M.B.) 1 Institute of Biochemical Plant Physiology, Heinrich Heine University, Düsseldorf 40225, Germany; a.hofmann@hhu.de (A.H.); somue106@uni-duesseldorf.de (S.M.); mareike.berleth@hhu.de (M.B.) 2 Center for Plant Molecular Biology (ZMBP), Eberhard Karls Universität Tübingen, Tübingen 72076, 1 Institute of Biochemical Plant Physiology, Heinrich Heine University, Düsseldorf 40225, Germany; a.hofmann@hhu.de (A.H.); somue106@uni-duesseldorf.de (S.M.); mareike.berleth@hhu.de (M.B.) 2 Center for Plant Molecular Biology (ZMBP), Eberhard Karls Universität Tübingen, Tübingen 72076, Germany; thomas.drechsler@zmbp.uni-tuebingen.de (T.D.); katharina.caesar@mwk.bwl.de (K.C.); leander.rohr@zmbp.uni-tuebingen.de (L.R.); klaus.harter@zmbp.uni-tuebingen.de (K.H.) * Correspondence: georg groth@hhu de; Tel : +49-211-811-2822 2 Center for Plant Molecular Biology (ZMBP), Eberhard Karls Universität Tübingen, Tübingen 72076, Germany; thomas.drechsler@zmbp.uni-tuebingen.de (T.D.); katharina.caesar@mwk.bwl.de (K.C.); leander.rohr@zmbp.uni-tuebingen.de (L.R.); klaus.harter@zmbp.uni-tuebingen.de (K.H.) * Correspondence: georg.groth@hhu.de; Tel.: +49-211-811-2822 † These authors contributed equally to this work. 1. Introduction Plants are sessile organisms that have to adapt to ﬂuctuating environmental conditions during their life cycle. To sense, respond, and adapt to a wide range of environments, stressors, and growth conditions, plants employ phosphorylation cascades that are already found in their basic genetic set-up in archaea, bacteria and fungi [1]. However, in contrast to the typical prokaryotic two-component phosphotransfer systems (TCS) involving a single His-Asp phosphotransfer process from a soluble or membrane-bound sensor histidine kinase to the receiver domain of a cognate response regulator protein, plants employ multi-step His-Asp phosphotransfer cascades (phosphorelays) Plants 2020, 9, 304; doi:10.3390/plants9030304 www.mdpi.com/journal/plants www.mdpi.com/journal/plants 2 of 17 Plants 2020, 9, 304 encompassing hybrid-type sensors that carry kinase and receiver domains within the same molecule, His phosphotransfer proteins, and response regulator proteins [2]. Altogether the genome of Arabidopsis thaliana contains 11 genes encoding histidine kinases (HK) [3,4]. Sequence analysis suggests that all members of the HK family have a modular basic structure consisting of an N-terminal transmembrane sensor domain followed by a catalytic transmitter domain and a receiver domain at the C-terminus. Biochemical, biophysical, and cellular studies show that the receptors form dimers and higher-molecular-weight oligomers at the membrane in their functional state [5–11]. Further studies demonstrate that ﬁve members of the Arabidopsis HK family encode receptors sensing the plant hormone ethylene (ETR1, ERS1, ETR2, ERS2, and EIN4) [12–14], whereas the other six encode non-ethylene receptor kinases (AHK1, AHK2, AHK3, CRE1/AHK4, CKI1, and CKI2/AHK5). Three of the non-ethylene receptors (AHK2, AHK3, and CRE1) have been identiﬁed as receptors for the plant hormone cytokinin [15–17]. The residual three HK receptors (AHK1, CKI1, and AHK5) show neither ethylene nor cytokinin-related activity and have been attributed to a variety of plant processes including osmoregulation [18], megagametogenesis [19], salt sensitivity [20], or stomatal responses [21], although the molecular trigger or ligand stimulating these activities is not known for all of these processes yet. While the overall architecture is conserved for all members of the Arabidopsis HK family, some of them lack histidine kinase activity due to a degenerate catalytic domain (ETR2, ERS2, and EIN4) or miss a receiver domain at their C-terminus (ERS1 and ERS2). Consequently, these isoforms cannot participate in a canonical phosphorelay, but may contribute to phosphotransfer to downstream proteins via the formation of heterodimers with other fully functional members of the Arabidopsis HK family. 1. Introduction Still, the most striking diﬀerence in the architecture of the Arabidopsis HK family is found in their sensor domain. For the ethylene receptor kinases this domain is transmembrane and contains a bound copper cofactor coordinated by three (ETR1 and ERS1) or four (ETR2, ERS2, and EIN4) transmembrane α-helices, whereas for the non-ethylene receptor kinases involved in cytokinin signaling the ligand binding domain (CHASE) is located in a loop connecting their two (AHK4), three (AHK3) or four (AHK2) transmembrane spans. Similarly, to the AHK4 cytokinin receptor prototype two of the non-plant hormone receptor HKs also contain two putative transmembrane helices (CKI1 and AHK1) in their N-terminal sensor domain, whereas sequence analysis of AHK5 indicates no obvious transmembrane element in this receptor kinase. Although AHK1 has been attributed to osmosensing [18], the molecular basis of plant osmosensing is still unknown. Biochemical and biophysical studies on puriﬁed receptors or their subdomains have substantially expanded our molecular understanding of ethylene and cytokinin sensing and signaling in the past [22–27]. Due to their low abundance in their natural host, recombinant production of the related proteins was a critical prerequisite for these studies. Hence, in order to expand our knowledge on the putative osmosensor AHK1, we have expressed this non-plant hormone transmembrane receptor HK in a bacterial system and puriﬁed the full-length recombinant protein from the bacterial host to homogeneity. Functional folding of the puriﬁed detergent-solubilized AHK1was veriﬁed by CD-spectroscopy, phosphorylation activity assays, and protein interaction studies with downstream Arabidopsis histidine-containing phosphotransfer proteins (AHPs). Consequently, the high-level expression and eﬃcient puriﬁcation of recombinant Arabidopsis osmosensor histidine kinase AHK1 reported in this work provide a critical milestone for further mechanistic and structural studies on a plant osmosensor candidate which might help to resolve plant osmoregulation and signaling on the molecular level. 2.1. Heterologous Expression and Puriﬁcation of AHK1 Sensor Kinase 2.1. Heterologous Expression and Puriﬁcation of AHK1 Sensor Kinase In this study we successfully expressed His-tagged recombinant full-length Arabidopsis histidine kinase AHK1 in the bacterial host E. coli BL21 (DE3). Systematic screening of culture conditions 3 of 17 osCholi e from Plants 2020, 9, 304 In sub 16 was ide identiﬁed a growth temperature of 16 ◦C after induction as the most suitable to obtain suﬃcient amounts of the recombinant plant histidine kinase at maximal stability. Further experiments revealed that translational errors leading to protein aggregation can be minimized by the addition of plasmid pRARE which provides rare codon tRNAs during bacterial expression. Extra addition of 2% (v/v) ethanol in the expression culture further reduced the tendency to form inclusion bodies. With these conditions and in culture medium Terriﬁc broth (TB) (Figure S1), high protein levels with minimal contaminations were obtained 5 h after induction (Figure 1a). The observed small contaminations at 30 kDa may be attributed to protein degradation or incomplete protein translation. Upon longer expression times these contaminations increased relatively to the target protein rendering these conditions less favorable for recombinant AHK1 production in the bacterial host. 230,000 x g and applied to nickel-nitrilotriacetic acid (Ni-NTA) immobilized metal affin chromatography (IMAC). Nonspecifically bound proteins were removed by washing the affin resin with 50 mM imidazole. Highly pure recombinant AHK1 was eluted at 100 mM imidaz Coomassie staining (Figure 1b) and Western blot analysis (Figure 1c) revealed a dominant sin band at approximately 130 kDa which correlated well to the sequence-based theoretical molecu mass of recombinant AHK1 of 138 kDa. While Coomassie staining of the purified sample showed obvious signs of contaminations, some faint background bands become visible in the Western b suggesting that minor protein degradation may have occurred before or during chromatograp purification in the presence of 0.015% (w/v) FosCholine-16. However, due to their low intensitie comparison to the band intensity of the full-length AHK1 membrane protein these backgrou t i t b l t d g (a) (b) (c) Figure 1. Expression and purification of recombinant His-tagged AHK1. (a) Expression of Arabido histidine kinase 1 (AHK1) in E. coli BL21 (DE3) containing the pRARE plasmid was checked on 1 SDS-polyacrylamide gel electrophoresis (SDS–PAGE). Samples from the expression were taken up induction with isopropyl β-d-1-thiogalactopyranoside (IPTG) (lane 1) and after 2, 4, and 5 respectively (lanes 2–4) and analyzed by Western blotting using anti-His Tag antibodies. 2.1. Heterologous Expression and Puriﬁcation of AHK1 Sensor Kinase (b) AH purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Left lane, protein mark right lane, 1 µg of purified protein. Protein bands were visualized by Coomassie staining. T apparent molecular mass of about 130 kDa of the purified protein corresponds to the theoret molecular mass of AHK1. (c) Purity and identity of the recombinant AHK1 was confirmed by West blotting using anti-His Tag antibodies. Figure 1. Expression and puriﬁcation of recombinant His-tagged AHK1. (a) Expression of Arabidopsis histidine kinase 1 (AHK1) in E. coli BL21 (DE3) containing the pRARE plasmid was checked on 10% SDS-polyacrylamide gel electrophoresis (SDS–PAGE). Samples from the expression were taken upon induction with isopropyl β-d-1-thiogalactopyranoside (IPTG) (lane 1) and after 2, 4, and 5 h, respectively (lanes 2–4) and analyzed by Western blotting using anti-His Tag antibodies. (b) AHK1 puriﬁed by nickel-nitrilotriacetic acid (Ni-NTA) aﬃnity chromatography. Left lane, protein marker; right lane, 1 µg of puriﬁed protein. Protein bands were visualized by Coomassie staining. The apparent molecular mass of about 130 kDa of the puriﬁed protein corresponds to the theoretical molecular mass of AHK1. (c) Purity and identity of the recombinant AHK1 was conﬁrmed by Western blotting using anti-His Tag antibodies. (c) (a) (b) (a) (c) Figure 1. Expression and purification of recombinant His-tagged AHK1. (a) Expression of Arabidop Figure 1. Expression and puriﬁcation of recombinant His-tagged AHK1. (a) Expression of Arabidopsis Figure 1. Expression and purification of recombinant His-tagged AHK1. (a) Expression of Arabido histidine kinase 1 (AHK1) in E. coli BL21 (DE3) containing the pRARE plasmid was checked on 1 SDS-polyacrylamide gel electrophoresis (SDS–PAGE). Samples from the expression were taken up induction with isopropyl β-d-1-thiogalactopyranoside (IPTG) (lane 1) and after 2, 4, and 5 respectively (lanes 2–4) and analyzed by Western blotting using anti-His Tag antibodies. (b) AH purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Left lane, protein mark right lane, 1 µg of purified protein. Protein bands were visualized by Coomassie staining. T apparent molecular mass of about 130 kDa of the purified protein corresponds to the theoret molecular mass of AHK1. (c) Purity and identity of the recombinant AHK1 was confirmed by West blotting using anti-His Tag antibodies. Figure 1. Expression and puriﬁcation of recombinant His-tagged AHK1. (a) Expression of Arabidopsis histidine kinase 1 (AHK1) in E. coli BL21 (DE3) containing the pRARE plasmid was checked on 10% SDS-polyacrylamide gel electrophoresis (SDS–PAGE). 2.1. Heterologous Expression and Puriﬁcation of AHK1 Sensor Kinase Samples from the expression were taken upon induction with isopropyl β-d-1-thiogalactopyranoside (IPTG) (lane 1) and after 2, 4, and 5 h, respectively (lanes 2–4) and analyzed by Western blotting using anti-His Tag antibodies. (b) AHK1 puriﬁed by nickel-nitrilotriacetic acid (Ni-NTA) aﬃnity chromatography. Left lane, protein marker; right lane, 1 µg of puriﬁed protein. Protein bands were visualized by Coomassie staining. The apparent molecular mass of about 130 kDa of the puriﬁed protein corresponds to the theoretical molecular mass of AHK1. (c) Purity and identity of the recombinant AHK1 was conﬁrmed by Western blotting using anti-His Tag antibodies. Figure 1. Expression and purification of recombinant His-tagged AHK1. (a) Expression of Arabido histidine kinase 1 (AHK1) in E. coli BL21 (DE3) containing the pRARE plasmid was checked on 1 SDS-polyacrylamide gel electrophoresis (SDS–PAGE). Samples from the expression were taken up induction with isopropyl β-d-1-thiogalactopyranoside (IPTG) (lane 1) and after 2, 4, and 5 respectively (lanes 2–4) and analyzed by Western blotting using anti-His Tag antibodies. (b) AH purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Left lane, protein mark right lane, 1 µg of purified protein. Protein bands were visualized by Coomassie staining. T apparent molecular mass of about 130 kDa of the purified protein corresponds to the theoret molecular mass of AHK1. (c) Purity and identity of the recombinant AHK1 was confirmed by West blotting using anti-His Tag antibodies. Figure 1. Expression and puriﬁcation of recombinant His-tagged AHK1. (a) Expression of Arabidopsis histidine kinase 1 (AHK1) in E. coli BL21 (DE3) containing the pRARE plasmid was checked on 10% SDS-polyacrylamide gel electrophoresis (SDS–PAGE). Samples from the expression were taken upon induction with isopropyl β-d-1-thiogalactopyranoside (IPTG) (lane 1) and after 2, 4, and 5 h, respectively (lanes 2–4) and analyzed by Western blotting using anti-His Tag antibodies. (b) AHK1 puriﬁed by nickel-nitrilotriacetic acid (Ni-NTA) aﬃnity chromatography. Left lane, protein marker; right lane, 1 µg of puriﬁed protein. Protein bands were visualized by Coomassie staining. The apparent molecular mass of about 130 kDa of the puriﬁed protein corresponds to the theoretical molecular mass of AHK1. (c) Purity and identity of the recombinant AHK1 was conﬁrmed by Western blotting using anti-His Tag antibodies. purified by nickel nitrilotriacetic acid (Ni NTA) affinity chromatography. Left lane, protein mark right lane, 1 µg of purified protein. Protein bands were visualized by Coomassie staining. 2.2. Size Exclusion Chromatography of the Recombinant AHK1 Osmosensor g p y f To evaluate homgeneity and monodispersity of the recombinant 2.2. Size Exclusion Chromatography of the Recombinant AHK1 Osmosensor To evaluate homgeneity and monodispersity of the recombinant To evaluate homgeneity and monodispersity of the recombinantly-produced osmosensor we performed a size exclusion chromatography (SEC). The SEC proﬁle shown in Figure 2a shows a distinct major peak at 17.27 mL elution volume. Peak shape and narrow distribution indicate monodispersity and homogeneity of the puriﬁed AHK1 sensor kinase. The small minor peaks observed at lower elution volume may be attributed to larger micelles or aggregates. In order to relate the elution volume on the gel ﬁltration column to the molecular mass of the puriﬁed full-length AHK1 sensor kinase, a number of protein standards (thyroglobuline 669 kDa, ferritin 440 kDa, aldolase 158 kDa, ovalbumin 43 kDa) were applied to the gel ﬁltration column at the same buﬀer conditions. Based on the calibration with these proteins (see Figure 2b) the main peak of the elution proﬁle corresponded to a molecular mass of 111 kDa. Assuming that the overall fold of the recombinant AHK1 resembles the globular form of the protein standards in the presence of 0.015% (w/v) FosCholine-16 and taking into account the molecular mass of the FosCholine micelle, the molecular mass of 111 kDa calculated from the SEC experiments implies that the puriﬁed recombinant AHK1 osmosensor was isolated in its monomeric form. performed a size exclusion chromatography (SEC). The SEC profile shown in Figure 2a shows a distinct major peak at 17.27 mL elution volume. Peak shape and narrow distribution indicate monodispersity and homogeneity of the purified AHK1 sensor kinase. The small minor peaks observed at lower elution volume may be attributed to larger micelles or aggregates. In order to relate the elution volume on the gel filtration column to the molecular mass of the purified full-length AHK1 sensor kinase, a number of protein standards (thyroglobuline 669 kDa, ferritin 440 kDa, aldolase 158 kDa, ovalbumin 43 kDa) were applied to the gel filtration column at the same buffer conditions. Based on the calibration with these proteins (see Figure 2b) the main peak of the elution profile corresponded to a molecular mass of 111 kDa. 2.2. Size Exclusion Chromatography of the Recombinant AHK1 Osmosensor g p y f To evaluate homgeneity and monodispersity of the recombinant Assuming that the overall fold of the recombinant AHK1 resembles the globular form of the protein standards in the presence of 0.015% (w/v) FosCholine-16 and taking into account the molecular mass of the FosCholine micelle, the molecular mass of 111 kDa calculated from the SEC experiments implies that the purified recombinant AHK1 osmosensor was isolated in its monomeric form. Figure 2. Size exclusion chromatogram of the recombinant AHK1 osmosensor (a). Experiments were performed on a Superose 10/300 column in 50 mM Tris, 300 mM NaCl, 10% (w/v) glycerol, 2.5 mM DTT, 0.015 % (w/v) FosCholine16, 0.002% (w/v) phenylmethylsulfonyl fluoride (PMSF) at pH 7.6 with 100 µL of purified AHK1. The molecular weight of the AHK1 osmosensor was calculated based on the slope of the calibration curve obtained with standard proteins. The distinct elution peak observed at 17.27 mL corresponds to a molecular weight of 111 kDa, indicating that AHK1 was isolated in the monomeric state. Calibration curve (b) derived from the four elution volumes of thyroglobuline, ferritin, aldolase, and ovalbumin with the elution volume of AHK1 (17.27 mL) indicated in red. Figure 2. Size exclusion chromatogram of the recombinant AHK1 osmosensor (a). Experiments were performed on a Superose 10/300 column in 50 mM Tris, 300 mM NaCl, 10% (w/v) glycerol, 2.5 mM DTT, 0.015% (w/v) FosCholine16, 0.002% (w/v) phenylmethylsulfonyl ﬂuoride (PMSF) at pH 7.6 with 100 µL of puriﬁed AHK1. The molecular weight of the AHK1 osmosensor was calculated based on the slope of the calibration curve obtained with standard proteins. The distinct elution peak observed at 17.27 mL corresponds to a molecular weight of 111 kDa, indicating that AHK1 was isolated in the monomeric state. Calibration curve (b) derived from the four elution volumes of thyroglobuline, ferritin, aldolase, and ovalbumin with the elution volume of AHK1 (17.27 mL) indicated in red. Figure 2. Size exclusion chromatogram of the recombinant AHK1 osmosensor (a). Experiments were performed on a Superose 10/300 column in 50 mM Tris, 300 mM NaCl, 10% (w/v) glycerol, 2.5 mM DTT, 0.015 % (w/v) FosCholine16, 0.002% (w/v) phenylmethylsulfonyl fluoride (PMSF) at pH 7.6 with 100 µL of purified AHK1. The molecular weight of the AHK1 osmosensor was calculated based on the slope of the calibration curve obtained with standard proteins. The distinct elution peak observed at 17.27 mL corresponds to a molecular weight of 111 kDa, indicating that AHK1 was isolated in the monomeric state. 2.2. Size Exclusion Chromatography of the Recombinant AHK1 Osmosensor g p y f To evaluate homgeneity and monodispersity of the recombinant Calibration curve (b) derived from the four elution volumes of thyroglobuline, ferritin, aldolase, and ovalbumin with the elution volume of AHK1 (17.27 mL) indicated in red. Figure 2. Size exclusion chromatogram of the recombinant AHK1 osmosensor (a). Experiments were performed on a Superose 10/300 column in 50 mM Tris, 300 mM NaCl, 10% (w/v) glycerol, 2.5 mM DTT, 0.015% (w/v) FosCholine16, 0.002% (w/v) phenylmethylsulfonyl ﬂuoride (PMSF) at pH 7.6 with 100 µL of puriﬁed AHK1. The molecular weight of the AHK1 osmosensor was calculated based on the slope of the calibration curve obtained with standard proteins. The distinct elution peak observed at 17.27 mL corresponds to a molecular weight of 111 kDa, indicating that AHK1 was isolated in the monomeric state. Calibration curve (b) derived from the four elution volumes of thyroglobuline, ferritin, aldolase, and ovalbumin with the elution volume of AHK1 (17.27 mL) indicated in red. 2.1. Heterologous Expression and Puriﬁcation of AHK1 Sensor Kinase T apparent molecular mass of about 130 kDa of the purified protein corresponds to the theoret molecular mass of AHK1. (c) Purity and identity of the recombinant AHK1 was confirmed by West blotting using anti-His Tag antibodies. c e o ce c c (N N ) y c o og p y e e, p o e e ; g e, µg of puriﬁed protein. Protein bands were visualized by Coomassie staining. The apparent molecular mass of about 130 kDa of the puriﬁed protein corresponds to the theoretical molecular mass of AHK1. (c) Purity and identity of the recombinant AHK1 was conﬁrmed by Western blotting using anti-His Tag antibodies. In subsequent systematic detergent screens (Figures S2 and S3, Table S1), 1% (w/v) FosCholine-16 was identiﬁed as most eﬃcient in solubilizing the recombinant plant histidine kinase from the bacterial membranes. Whole cell E. coli lysate solubilized this way was clariﬁed by centrifugation at 230,000× g and applied to nickel-nitrilotriacetic acid (Ni-NTA) immobilized metal aﬃnity chromatography (IMAC). Nonspeciﬁcally bound proteins were removed by washing the aﬃnity resin with 50 mM imidazole. Highly pure recombinant AHK1 was eluted at 100 mM imidazole. Coomassie staining (Figure 1b) and Western blot analysis (Figure 1c) revealed a dominant single band at approximately 130 kDa which correlated well to the sequence-based theoretical molecular mass of recombinant AHK1 of 138 kDa. While Coomassie staining of the puriﬁed sample showed no obvious signs of contaminations, some faint background bands become visible in the Western blot, suggesting that minor protein degradation may have occurred before or during chromatographic puriﬁcation in the presence of 0.015% (w/v) FosCholine-16. However, due to their low intensities in comparison to the band intensity of the full-length AHK1 membrane protein these background contaminants can be neglected. Plants 2020, 9, 304 Plants 2019, 8, 4 of 17 4 of 18 2.3. Circular Dichroism of Purified Recombinant AHK1 Osmosensor 2.3. Circular Dichroism of Puriﬁed Recombinant AHK1 Osmosensor The secondary structures of puriﬁed recombinant AHK1. Deconvolution results from CD spectra acquired with puriﬁed recombinant AHK1 by algorithms SELCON3, CONTINLL, and CDSSTR are shown together with its sequence-based in-silico secondary structures predicted by SOPMA. (a) (b) (b) (a) Figure 3. Circular dichroism of recombinant AHK1. (a) Circular dichroism spectrum of recombinant AHK1 shows minima at 208–210 nm and around 220 nm which are typical for α-helical structures. The spectrum has no significant negative band below 200 nm which is indicative for unordered polypeptide chains. (b) The secondary structure content derived from spectrum deconvolution specifies that 72% of AHK1 are present in a defined secondary structure and adopt either an α-helical, Figure 3. Circular dichroism of recombinant AHK1. (a) Circular dichroism spectrum of recombinant AHK1 shows minima at 208–210 nm and around 220 nm which are typical for α-helical structures. The spectrum has no signiﬁcant negative band below 200 nm which is indicative for unordered polypeptide chains. (b) The secondary structure content derived from spectrum deconvolution speciﬁes that 72% of AHK1 are present in a deﬁned secondary structure and adopt either an α-helical, β-sheet or β-turn fold. β-sheet or β-turn fold. Table 1. The secondary structures of purified recombinant AHK1. Deconvolution results from CD spectra acquired with purified recombinant AHK1 by algorithms SELCON3 CONTINLL and Table 1. The secondary structures of puriﬁed recombinant AHK1. Deconvolution results from CD spectra acquired with puriﬁed recombinant AHK1 by algorithms SELCON3, CONTINLL, and CDSSTR are shown together with its sequence-based in-silico secondary structures predicted by SOPMA. p q p y g CDSSTR are shown together with its sequence-based in-silico secondary structures predicted by SOPMA. Algorithm % Helical Content % Beta Strand Content % Turn Content % Unordered Content SELCON3 26,8 20,0 21,9 27,4 CONTINLL 31,2 21,8 20,0 27,0 CDSSTR 34 9 20 4 17 7 26 7 Algorithm % Helical Content % Beta Strand Content % Turn Content % Unordered Content SELCON3 26.8 20.0 21.9 27.4 CONTINLL 31.2 21.8 20.0 27.0 CDSSTR 34.9 20.4 17.7 26.7 GOR4 prediction 34.9 19.4 0 45,7 SOPMA prediction 39.4 15.5 3.9 41.3 2.4. Dimerization Studies of AHK1 Sensor Kinase CONTINLL 31,2 CDSSTR 34 9 2.4. Dimerization Studies of AHK1 Sensor Kinase CONTINLL 31,2 CDSSTR 34 9 2.4. 2.3. Circular Dichroism of Purified Recombinant AHK1 Osmosensor 2.3. Circular Dichroism of Puriﬁed Recombinant AHK1 Osmosensor Algorithm % Helical Content % Beta Strand Content % Turn Content % Unordered Content SELCON3 26,8 20,0 21,9 27,4 CONTINLL 31,2 21,8 20,0 27,0 CDSSTR 34,9 20,4 17,7 26,7 GOR4 prediction 34,9 19,4 0 45,7 Figure 3. Circular dichroism of recombinant AHK1. (a) Circular dichroism spectrum of recombinant AHK1 shows minima at 208–210 nm and around 220 nm which are typical for α-helical structures. The spectrum has no signiﬁcant negative band below 200 nm which is indicative for unordered polypeptide chains. (b) The secondary structure content derived from spectrum deconvolution speciﬁes that 72% of AHK1 are present in a deﬁned secondary structure and adopt either an α-helical, β-sheet or β-turn fold. Table 1. The secondary structures of puriﬁed recombinant AHK1. Deconvolution results from CD spectra acquired with puriﬁed recombinant AHK1 by algorithms SELCON3, CONTINLL, and CDSSTR are shown together with its sequence-based in-silico secondary structures predicted by SOPMA. Algorithm % Helical Content % Beta Strand Content % Turn Content % Unordered Content SELCON3 26.8 20.0 21.9 27.4 CONTINLL 31.2 21.8 20.0 27.0 CDSSTR 34.9 20.4 17.7 26.7 GOR4 prediction 34.9 19.4 0 45,7 SOPMA prediction 39.4 15.5 3.9 41.3 Dimerization Studies of AHK1 Sensor Kinase Dimerization of full length AHK1 was analyzed by microscale thermophoresis (M asurements. Changes in initial ﬂuorescence were detected upon addition of increasing concentrati (a) (b) Figure 3. Circular dichroism of recombinant AHK1. (a) Circular dichroism spectrum of recombinant AHK1 shows minima at 208–210 nm and around 220 nm which are typical for α-helical structures. The spectrum has no significant negative band below 200 nm which is indicative for unordered polypeptide chains. (b) The secondary structure content derived from spectrum deconvolution specifies that 72% of AHK1 are present in a defined secondary structure and adopt either an α-helical, β-sheet or β-turn fold. Table 1. The secondary structures of purified recombinant AHK1. Deconvolution results from CD spectra acquired with purified recombinant AHK1 by algorithms SELCON3 CONTINLL and Figure 3. Circular dichroism of recombinant AHK1. (a) Circular dichroism spectrum of recombinant AHK1 shows minima at 208–210 nm and around 220 nm which are typical for α-helical structures. The spectrum has no signiﬁcant negative band below 200 nm which is indicative for unordered polypeptide chains. (b) The secondary structure content derived from spectrum deconvolution speciﬁes that 72% of AHK1 are present in a deﬁned secondary structure and adopt either an α-helical, β-sheet or β-turn fold. Table 1. 2.3. Circular Dichroism of Purified Recombinant AHK1 Osmosensor 2.3. Circular Dichroism of Puriﬁed Recombinant AHK1 Osmosensor f f Purified AHK1 was analysed by circular dichroism (CD) spectroscopy to demonstrate functional folding of the recombinant protein and to exclude that heterologous expression and detergent solubilization have resulted in improperly-folded or partially-unfolded protein. The CD spectrum obtained on this sample (Figure 3a) shows minima at 205 nm and around 220 nm. Together with the maximum at 195 nm and the observed positive absorbance at wavelengths below 200 nm these minima are characteristic for a well-folded highly α-helical protein [28,29]. For a detailed analysis of secondary structure content, the CD spectrum was deconvoluted with three different algorithms (SELCON3, CONTINLL, and CDSSTR [30]) and the SMP56 membrane protein reference set (Table 1). All three algorithms gave similar results which are summarized in Table 1. However, as no experimental structural data are available for AHK1 yet, secondary structure contents obtained from CD measurements were compared to sequence-based in-silico secondary structures predicted by Puriﬁed AHK1 was analysed by circular dichroism (CD) spectroscopy to demonstrate functional folding of the recombinant protein and to exclude that heterologous expression and detergent solubilization have resulted in improperly-folded or partially-unfolded protein. The CD spectrum obtained on this sample (Figure 3a) shows minima at 205 nm and around 220 nm. Together with the maximum at 195 nm and the observed positive absorbance at wavelengths below 200 nm these minima are characteristic for a well-folded highly α-helical protein [28,29]. For a detailed analysis of secondary structure content, the CD spectrum was deconvoluted with three diﬀerent algorithms (SELCON3, CONTINLL, and CDSSTR [30]) and the SMP56 membrane protein reference set (Table 1). All three algorithms gave similar results which are summarized in Table 1. However, as no experimental structural data are available for AHK1 yet, secondary structure contents obtained from CD measurements were compared to sequence-based in-silico secondary structures predicted by 5 of 17 5 of 18 Plants 2020, 9, 304 Plants 2019, 8, GOR4 [31] and SOPMA [32]. Data from spectroscopic analysis (Figure 3b) and in silico predictions (Table 1) agree well, with GOR4 resembling precisely the experimental data for the helical content. They specify that most of the recombinant protein adopts a well-deﬁned secondary structure with only 25% of the sequence existing in an unstructured conformation which might represent the ﬂexible loops connecting the well-deﬁned transmembrane sensor, and catalytic topological domains. 2.3. Circular Dichroism of Purified Recombinant AHK1 Osmosensor 2.3. Circular Dichroism of Puriﬁed Recombinant AHK1 Osmosensor Thus, the puriﬁed recombinant AHK1 is likely to reﬂect the native fold and conformation of the plant histidine kinase. GOR4 [31]and SOPMA [32]. Data from spectroscopic analysis (Figure 3b) and in silico predictions (Table 1) agree well, with GOR4 resembling precisely the experimental data for the helical content. They specify that most of the recombinant protein adopts a well-defined secondary structure with only 25% of the sequence existing in an unstructured conformation which might represent the flexible loops connecting the well-defined transmembrane sensor, and catalytic topological domains. Thus, the purified recombinant AHK1 is likely to reflect the native fold and conformation of the plant histidine kinase. (a) Figure 3. Circular dichroism of recombina AHK1 shows minima at 208–210 nm and The spectrum has no significant negativ polypeptide chains. (b) The secondary specifies that 72% of AHK1 are present in a β-sheet or β-turn fold. Table 1. The secondary structures of pur spectra acquired with purified recombin CDSSTR are shown together with its seq SOPMA. Algorithm % Helical Content SELCON3 26,8 CONTINLL 31,2 CDSSTR 34,9 GOR4 Figure 3. Circular dichroism of recombina AHK1 shows minima at 208–210 nm and ar spectrum has no signiﬁcant negative band b chains. (b) The secondary structure conten AHK1 are present in a deﬁned secondary st Table 1. The secondary structures of puri spectra acquired with puriﬁed recombinan are shown together with its sequence-base Algorithm % Helical Content % SELCON3 26.8 CONTINLL 31.2 CDSSTR 34.9 GOR4 prediction 34.9 SOPMA prediction 39.4 2.4. Dimerization Studies of AHK1 Sensor Kin Dimerization of full length AHK1 (a) (b) Figure 3. Circular dichroism of recombinant AHK1. (a) Circular dichroism spectrum of recombinant AHK1 shows minima at 208–210 nm and around 220 nm which are typical for α-helical structures. The spectrum has no significant negative band below 200 nm which is indicative for unordered polypeptide chains. (b) The secondary structure content derived from spectrum deconvolution specifies that 72% of AHK1 are present in a defined secondary structure and adopt either an α-helical, β-sheet or β-turn fold. Table 1. The secondary structures of purified recombinant AHK1. Deconvolution results from CD spectra acquired with purified recombinant AHK1 by algorithms SELCON3, CONTINLL, and CDSSTR are shown together with its sequence-based in-silico secondary structures predicted by SOPMA. 2.3. Circular Dichroism of Purified Recombinant AHK1 Osmosensor 2.3. Circular Dichroism of Puriﬁed Recombinant AHK1 Osmosensor Dimerization Studies of AHK1 Sensor Kinase NTINLL 31,2 21,8 20,0 27,0 DSSTR 34 9 20 4 17 7 26 7 merization Studies of AHK1 Sensor Kinase CDSSTR 34,9 20,4 17,7 26,7 GOR4 prediction 34,9 19,4 0 45,7 SOPMA prediction 39,4 15,5 3,9 41,3 2.4. Dimerization Studies of AHK1 Sensor Kinase Dimerization of full length AHK1 was analyzed by microscale thermophoresis (MST) measurements. Changes in initial fluorescence were detected upon addition of increasing concentrations of unlabeled full length AHK1 (0.4–12 µM) to 40 nM AHK1 labeled with the fluorophore AlexaFluor488-NHS. The resulting binding curve (Figure 4a) reflects tight binding of the monomers with an apparent dissociation constant (Kd) of 208 +/− 48 nM. The observed high affinity binding between AHK1 monomers resulting in the formation of the dimer present at physiological conditions further supports in vitro functional folding of the expressed and purified full-length AHK1 osmosensor The negative control with chemically denatured AHK1 showed no interaction as Dimerization of full length AHK1 was analyzed by microscale thermophoresis (MST) measurements. Changes in initial ﬂuorescence were detected upon addition of increasing concentrations of unlabeled full length AHK1 (0.4–12 µM) to 40 nM AHK1 labeled with the ﬂuorophore AlexaFluor488-NHS. The resulting binding curve (Figure 4a) reﬂects tight binding of the monomers with an apparent dissociation constant (Kd) of 208 +/−48 nM. The observed high aﬃnity binding between AHK1 monomers resulting in the formation of the dimer present at physiological conditions further supports in vitro functional folding of the expressed and puriﬁed full-length AHK1 osmosensor. The negative control with chemically denatured AHK1 showed no interaction as expected (see Figure 4a). To corroborate the in vitro results in vivo, we performed a mating-based split-ubiquitin system (mbSUS) assay in yeast [33] and Förster resonance energy transfer (FRET)-ﬂuorescence lifetime imaging microscopy (FLIM) study in transiently-transformed Nicotiana benthamiana epidermal leaf cells [34]. For the mbSUS assay, full-length AHK1 and control proteins were expressed as Nub or Cub fusion in yeast. After mating, the presence of the plasmids was veriﬁed by growth of yeast cells on synthetic complete (SC) medium containing adenine and histidine (SC + Ade, His), whereas the putative 6 of 17 Plants 2020, 9, 304 interaction of AHK1 was assayed by growth on SC medium containing 50 µM Met, which enhances the stringency of the interaction test (Figure 4b). 2.3. Circular Dichroism of Purified Recombinant AHK1 Osmosensor 2.3. Circular Dichroism of Puriﬁed Recombinant AHK1 Osmosensor AHK1-Cub together with the negative control NubG (Nub mutant to avoid reassembling) did not show complementation of yeast growth, whilst the growth in the presence of the positive control NubWT (wild type Nub) proved the eﬀectiveness of the assay. A clear complementation of yeast growth was observed in the presence of AHK1-Cub and AHK1-NubA (Figure 4b), demonstrating AHK1 homomerization in vivo. To verify the results of the MST and mbSUS studies, we performed a (FLIM) analysis. AHK1-GFP was transiently expressed in tobacco (Nicotiana benthamiana) epidermal leaf cells either alone (negative control) or with AHK1-mCherry. The AHK1-GFP-mCherry served as positive control. All fusion proteins (co-)localized to the cellular periphery suggesting that AHK1 is a plasma membrane-resident protein in plant cells (Figure 4c). In the presence of AHK1-mCherry the ﬂuorescence lifetime of AHK1-GFP decreased signiﬁcantly indicating energy transfer between the ﬂuorophores and, thus, very close spatial proximity/homomerization of the AHK1 monomers (Figure 4d). The AHK1-GFP-mCherry positive control exhibited the expected very strong reduction of the ﬂuorescence lifetime, as the donor and acceptor ﬂuorophores were directly linked in the fusion protein (Figure 4d). Plants 2019, 8, x FOR PEER REVIEW expected (see Figure 4a). To corroborate the in vitro results in vivo, we performed a mating-b split-ubiquitin system (mbSUS) assay in yeast [33] and Förster resonance energy transfer (FR fluorescence lifetime imaging microscopy (FLIM) study in transiently-transformed Nic benthamiana epidermal leaf cells [34]. For the mbSUS assay, full-length AHK1 and control pro 2.5. Heterologous Expression and Puriﬁcation of the AHP2 Transfer Protein were expressed as Nub or Cub fusion in yeast. After mating, the by growth of yeast cells on synthetic complete (SC) medium co The AHK1 phosphorelay downstream binding partner AHP2 identiﬁed in previous studies [35,36] was overexpressed as GST-tagged fusion protein in E. coli BL21 (DE3)-Gold cells overnight at 16 ◦C (Figure 5a) and puriﬁed to apparent homogeneity from bacterial extracts using glutathione-S-transferase sepharose (GE Healthcare). A single band on SDS–PAGE conﬁrmed the homogeneity of the recombinant protein (Figure 5b). Identity of AHP2-GST was conﬁrmed with anti-GST antibody (Figure 5c). Ade, His), whereas the putative interaction of AHK1 was assayed by growth on SC me containing 50 µM Met, which enhances the stringency of the interaction test (Figure 4b). AHK1 together with the negative control NubG (Nub mutant to avoid reassembling) did not complementation of yeast growth, whilst the growth in the presence of the positive control Nu (wild type Nub) proved the effectiveness of the assay. A clear complementation of yeast growth observed in the presence of AHK1 Cub and AHK1 NubA (Figure 4b) demonstrating A 2.6. Interaction Studies of AHK1 Sensor Kinase with AHP2 Phosphotransfer Protein homomerization in vivo. To verify the results of the MST and mbSUS stu Confocal images were taken 3 days after Agrobacterium transformation; bars: 10 µm. (d) FRET-FLIM analysis of the AHK1-GFP/AHK1-mCherry association in N. benthamiana epidermal leaf cells 3 days after Agrobacterium infection. The AHK1-GFP fusion serves as donor-only control and AHK1-GFP-mCherry fusion as positive control. Center lines in the boxplot show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; the whiskers extend to minimum and maximum values. The details of the statistical evaluation are provided in Material and Methods; asterisks indicate significant differences (: p ˂ 0.02; : p ˂ 0.01; *: p ˂ 0.001). Figure 4. AHK1 forms homodimers in vitro and in vivo. (a) Microscale thermophoresis (MST) interaction studies of ﬂuorescently labeled AHK1 sensor kinase with unlabeled AHK1. Relative changes observed in thermophoresis reﬂecting binding of the monomers (•) in the functional dimer were ﬁtted according to a one-binding-site model. The related binding curve indicates a Kd value of 208 +/−48 nM for AHK1-AHK1 homodimer formation. Data for chemically denatured AHK1 (△) shows no changes in thermophoresis. Data represent the mean of three independent measurements +/− standard deviation. (b) Yeast mating-based split-ubiquitin system (mbSUS) assay with AHK1-Cub and the indicated Nub fusion proteins. Yeast cells transfected with the corresponding expression constructs were grown at diﬀerent OD600nm and 30 ◦C on either non-interaction-selecting medium (SC + Ade, His, left) for 1 day or interaction-selecting medium (SC + 50 µM Met, right) for 4 days. (c) Co-localization of AHK1-GFP and AHK1-mCherry in the plasma membrane of transiently-transformed N. benthamiana epidermal leaf cells. Confocal images were taken 3 days after Agrobacterium transformation; bars: 10 µm. (d) FRET-FLIM analysis of the AHK1-GFP/AHK1-mCherry association in N. benthamiana epidermal leaf cells 3 days after Agrobacterium infection. The AHK1-GFP fusion serves as donor-only control and AHK1-GFP-mCherry fusion as positive control. Center lines in the boxplot show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; the whiskers extend to minimum and maximum values. The details of the statistical evaluation are provided in Material and Methods; asterisks indicate signiﬁcant diﬀerences (: p < 0.02; *: p < 0.01; : p < 0.001). Figure 4. AHK1 forms homodimers in vitro and in vivo. (a) Microscale thermophoresis (MST) interaction studies of fluorescently labeled AHK1 sensor kinase with unlabeled AHK1. 2.6. Interaction Studies of AHK1 Sensor Kinase with AHP2 Phosphotransfer Protein homomerization in vivo. To verify the results of the MST and mbSUS stu Relative changes observed in thermophoresis reflecting binding of the monomers (●) in the functional dimer were fitted according to a one-binding-site model. The related binding curve indicates a Kd value of 208 +/- 48 nM for AHK1-AHK1 homodimer formation. Data for chemically denatured AHK1 () shows no changes in thermophoresis. Data represent the mean of three independent measurements +/- standard deviation. (b) Yeast mating-based split-ubiquitin system (mbSUS) assay with AHK1-Cub and the indicated Nub fusion proteins. Yeast cells transfected with the corresponding expression constructs were grown at different OD600nm and 30 °C on either non-interaction-selecting medium (SC + Ade, His, left) for 1 day or interaction-selecting medium (SC + 50 µM Met, right) for 4 days. (c) Co- localization of AHK1-GFP and AHK1-mCherry in the plasma membrane of transiently-transformed N. benthamiana epidermal leaf cells. Confocal images were taken 3 days after Agrobacterium transformation; bars: 10 µm. (d) FRET-FLIM analysis of the AHK1-GFP/AHK1-mCherry association in N. benthamiana epidermal leaf cells 3 days after Agrobacterium infection. The AHK1-GFP fusion serves as donor-only control and AHK1-GFP-mCherry fusion as positive control. Center lines in the boxplot show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; the whiskers extend to minimum and maximum values. The details of the statistical evaluation are provided in Material and Methods; asterisks indicate significant differences (: p ˂ 0.02; *: p ˂ 0.01; : p ˂ 0.001). Figure 4. AHK1 forms homodimers in vitro and in vivo. (a) Microscale thermophoresis (MST) interaction studies of ﬂuorescently labeled AHK1 sensor kinase with unlabeled AHK1. Relative changes observed in thermophoresis reﬂecting binding of the monomers (•) in the functional dimer were ﬁtted according to a one-binding-site model. The related binding curve indicates a Kd value of 208 +/−48 nM for AHK1-AHK1 homodimer formation. Data for chemically denatured AHK1 (△) shows no changes in thermophoresis. Data represent the mean of three independent measurements +/− standard deviation. (b) Yeast mating-based split-ubiquitin system (mbSUS) assay with AHK1-Cub and the indicated Nub fusion proteins. Yeast cells transfected with the corresponding expression constructs were grown at diﬀerent OD600nm and 30 ◦C on either non-interaction-selecting medium (SC + Ade, His, left) for 1 day or interaction-selecting medium (SC + 50 µM Met, right) for 4 days. (c) Co-localization of AHK1-GFP and AHK1-mCherry in the plasma membrane of transiently-transformed N. benthamiana epidermal leaf cells. 2.6. Interaction Studies of AHK1 Sensor Kinase with AHP2 Phosphotransfer Protein homomerization in vivo. To verify the results of the MST and mbSUS stu MST was used to monitor and quantify binding of histidine phosphotransfer protein AHP2 to ﬂuorescently labeled full-length AHK1 sensor kinase. Changes observed in thermophoresis at diﬀerent concentrations of AHP2-GST added to 40 nM AHK1 labeled with ﬂuorescent dye AlexaFluor488-NHS were plotted as function of the AHP2 concentrations (0.4 nM–12.5 µM) used in the titration series (see Figure 6). The related binding curve corresponds to an apparent dissociation constant (Kd) of 317 +/− 62 nM. This relatively low value provides evidence of a high aﬃnity interaction between AHK1 and AHP2 and further supports functional folding of the puriﬁed recombinant full-length sensor kinase. Notably, a negative control using chemically denatured AHK1 shows no interaction with AHP2. analysis. AHK1-GFP was transiently expressed in tobacco (Nicotiana benthamiana) epidermal leaf either alone (negative control) or with AHK1-mCherry. The AHK1-GFP-mCherry served as pos control. All fusion proteins (co-)localized to the cellular periphery suggesting that AHK1 is a pl membrane-resident protein in plant cells (Figure 4c). In the presence of AHK1-mCherry fluorescence lifetime of AHK1-GFP decreased significantly indicating energy transfer between fluorophores and, thus, very close spatial proximity/homomerization of the AHK1 monomers (Fi 4d). The AHK1-GFP-mCherry positive control exhibited the expected very strong reduction o fluorescence lifetime, as the donor and acceptor fluorophores were directly linked in the fu protein (Figure 4d). (a) Figure 4. Cont. Figure 4. Cont. 7 of 17 of 18 Plants 2020, 9, 304 Figure 4. AHK1 forms homodimers in vitro and in vivo. (a) Microscale thermophoresis (MST) interaction studies of fluorescently labeled AHK1 sensor kinase with unlabeled AHK1. Relative changes observed in thermophoresis reflecting binding of the monomers (●) in the functional dimer were fitted according to a one-binding-site model. The related binding curve indicates a Kd value of 208 +/- 48 nM for AHK1-AHK1 homodimer formation. Data for chemically denatured AHK1 () shows no changes in thermophoresis. Data represent the mean of three independent measurements +/- standard deviation. (b) Yeast mating-based split-ubiquitin system (mbSUS) assay with AHK1-Cub and the indicated Nub fusion proteins. Yeast cells transfected with the corresponding expression constructs were grown at different OD600nm and 30 °C on either non-interaction-selecting medium (SC + Ade, His, left) for 1 day or interaction-selecting medium (SC + 50 µM Met, right) for 4 days. (c) Co- localization of AHK1-GFP and AHK1-mCherry in the plasma membrane of transiently-transformed N. benthamiana epidermal leaf cells. 2.6. Interaction Studies of AHK1 Sensor Kinase with AHP2 Phosphotransfer Protein homomerization in vivo. To verify the results of the MST and mbSUS stu coli BL21 (DE3) Gold cells wa analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blotting Protein expression was monitored upon induction with IPTG (lane 1), after 2 h, 4 h, and overnigh (lanes 2 and 4, respectively) and detected with an anti-GST antibody. (b) The recombinant AHP2 GST, purified by GST-affinity chromatography, migrates on the SDS gel with an apparent molecula mass of 44 kDa corresponding to the estimated theoretical molecular mass. Protein bands were visualized by colloidal Coomassie staining. (c) GST-tagged AHP2 was purified by GST-affinity chromatography, separated by SDS-PAGE and visualized by immunoblotting using an anti-GST Figure 5. Expression and puriﬁcation of recombinant GST-tagged AHP2. (a) Expression of GST-fusion protein of Arabidopsis histidine phosphotransfer protein 2 (AHP2) in E. coli BL21 (DE3) Gold cells was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blotting. Protein expression was monitored upon induction with IPTG (lane 1), after 2 h, 4 h, and overnight (lanes 2 and 4, respectively) and detected with an anti-GST antibody. (b) The recombinant AHP2-GST, puriﬁed by GST-aﬃnity chromatography, migrates on the SDS gel with an apparent molecular mass of 44 kDa corresponding to the estimated theoretical molecular mass. Protein bands were visualized by colloidal Coomassie staining. (c) GST-tagged AHP2 was puriﬁed by GST-aﬃnity chromatography, separated by SDS-PAGE and visualized by immunoblotting using an anti-GST antibody. Plants 2019, 8, x FOR PEER REVIEW 9 antibody. .6. Interaction Studies of AHK1 Sensor Kinase with AHP2 Phosphotransfer Protein MST was used to monitor and quantify binding of histidine phosphotransfer protein AH luorescently labeled full-length AHK1 sensor kinase. Changes observed in thermophore ifferent concentrations of AHP2-GST added to 40 nM AHK1 labeled with fluorescent AlexaFluor488-NHS were plotted as function of the AHP2 concentrations (0.4 nM–12.5 µM) us he titration series (see Figure 6). The related binding curve corresponds to an apparent dissoci onstant (Kd) of 317 +/− 62 nM. This relatively low value provides evidence of a high af nteraction between AHK1 and AHP2 and further supports functional folding of the pu ecombinant full-length sensor kinase. Notably, a negative control using chemically denatured A hows no interaction with AHP2. Figure 6. Microscale thermophoresis (MST) interaction studies of fluorescently-labeled AHK1 sensor kinase with GST-fusion of AHP2. Relative changes observed thermophoresis reflecting binding of the two protein partners (●) at different concentrations of AHP2-GST were fitted according to a one- binding-site model. 2.6. Interaction Studies of AHK1 Sensor Kinase with AHP2 Phosphotransfer Protein homomerization in vivo. To verify the results of the MST and mbSUS stu Confocal images were taken 3 days after Agrobacterium transformation; bars: 10 µm. (d) FRET-FLIM analysis of the AHK1-GFP/AHK1-mCherry association in N. benthamiana epidermal leaf cells 3 days after Agrobacterium infection. The AHK1-GFP fusion serves as donor-only control and AHK1-GFP-mCherry fusion as positive control. Center lines in the boxplot show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; the whiskers extend to minimum and maximum values. The details of the statistical evaluation are provided in Material and Methods; asterisks indicate signiﬁcant diﬀerences (: p < 0.02; *: p < 0.01; **: p < 0.001). 8 of 17 antibody Plants 2020, 9, 304 the recombin (Fi 5 ) Figure 5c). (a) (b) (c) Figure 5. Expression and purification of recombinant GST-tagged AHP2. (a) Expression of GST-fusion protein of Arabidopsis histidine phosphotransfer protein 2 (AHP2) in E. coli BL21 (DE3) Gold cells was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blotting. Protein expression was monitored upon induction with IPTG (lane 1), after 2 h, 4 h, and overnight (lanes 2 and 4, respectively) and detected with an anti-GST antibody. (b) The recombinant AHP2- GST, purified by GST-affinity chromatography, migrates on the SDS gel with an apparent molecular mass of 44 kDa corresponding to the estimated theoretical molecular mass. Protein bands were visualized by colloidal Coomassie staining. (c) GST-tagged AHP2 was purified by GST-affinity chromatography, separated by SDS-PAGE and visualized by immunoblotting using an anti-GST Figure 5. Expression and puriﬁcation of recombinant GST-tagged AHP2. (a) Expression of GST-fusion protein of Arabidopsis histidine phosphotransfer protein 2 (AHP2) in E. coli BL21 (DE3) Gold cells was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blotting. Protein expression was monitored upon induction with IPTG (lane 1), after 2 h, 4 h, and overnight (lanes 2 and 4, respectively) and detected with an anti-GST antibody. (b) The recombinant AHP2-GST, puriﬁed by GST-aﬃnity chromatography, migrates on the SDS gel with an apparent molecular mass of 44 kDa corresponding to the estimated theoretical molecular mass. Protein bands were visualized by colloidal Coomassie staining. (c) GST-tagged AHP2 was puriﬁed by GST-aﬃnity chromatography, separated by SDS-PAGE and visualized by immunoblotting using an anti-GST antibody. Plants 2019, 8, x FOR PEER REVIEW 9 (b) (c) (a) (c) (b) (a) Figure 5. Expression and purification of recombinant GST-tagged AHP2. (a) Expression of GST-fusion protein of Arabidopsis histidine phosphotransfer protein 2 (AHP2) in E. 2.6. Interaction Studies of AHK1 Sensor Kinase with AHP2 Phosphotransfer Protein homomerization in vivo. To verify the results of the MST and mbSUS stu Data for chemically denatured AHK1 (△) which show no changes in thermophoresis are given for comparison. Data represent the mean of seven independent measurements +/−standard deviation. For control with denatured protein duplicates were taken for each concentration. 2.6. Interaction Studies of AHK1 Sensor Kinase with AHP2 Phosphotransfer Protein homomerization in vivo. To verify the results of the MST and mbSUS stu From the related binding curve a Kd value of 317 +/− 62 nM was obtained for the AHK1-AHP2 complex formation. Data for chemically denatured AHK1 () which show no changes in thermophoresis are given for comparison. Data represent the mean of seven independent measurements +/− standard deviation. For control with denatured protein duplicates were taken for each concentration Figure 6. Microscale thermophoresis (MST) interaction studies of ﬂuorescently-labeled AHK1 sensor kinase with GST-fusion of AHP2. Relative changes observed thermophoresis reﬂecting binding of the two protein partners (•) at diﬀerent concentrations of AHP2-GST were ﬁtted according to a one-binding-site model. From the related binding curve a Kd value of 317 +/−62 nM was obtained for the AHK1-AHP2 complex formation. Data for chemically denatured AHK1 (△) which show no changes in thermophoresis are given for comparison. Data represent the mean of seven independent measurements +/−standard deviation. For control with denatured protein duplicates were taken for each concentration. dies of AHK1 Sensor Kinase with AHP2 Phosphotransfer Protei d to monitor and quantify binding of histidine phosphotr led full-length AHK1 sensor kinase. Changes observed rations of AHP2-GST added to 40 nM AHK1 labeled HS were plotted as function of the AHP2 concentrations (0 (see Figure 6). The related binding curve corresponds to a 317 +/− 62 nM. This relatively low value provides evid en AHK1 and AHP2 and further supports functional f ength sensor kinase. Notably, a negative control using chem on with AHP2. Figure 6. Microscale thermophoresis (MST) interaction studies of fluorescently-labeled AHK1 sensor kinase with GST-fusion of AHP2. Relative changes observed thermophoresis reflecting binding of the two protein partners (●) at different concentrations of AHP2-GST were fitted according to a one- binding-site model. From the related binding curve a Kd value of 317 +/− 62 nM was obtained for the AHK1-AHP2 complex formation. Data for chemically denatured AHK1 () which show no changes in thermophoresis are given for comparison. Data represent the mean of seven independent measurements +/− standard deviation. For control with denatured protein duplicates were taken for Figure 6. Microscale thermophoresis (MST) interaction studies of ﬂuorescently-labeled AHK1 sensor kinase with GST-fusion of AHP2. Relative changes observed thermophoresis reﬂecting binding of the two protein partners (•) at diﬀerent concentrations of AHP2-GST were ﬁtted according to a one-binding-site model. From the related binding curve a Kd value of 317 +/−62 nM was obtained for the AHK1-AHP2 complex formation. 2.6. Interaction Studies of AHK1 Sensor Kinase with AHP2 Phosphotransfer Protein homomerization in vivo. To verify the results of the MST and mbSUS stu Data for chemically denatured AHK1 (△) which show no changes in thermophoresis are given for comparison. Data represent the mean of seven independent measurements +/−standard deviation. For control with denatured protein duplicates were taken for each concentration. Figure 6. Microscale thermophoresis (MST) interaction studies of fluorescently-labeled AHK1 sensor kinase with GST-fusion of AHP2. Relative changes observed thermophoresis reflecting binding of the two protein partners (●) at different concentrations of AHP2-GST were fitted according to a one- binding-site model. From the related binding curve a Kd value of 317 +/− 62 nM was obtained for the AHK1-AHP2 complex formation. Data for chemically denatured AHK1 () which show no changes in thermophoresis are given for comparison. Data represent the mean of seven independent measurements +/− standard deviation. For control with denatured protein duplicates were taken for Figure 6. Microscale thermophoresis (MST) interaction studies of ﬂuorescently-labeled AHK1 sensor kinase with GST-fusion of AHP2. Relative changes observed thermophoresis reﬂecting binding of the two protein partners (•) at diﬀerent concentrations of AHP2-GST were ﬁtted according to a one-binding-site model. From the related binding curve a Kd value of 317 +/−62 nM was obtained for the AHK1-AHP2 complex formation. Data for chemically denatured AHK1 (△) which show no changes in thermophoresis are given for comparison. Data represent the mean of seven independent measurements +/−standard deviation. For control with denatured protein duplicates were taken for each concentration. Figure 6. Microscale thermophoresis (MST) interaction studies of fluorescently-labeled AHK1 sensor kinase with GST-fusion of AHP2. Relative changes observed thermophoresis reflecting binding of the two protein partners (●) at different concentrations of AHP2-GST were fitted according to a one- binding-site model. From the related binding curve a Kd value of 317 +/− 62 nM was obtained for the AHK1-AHP2 complex formation. Data for chemically denatured AHK1 () which show no changes in thermophoresis are given for comparison. Data represent the mean of seven independent measurements +/− standard deviation. For control with denatured protein duplicates were taken for Figure 6. Microscale thermophoresis (MST) interaction studies of ﬂuorescently-labeled AHK1 sensor kinase with GST-fusion of AHP2. Relative changes observed thermophoresis reﬂecting binding of the two protein partners (•) at diﬀerent concentrations of AHP2-GST were ﬁtted according to a one-binding-site model. From the related binding curve a Kd value of 317 +/−62 nM was obtained for the AHK1-AHP2 complex formation. each concentration. 2.7. AHK1 Acts as Phospho-His and/or Phospho-Asp Phosphatase In Vitro 2.7. AHK1 Acts as Phospho-His and/or Phospho-Asp Phosphatase In Vitro To test whether AHK1 has enzymatic activity, we performed auto-phosphorylation assays using γ-33P-ATP. However, no auto-modification of AHK1 with 33phosphate was observed. This suggested that AHK1 is not an active His kinase in the absence of the yet unknown ligand. However, as reported To test whether AHK1 has enzymatic activity, we performed auto-phosphorylation assays using γ-33P-ATP. However, no auto-modiﬁcation of AHK1 with 33phosphate was observed. This suggested that AHK1 is not an active His kinase in the absence of the yet unknown ligand. However, as reported for the cytokinin receptor AHK4 [37], plant histidine kinases can also act as P-His or P-Asp phosphatases by 9 of 17 Plants 2020, 9, 304 forcing a backward ﬂux of phosphoryl residues within a given phosphorelay system. To establish such an phosphorelay system in vitro, we ﬁrst tested the capacity of the (His)6-tagged output domain of the soluble Arabidopsis thaliana histidine kinase 5 (AHK5output; amino acid 343 to 922), which contains the His kinase-typical HisKA, HATPAse c and the receiver domains [21] to auto-phosphorylate and to transfer the phosphoryl group to GST-tagged AHP2. When incubated with γ-33P-ATP, AHK5output clearly displayed incorporation of 33phosphate (Figure 7, lane 1), demonstrating its auto-phosphorylation capacity. When GST-tagged AHP2 was present in the phosphorylation reaction, AHK5output was able to transfer 33P-labeled phosphoryl residues to the phosphotransfer protein, as shown by the enhanced labelling of AHP2 against background and, in parallel, by the reduction of AHK5output auto-labelling (Figure 7, compare lanes 2 and 5). In the presence of AHK1, however, neither auto-phosphorylation of AHK5output (Figure 7, lane 3) nor the background-caused and AHK5output-caused enhanced labelling of AHP2 (Figure 7, lanes 4 and 6) were observed anymore. These results indicate that AHK1 is able to empty the AHK5output-to-AHP2 phosphorelay system from phosphoryl residues and, therefore, acts as P-His and/or P-Asp phosphatase in vitro. Plants 2019, 8, x FOR PEER REVIEW 10 of 18 Figure 7. AHK1 functions as phospho-His and/or phospho-Asp phosphatase in vitro. (a) Autoradiogram of the phosphorylation experiments performed with (His)6-tagged AHK1 (AHK1), (His)6-tagged AHK5output (AHK5output) and GST-tagged AHP2 (AHP2) in the presence of γ-33P- ATP. Lane 1: AHK5output; lane 2: AHK5output and AHP2; lane 3: AHK1, AHK5output; lane 4: AHK1, AHK5output, AHP2; lane 5: AHP2; lane 6: AHK1, AHP2. Phosphorylation reactions were run at room temperature for 20 min. 3. Discussion 3. Discussion Despite the large number of histidine kinases participating in two-component signal transfer processes and their counterparts in plants involved in multi-step phosphorelay signaling, our information on thermodynamics and kinetics that determine the interaction of these sensor kinases with their downstream histidine phosphotransfer proteins as well as the conformational changes triggering signal transfer activity in the phoshorelay is still sparse. This is partly due to the fact that most of these sensor kinases are integral membrane proteins which are hardly accessible to biochemical characterization. Thus, not surprisingly, most of the published interaction studies are based on yeast-two-hybrid analysis allowing qualitative assessments on the interaction, although two systems have been addressed in a quantitative way in biophysical studies by fluorescence polarization [23] assay or surface plasmon resonance (SPR) experiments [38] in the past. Among the five receptor histidine kinases described in Arabidopsis AHK1 takes a special place Despite the large number of histidine kinases participating in two-component signal transfer processes and their counterparts in plants involved in multi-step phosphorelay signaling, our information on thermodynamics and kinetics that determine the interaction of these sensor kinases with their downstream histidine phosphotransfer proteins as well as the conformational changes triggering signal transfer activity in the phoshorelay is still sparse. This is partly due to the fact that most of these sensor kinases are integral membrane proteins which are hardly accessible to biochemical characterization. Thus, not surprisingly, most of the published interaction studies are based on yeast-two-hybrid analysis allowing qualitative assessments on the interaction, although two systems have been addressed in a quantitative way in biophysical studies by ﬂuorescence polarization [23] assay or surface plasmon resonance (SPR) experiments [38] in the past. Among the five receptor histidine kinases described in Arabidopsis, AHK1 takes a special place for its potential involvement in osmoregulation and its role as a positive regulator in drought response [39], although the ligand of this receptor has remained unknown so far. In this study, to the best of our knowledge, AHK1 has been successfully produced heterologously in the enterobacterium E. coli in a functional state for the first time. Successful expression in a bacterial host turned out to be critically dependent on the presence of rare tRNAs. each concentration. 2.7. AHK1 Acts as Phospho-His and/or Phospho-Asp Phosphatase In Vitro Thereafter, the proteins were separated by SDS-PAGE and the gel exposed to a phospho-imaging plate overnight. (b) Coomassie-stained SDS-PAGE gel showing the presence of the recombinant proteins in the reactions indicated in (a). M: protein size marker. Figure 7. AHK1 functions as phospho-His and/or phospho-Asp phosphatase in vitro. (a) Autoradiogram of the phosphorylation experiments performed with (His)6-tagged AHK1 (AHK1), (His)6-tagged AHK5output (AHK5output) and GST-tagged AHP2 (AHP2) in the presence of γ-33P-ATP. Lane 1: AHK5output; lane 2: AHK5output and AHP2; lane 3: AHK1, AHK5output; lane 4: AHK1, AHK5output, AHP2; lane 5: AHP2; lane 6: AHK1, AHP2. Phosphorylation reactions were run at room temperature for 20 min. Thereafter, the proteins were separated by SDS-PAGE and the gel exposed to a phospho-imaging plate overnight. (b) Coomassie-stained SDS-PAGE gel showing the presence of the recombinant proteins in the reactions indicated in (a). M: protein size marker. Figure 7. AHK1 functions as phospho-His and/or phospho-Asp phosphatase in vitro. (a) Figure 7. AHK1 functions as phospho-His and/or phospho-Asp phosphatase in vitro. Figure 7. AHK1 functions as phospho-His and/or phospho-Asp phosphatase in vitro. (a) Autoradiogram of the phosphorylation experiments performed with (His)6-tagged AHK1 (AHK1), (His)6-tagged AHK5output (AHK5output) and GST-tagged AHP2 (AHP2) in the presence of γ-33P- ATP. Lane 1: AHK5output; lane 2: AHK5output and AHP2; lane 3: AHK1, AHK5output; lane 4: AHK1, AHK5output, AHP2; lane 5: AHP2; lane 6: AHK1, AHP2. Phosphorylation reactions were run at room temperature for 20 min. Thereafter, the proteins were separated by SDS-PAGE and the gel exposed to a phospho-imaging plate overnight. (b) Coomassie-stained SDS-PAGE gel showing the presence of the recombinant proteins in the reactions indicated in (a). M: protein size marker. Figure 7. AHK1 functions as phospho-His and/or phospho-Asp phosphatase in vitro. (a) Autoradiogram of the phosphorylation experiments performed with (His)6-tagged AHK1 (AHK1), (His)6-tagged AHK5output (AHK5output) and GST-tagged AHP2 (AHP2) in the presence of γ-33P-ATP. Lane 1: AHK5output; lane 2: AHK5output and AHP2; lane 3: AHK1, AHK5output; lane 4: AHK1, AHK5output, AHP2; lane 5: AHP2; lane 6: AHK1, AHP2. Phosphorylation reactions were run at room temperature for 20 min. Thereafter, the proteins were separated by SDS-PAGE and the gel exposed to a phospho-imaging plate overnight. (b) Coomassie-stained SDS-PAGE gel showing the presence of the recombinant proteins in the reactions indicated in (a). M: protein size marker. 3. Discussion 3. Discussion Furthermore, expression at a low temperature (16 °C), short expression time (5 h) and the addition of 2% (v/v) of ethanol were proven to be crucial Among the ﬁve receptor histidine kinases described in Arabidopsis, AHK1 takes a special place for its potential involvement in osmoregulation and its role as a positive regulator in drought response [39], although the ligand of this receptor has remained unknown so far. In this study, to the best of our knowledge, AHK1 has been successfully produced heterologously in the enterobacterium E. coli in a functional state for the ﬁrst time. Successful expression in a bacterial host turned out to be critically dependent on the presence of rare tRNAs. Furthermore, expression at a low temperature (16 ◦C), 10 of 17 Plants 2020, 9, 304 short expression time (5 h) and the addition of 2% (v/v) of ethanol were proven to be crucial for a successful expression of the full-length integral membrane protein. The critical importance of these parameters for a successful recombinant production of the sensor kinase probably lies in the fact that higher expression level will result in intracellular protein aggregation, non-functional protein, or negative interference with bacterial TCSs. Hence, in order to obtain the recombinant sensor kinase in a functional state for further physiological studies, strict monitoring of the heterologous expression is crucial. Solubilization and puriﬁcation of AHK1 from the bacterial membranes was possible with the lipid-like detergent FosCholine-16 at high protein purity and yield leaving only minor background contaminants in the sample puriﬁed by single-step aﬃnity chromatography. In order to conﬁrm the high purity and monodispersity of the AHK1 produced, the solubilized and puriﬁed membrane protein was subjected to gel ﬁltration. The resulting SEC proﬁle conﬁrms that only minor contaminants were present, which due to fact that they elute at a lower elution volume, might represent aggregates, larger oligomers of solubilized AHK1, or larger micelle-protein complexes. The apparent molecular weight of 111 kDa, calculated for the protein eluting at the main peak is somewhat lower than the sequence-based molecular mass expected for the full-length AHK1 sensor kinase. The deviation from the theoretical molecular mass might be related to detergent eﬀects on the column matrix or a non-globular fold of the puriﬁed membrane receptor. Functional folding of recombinant AHK1 was veriﬁed by CD spectroscopy and by its ability to interact in vitro with the downstream phosphorelay element AHP2 [36]. 3. Discussion 3. Discussion Still, the apparent size observed for the puriﬁed recombinant AHK1 suggests that the protein elutes in a monomeric state. At ﬁrst glance, this result appeared to be in disagreement with the dimeric or higher oligomeric states observed for other plant histidine kinases such as the receptors of the plant hormones ethylene and cytokinin [40]. Nonetheless, monomeric states of sensor histidine kinases have been also observed in solution and crystals in the past, as in the case of chemoreceptor Tlp1 from Campylobacter jejuni [41]. Remarkably, despite the apparent monomeric state under size exclusion chromatography conditions, the functionality of AHK1 in terms of potential to form homodimers could be shown in MST. The homomeric aﬃnities of the AHK1 osmosensor strongly resemble the aﬃnities observed for other sensor histidine kinases like AtETR1 and AtETR2 [7]. The same experimental setup and method, under which a dissociation constant of 208 nM was observed in this study for AHK1, showed values of 326 nM and 96 nM for AtETR1 and AtETR2 respectively [7]. The in vitro MST results of AHK1 homomerization were conﬁrmed by two independent in vivo approaches, namely yeast mbSUS and in planta FRET-FLIM. The latter technique, in combination with confocal microscopy, demonstrates that AHK1 homomerization takes places in the plasma membrane of plant cells. MST additionally revealed a high aﬃnity interaction of AHK1 with AHP2. The dissociation constant of 300 nM for the AHK1-AHP2 interaction is in the same Kd range as observed for the interaction of other histidine kinases with their cognate histidine-containing phosphotransfer proteins (HPs). Interaction of the ethylene receptor ETR1 and the histidine phosphotransfer protein AHP1 is characterized by a Kd of 1.5 µM [23], aﬃnities for sensor kinase AHK5 with its three interactors AHP1, AHP2, and AHP3 are in the range of 2.7 to 4.4 µM [38]. Still, the aﬃnity of AHK1 to AHP2 is the tightest interaction observed for this type of signaling system known to date indicating strong interaction and high stability of the related phosphorelay complex. The puriﬁed AHK1 protein also enabled us to perform in vitro auto-phosphorylation assays. However, no auto-phosphorylation was observed which we attribute to the missing AHK1 ligand in the phosphorylation reactions. However, this did not exclude the possibility that ligand-less AHK1 may act as P-His and/or P-Asp phosphatase, as it was, for instance, described for the cytokinin receptor AHK4 in the absence of the hormonal ligand [37]. 4.1. Cloning AHK1 was cloned into the pET16b-H10-TEV vector for expression using Gibson assembly [42]. The cDNA coding for AHP2 was cloned into a pGEX-6P-1 vector to generate a GST-fusion construct. The cDNA encoding AHK5output was cloned into the pET-DEST42 vector to generate a C-terminal (His)6 fusion construct. ( ) The following oligonucleotides were used in the process: AHK5output-F 5‘-GGGGACAAGTTTGTACAAAAAAGCAGGCT gtatggataacgctgtgagaaaggc-3‘ AHK5output-R 5′-GGGGACGACTTTGTACAAGAAAGCTGGGT tgtgcaaatactgttgcaaacactc-3′ ColE1-F 5′-ggagcgaacgacctacaccgaactgagatacctacagcg-3‘ gib_pET16b-TEV-rev 5′-ATGTCCCTGAAAATACAG-3‘ gib-pET16b-AHK1-for 5′-ctgtattttcagggacatATGCGAGGAGATAGCTTC-3‘ gib-pET16b-AHK1-rev 5′-gttagcagccggatccttaAGCGGACAATGAAGTTTG-3‘ gib_pET16b-TEV-for 5′-TAAGGATCCGGCTGCTAAC-3‘ ColE1-R 5′-cgctgtaggtatctcagttcggtgtaggtcgttcgctcc -3‘ 3. Discussion 3. Discussion To test this possibility, we successfully established an in vitro phosphorelay system consisting of the active output domain of soluble AHK5 (AHK5output) and AHP2. AHK5output was not only able to auto-phosphorylate but also to transfer phosphoryl residues onto AHP2. Notably, AHP2 showed background labelling in the absence of AHK5output, which we attribute to the activity of an E. coli His kinase contaminating the AHP2 sample. However, in the presence of 11 of 17 Plants 2020, 9, 304 AHK1, the 33P-auto-labelling of AHK5output and 33P-labelling of AHP2 were no longer detectable. This observation can be explainable by an AHK1-intrinsic phospho-His and/or phospho-Asp phosphatase activity, which, on the one hand, relays the phosphoryl residues directly from AHK5output, and on the other hand, is also able to relay phosphoryl residues from phosphorylated AHP2 on itself. Finally, AHK1 must lose its phosphoryl residues rapidly with a short halftime, eventually emptying the γ-33P-ATP pool in the reaction mixture. Interestingly, we were never able to observe a heteromer formation of full length AHK5 and AHK5output with AHK1 via mbSUS and FRET-FLIM. However, the association of kinases and phosphatases with their target substrates can be very transient, impeding the detection of interaction by these and other (e.g., biochemical) approaches. As AHK1 interacts with AHP2 strongly, it is very likely that AHK1 preferentially relays the phosphoryl residues from AHP2 on itself and not from AHK5output directly. However, we cannot entirely exclude the possibility that AHK1 also competes with AHK5output for the availability of γ-33P-ATP directly. With the puriﬁcation protocol of AHK1 presented in this work a tool is provided to gain further in-depth knowledge on phosphorelay histidine kinases and their HPs, their interactions and physiological relevance. Moreover, with the in vitro AHK1-(AHK5output)-AHP2 phosphorelay system it now seems possible to determine the molecular mechanism of AHK1 sensing and the identity of the signaling molecule triggering AHK1 activity in further biochemical studies. Naturally, structural studies on the isolated AHK1 or the AHK1-AHP2 complex are possible, too. Here, the high purity and monodispersity of AHK1 opens systematic crystallization studies of the receptor, hopefully resulting in the ﬁrst high-resolution structure of an integral membrane plant receptor histidine kinase. 4. Materials and Methods 4.1. Cloning 4.3. Puriﬁcation of Recombinant Proteins 4.3. Puriﬁcation of Recombinant Proteins During the whole puriﬁcation, the samples were kept on ice or at 4 ◦C. The cell pellet of the membrane-bound protein AHK1 was resuspended in 5 mL lysis buﬀer L (50 mM Tris, 300 mM NaCl, 10% (w/v) glycerol, 5 mM DTT, 0.002% (w/v) phenylmethylsulfonyl ﬂuoride (PMSF), DNaseI, pH 7.6) per g of cell pellet. In each 50 mL of lysis buﬀer one tablet of protease inhibitor (cOmplete ULTRA, EDTA-free, Roche, Basel, Switzerland) was added. The lysis was performed using a cell disruption system (Constant Systems) at 2.4 kbar. For ﬁrst removal of unwanted cell debris and inclusion bodies, the lysate was centrifuged at 14,000 × g for 30 min. To collect the membrane fractions, the supernatant was centrifuged for another 30 min at 40,000 × g. After resuspension, equal distribution, and centrifugation at 34,000 × g for 30 min, the membrane pellets were frozen in liquid nitrogen and stored at −80 ◦C. AHK1 was solubilized in 1% (w/v) FosCholine-16 in buﬀer A (50 mM Tris, 300 mM NaCl, 10% (w/v) glycerol, 2.5 mM DTT, 0.002% (w/v) PMSF, pH 7.6) for 1 h at 700 rpm. After centrifugation at 230,000 × g for 30 min, another 0.002% (w/v) PMSF were added to the supernatant, which was loaded onto a 5 mL Ni-NTA HisTrap FF column (GE Healthcare, Chicago, IL, USA) using an ÄKTAPrime Plus system (GE Healthcare). Prior to loading, the column was equilibrated with at least 10 bed volumes of buﬀer A with 0.015% (w/v) FosCholine-16. A washing step was performed with buﬀer A containing 50 mM imidazole and elution followed with buﬀer A at 100 mM imidazole. After pooling and concentrating the samples by centrifugation with a 50 kDa Amicon Ultra-15 tube (Merck Millipore, Darmstadt, Germany), the buﬀer was exchanged using a PD 10 or PD MiniTrap G-25 desalting column (GE Healthcare). The AHP2 cell pellets were resuspended with PBS buﬀer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) with 0.002% (w/v) PMSF, lysed in two cycles using a pre-cooled French pressure cell (Thermo, Waltham, MA, USA) with approximately 1 kbar (14,000 psi). After ultracentrifugation at 230,000 × g for 1 h, the supernatant was loaded on an equilibrated GSTrap HP 5 mL column (GE Healthcare Life Sciences) using an ÄKTAPrime Plus system. 4.2. Expression of Recombinant Proteins For the expression of AHK1 the E. coli strain BL21 (DE3), containing the pRARE plasmid (Novagen, Darmstadt, Germany), which encodes for rare tRNAs, was used. The overnight pre-culture was inoculated with a colony picked from a 1.5% agar plate and grown in 2YT medium (5 g/L NaCl, 10 g/L yeast extract, and 16 g/L peptone) containing ampicillin (100 µg/mL) and chloramphenicol (34 µg/mL) as selection markers, at 37 ◦C and 180 rpm. The expression culture was inoculated to an optical density (OD) of 0.15 measured at 600 nm in Terriﬁc broth (TB) medium (5 g/L glycerol, 24 g/L yeast extract, 12 g/L peptone, 1.82 g/L KH2PO4, 19.76 g/L K2HPO4, and 100 µg/mL ampicillin) containing 2% (v/v) 12 of 17 Plants 2020, 9, 304 ethanol. At an OD600 of 0.3–0.35 at 37 ◦C, cultures were cooled down to 30 ◦C until they reach an optical density of 0.4–0.45, where they were cooled down to their ﬁnal expression temperature of 16 ◦C. The induction with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was performed at an OD600 of 0.6. Following an incubation at 16 ◦C and 180 rpm, the cells were harvested after 5 h at 7500 × g and 4 ◦C for 15 min. The cell pellets were then frozen in liquid nitrogen and stored at −20 ◦C. In contrast to AHK1, AHP2 was transformed into BL21 (DE3) Gold cells, expressed in 2YT medium containing ampicillin (100 µg/mL) and 2% (v/v) ethanol. After reaching an OD600 of 0.4–0.5 the cultures were cooled down to 16 ◦C and induced at an OD600 of 0.8 with 0.1 mM IPTG. The cells were grown overnight and harvested as described above. g For the expression of AHK5output the E. coli strain BL21 (DE3) was grown using Terriﬁc broth (TB) medium. The LB overnight culture was diluted with TB medium to an OD600nm of approximately 0.4. The cells were further grown for approximately 4 h at 37 ◦C until they reached the logarithmic phase at an OD600nm of 0.6. Protein expression was induced by addition of 0.5 mM IPTG and cells were incubated for 20 h at 20 ◦C and harvested as described above. Expression success was analyzed by SDS-PAGE and semidry Western blotting and electrogenerated chemiluminescence (ECL) detection after immunostaining with the indicated antibodies. 4.3. Puriﬁcation of Recombinant Proteins 4.5. Circular Dichroism Spectroscopy For CD spectroscopy the protein was rebuﬀered to the CD buﬀer (50 mM K2HPO4/KH2PO4, 0.015% (w/v) FosCholine-16, pH 7.6). Spectra were recorded with a protein concentration of 0.1 mg/mL in 200 µL in a JASCO J-810 CD spectropolarimeter at room temperature. In the wavelength range between 190 nm and 260 nm, 20 spectra were accumulated. Deconvolution was performed with the CDPRO suite [30]. SMP65 was used as a reference data set which included 65 membrane proteins. The SOPMA analysis was performed with the full-length sequence of H10-TEV-AHK1 [32]. 4.4. Size Exclusion Chromatography Gel ﬁltration was performed on a 10/300 Superose 6 Increase column by GE Healthcare Life Sciences using an ÄKTAPrime Plus system. At a ﬂow rate of 0.1 mL min-1, 100 µg of protein in a volume of 50 µL were applied to the column via a 100 µL loop. The system was equilibrated in buﬀer A with 0.015% (w/v) FosCholine-16 (see 4.3). Column calibration was performed as a four-point calibration with thyroglobuline (669 kDa, bovine thyroid), ferritin (440 kDa, horse spleen), aldolase (158 kDa, rabbit muscle), and ovalbumin (43 kDa, hen egg) according to the instructions from the Gel Filtration Calibration Kit HMW (GE Healthcare Life Sciences). 4.3. Puriﬁcation of Recombinant Proteins Whereas equilibration and washing were performed with at least ﬁve bed volumes with the PBS buﬀer, the protein was released by competitive binding with elution buﬀer E (50 mM Tris, 10 mM reduced glutathione, pH 8.0). Concentrating (10 kDa cut-oﬀ) and buﬀer exchange to storage buﬀer (50 mM Tris, 300 mM NaCl, pH 7.6) was done as described. For storage, aliquots containing 20% (w/v) glycerol were shock-frozen and kept at −80 ◦C. p The AHK5output-(His)6 expressing E. coli cells were harvested by centrifugation at 4000 g for 5 min and resuspended in 10 mL pre-cooled NPI-10 buﬀer (50 mM NaH2PO4·H2O, 300 mM NaCl, 10 mM imidazole, pH 8.0) containing a proteinase inhibitor cocktail (Bimake). Glass beads (diameter: 13 of 17 Plants 2020, 9, 304 0.25–0.5 mm) were added and the cells were lysed by vortexing at 4 ◦C for 10 min. The lysate was centrifuged at 4 ◦C and 15000 × g for 40 min and the supernatant subjected to native protein puriﬁcation using Ni-NTA Superﬂow Columns according to the manufacturer’s protocol (QIAGEN). Puriﬁcation was veriﬁed by SDS-PAGE and Western blotting. 4.6. Fluorescent Labeling of Puriﬁed Protein To enable the measurement of binding studies using microscale thermophoresis, AHK1 was labeled with the ﬂuorophore AlexaFluor488-NHS (Invitrogen, Thermo Fisher, Waltham, MA, USA). Therefore, the protein was incubated with 2.5 × the amount of the ﬂuorophore in a total volume of 500 µL labeling buﬀer (50 mM K2HPO4/KH2PO4, 300 mM NaCl, 0.015% (w/v) FosCholine-16, pH 7.6) for 30 min in the dark during gentle agitation. The following buﬀer change to MST buﬀer 1 (50 mM Tris, 300 mM NaCl, 0.015% (w/v) FosCholine-16, pH 7.6) was performed using a PD Mini desalting column. After measuring the protein concentration and labeling eﬃciency, 20% (w/v) glycerol was added and aliquots for the MST measurement were frozen in liquid nitrogen. 4.9. Analytical Methods Protein concentrations were determined by the bicinchoninic acid assay (Perbio Science, Bonn, Germany) for the AHK1 sensor kinase and by the Bio-Rad protein assay (Bio-Rad, München, Germany) for AHP2-GST using bovine serum albumin as a standard. Puriﬁcation of recombinant proteins was examined by SDS-PAGE as described by Laemmli [43] and Schägger and Jagow [44]. Proteins were separated on 10–12% polyacrylamide gels and visualized by colloidal Coomassie staining according to Kang et al. [45,46]. Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/3/304/s1, Figure S1. Expression of His-tagged AHK1 in BL21 (DE3) E. coli cells containing the pRARE plasmid under diﬀerent growth conditions. For analysis, the samples were separated on a 10% SDS-PAGE gel and identiﬁed after Western blotting with an anti-his-antibody; (a) The cells were grown in 2YT medium at 30 ◦C after induction with 0.5 mM IPTG. Samples were taken every hour and show a strong signal for possible degradation bands at around 35 kDa. (b) E. coli cells were grown in 2YT medium at 16 ◦C and an addition of 2% ethanol. Samples were taken every 2 h after induction with 0.5 mM IPTG and show only a faint band after 4 h. (c) The protein expression in TB medium at 16 ◦C and the additional 2% (v/v) ethanol was sampled after 0 h, 2 h, 4 h and 5 h following induction with 0.5 mM IPTG. Under these conditions the most prominent protein bands with the least degradation or incomplete translated proteins were detected; Figure S2. Detergent screening for solubilization of AHK1 in 100 µL. P resembles the pellet directly after resuspension in buﬀer A, which was subsequently incubated with the ×-fold critical micell concentration (cmc) of the stated detergent for 1 h at 4 ◦C and 700 rpm. Detergents were selected from the JBScreen Detergent set (Jena Bioscience). The pellet (P) and supernatant (SN) after centrifugation at 230,000 × g for 30 min were analyzed using SDS-PAGE and Western blotting; (a) The detergents C12E8, Cymal-6 and LMNG were used at a ﬁnal concentration of 7.5 × cmc, whereas OG was used in a lower concentration of 3 × cmc and Fos12 at 4.5 × cmc. Fos12 is the only detergent showing a band of AHK1 in the supernatant fraction. (b) Whereas HTG was tested with the 3 × cmc, the other detergents ANAPOE-X-114, LDAO and Fos16 were tested with the 7.5 × cmc. 4.7. Interaction Studies by MST, mbSUS, and FRET-FLIM Analyses To verify the functionality of the puriﬁed proteins, the binding of AHK1 to AHP2 was measured. Therefore, the Monolith NT 115 and standard capillaries (NanoTemper Technologies, München, Germany) were used. Furthermore, the MST power was set to 40% and the LED power to 30%. The binding study was performed in seven measurements, whereas the negative control with denatured protein in a duplicate. The labeled AHK1 was diluted to a ﬁnal measuring concentration of 40 nM using MST buﬀer 2 (50 mM HEPES, 300 mM NaCl, 0.015% (w/v) FosCholine-16.5% (w/v) glycerol, pH 7.6). The dilution series of AHP2 from 12.5 µM to 0.4 nM was done using MST buﬀer 3 (50 mM Tris, 300 mM NaCl, 5% (w/v) glycerol, pH 7.6). The labeled AHK1 was incubated with the non-labeled AHP2 in a ratio of 1:1 resulting in the prior concentrations. After an incubation of 5 min in the dark, the samples were loaded into the glass capillaries for the measurement. For the negative controls, a denaturing buﬀer D1 (4% (w/v) SDS, 80 mM DTT in MST buﬀer 2 containing only 0.0075% (w/v) FosCholine-16 in total) was added to the mixed proteins of interest. As evaluation strategy T-Jump was used. Dimerization of AHK1 was analyzed by MST measurements, using 40% MST power and 70% LED power. The labeled AHK1 was treated as described before. The interacting unlabeled AHK1 was diluted in a series from 12 µM to 0.4 nM in MST buﬀer 4 (50 mM Tris, 300 mM NaCl, 0.015% (w/v) FosCholine-16, 20% (w/v) glycerol, pH 7.6). After mixing the samples in a ratio of 1:1 and incubation in 14 of 17 Plants 2020, 9, 304 the dark for 5 min, they were loaded into the standard capillaries and measured. For the triplicate of the negative control, denaturing buﬀer D2 (4% (w/v) SDS, 80 mM DTT, 156 mM NaCl, 26 mM Tris, 0.015% (w/v) FosCholine-16, 12.5% (w/v) glycerol, pH 7.6) was added in a ratio of 1:1 to the prior mixed interacting proteins. For binding analysis, initial ﬂuorescence was used. Yeast mbSUS studies were performed as described before [33]. For confocal imaging and FRET-FLIM analyses, full-length AHK1 was expressed as C-terminal GFP and C-terminal mCherry fusion in Agrobacterium tumefaciens-mediated transiently-transformed Nicotiana benthamiana epidermal leaf cells according to [34]. Confocal imaging and ﬂuorescence life measurements were done as described before [34]. 4.8. In Vitro Phosphorylation and Phosphorelay Assays Approximately 3 µg of AHK1-(His)6, AHK5output-(His)6, and AHP2-GST were incubated at the indicated combinations (see Figure 7) with 1.1 µl γ-33P-ATP (3700 MBq/mL; Hartmann Analytics, Braunschweig, Germany) in a ﬁnal volume of 30.1 µl TEDG buﬀer (50 mM Tris, 0.5 mM EDTA, 2 mM DTT, 50 mM KCl, 5 mM MgCl2, 10% glycerol, pH 8.0) at room temperature for 30 min. The reaction was stopped by addition of 5 µl of SDS-PAGE sample buﬀer. The samples were analyzed by SDS-PAGE and the gel was subsequently imaged overnight using a radiosensitive phospho-imaging plate. Detection was carried out using the BAS cassette of the Fluorescence Laser Imaging Scanner FLA-3000/300G according to the manufacturer´s instructions (FUJIFILM). A Coomassie-stained SDS-PAGE gel loaded in parallel with the same samples but in the absence of γ-33P-ATP was used as a loading control. 4.7. Interaction Studies by MST, mbSUS, and FRET-FLIM Analyses The statistical analysis was carried out with JMP 14 software available at https://www.jmp.com. The homogeneity of variance was tested with a Brown–Forsythe test. A non-parametric Kruskal–Wallis test was then performed followed by a Dunn’s post hoc test. The boxplot representation was designed with http://shiny.chemgrid.org/boxplotr/. 4.9. Analytical Methods All authors have read and agreed to the published version of the manuscript. Funding: Research in Tübingen was funded by the Deutsche Forschungsgemeinschaft (DFG; SFB 1101, pro B05) to K.H. Research in Düsseldorf was funded by the DFG (project 267205415 and SFB 1208, project B06) to Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Wuichet, K.; Cantwell, B.J.; Zhulin, I.B. Evolution and phyletic distribution of two-component signal transduction systems. Curr. Opin. Microbiol. 2010, 13, 219–225. [CrossRef] [PubMed] 2. Grebe, T.W.; Stock, J.B. The histidine protein kinase superfamily. Adv. Microb. Physiol 1999, 41, 139–227. [CrossRef] [PubMed] 3. Urao, T.; Yamaguchi-Shinozaki, K.; Shinozaki, K. Plant histidine kinases: An emerging picture of two-component signal transduction in hormone and environmental responses. Sci STKE 2001, 2001, re18. [CrossRef] [PubMed] 4. Hwang, I.; Chen, H.C.; Sheen, J. Two-component signal transduction pathways in Arabidopsis. Plant Physiol. 2002, 129, 500–515. [CrossRef] [PubMed] 5. 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The pellet (P) and supernatant (SN) after centrifugation at 230,000 × g for 30 min were analyzed using SDS-PAGE and Western blotting; (a) The detergents Na-cholate and 15 of 17 15 of 17 Plants 2020, 9, 304 CHAPS were used with 2% (w/v), whereas Fos16, DDM and Tween-20 with 1% (w/v). Fos16 is the only detergent showing a band of AHK1 in the supernatant fraction. (b) Whilst Fos12 and LDAO were tested with 1% (w/v), Fos10 due to its higher cmc was analyzed at a ﬁnal concentration of 2% (w/v). Both FosCholines show a distinct band in the supernatant, speaking for its solubilization. LDAO also shows a more prominent band in the supernatant, but also one remaining in the pellet, suggesting still incomplete solubilization even at a higher concentration than before. Table S1. Analyzed detergents and their properties. Author Contributions: Conceptualization, G.G. and K.H.; methodology, A.H., M.B., T.D. and K.C.; validation, A.H., S.M., G.G, T.D., L.R. and K.H.; formal analysis, A.H., S.M., M.B., L.R. and K.H.; investigation, A.H., S.M., M.B., T.D. and K.C.; resources, G.G. and K.H.; data curation, A.H., K.C. and L.R.; writing A.H., S.M., G.G. and K.H.; visualization, S.M., A.M., T.D., K.C. and L.R.; supervision, G.G. and K.H.; project administration, G.G. and K.H.; funding acquisition, G.G. and K.H. All authors have read and agreed to the published version of the manuscript. 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https://openalex.org/W2279274877	https://eprints.soton.ac.uk/388560/1/S0007114515005073a.pdf	English	null	Composition differences between organic and conventional meat: a systematic literature review and meta-analysis	British journal of nutrition	2,016	cc-by	19,454	British Journal of Nutrition (2016), 115, 994–1011 doi:10.1017/S0007114515005073 British Journal of Nutrition (2016), 115, 994–1011 © The Authors 2016. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. itish Jou nal of Nut ition ( 0 6), 5, 99 0 © The Authors 2016. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. © The Authors 2016. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Composition differences between organic and conventional meat: a systematic literature review and meta-analysis Burdge13, Smaragda Sotiraki14, Alexandros Stefanakis14, Halil Yolcu1,15, Sokratis Stergiadis1,16, Eleni Chatzidimitriou1, Gillian Butler1, Gavin Stewart1 and Carlo Leifert1* 1Nafferton Ecological Farming Group (NEFG), School of Agriculture, Food and Rural Development, Newcastle University, Nafferton Farm, Stocksﬁeld, Northumberland NE43 7XD, UK 2School of Agriculture Food and Rural Development Human Nutrition Research Centre Newcastle University Agriculture ood and Rural Development, Human Nutrition Research Centre, Newcastle University, Agriculture ewcastle upon Tyne NE1 7RU, UK 2School of Agriculture, Food and Rural Development, Human Nutrition Research Centre, Newca Building, Kings Road, Newcastle upon Tyne NE1 7RU, UK 3School of Biology, Newcastle University, Ridley Building, Newcastle upon Tyne NE1 7RU, UK 4B b k C l i S i 90063 T R d E i OR 97828 USA 5Food and Agriculture Division – Grassland and Forage, Norwegian Institute of Bioeconomy Research (NIBIO), Gunnars veg 6, N-6630 Tingvoll, Norway 5Food and Agriculture Division – Grassland and Forage, Norwegian Institute of Bioeconomy Research (NIBIO), Gunnars veg 6, N-6630 Tingvoll, Norway 6 6Department of Dietetics, Faculty of Human Nutrition and Consumer Sciences, Warsaw University of Life Sciences, Nowoursynowska 159c, 02-776 Warsaw, Poland 6Department of Dietetics, Faculty of Human Nutrition and Consumer Sciences, Warsaw University of Life Sciences, Nowoursynowska 159c, 02-776 Warsaw, Poland 7Department of Functional and Organic Food and Commodities, Faculty of Human Nutrition and Consumer Sciences, Warsaw University of Life Sciences, Nowoursynowska 159c, 02-776 Warsaw, Poland 7Department of Functional and Organic Food and Commodities, Faculty of Human Nutrition and Consumer Sciences, Warsaw University of Life Sciences, Nowoursynowska 159c, 02-776 Warsaw, Poland 8Department of Animal Physiology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland 9Department of Animal Medicine, Production and Health, University of Padua, Viale dell’ Università 19, 35020 Legnaro, Italy 10Department of Food Science – Food Chemistry & Technology, Aarhus University, Blichers Allé 20, Building F20/8845, 8830 l k p f y gy, y f gy, y f , , 9 , 9Department of Animal Medicine, Production and Health, University of Padua, Viale dell’ Università 19, 35020 Legnaro, Italy 10Department of Food Science – Food Chemistry & Technology, Aarhus University, Blichers Allé 20, Building F20/8845, 8830 Tjele, Denmark p f , , y f , , g , y 10Department of Food Science – Food Chemistry & Technology, Aarhus University, Blichers Allé 20, Building F20/8845, 8830 Tjele, Denmark 12Institute of Genetics and Animal Breeding, Polish Academy of Science, Jastrzębiec, Postępu 36, 05-552 Magdalenka, Poland 12Institute of Genetics and Animal Breeding, Polish Academy of Science, Jastrzębiec, Postępu 36, 05-552 Magdalenka, Poland 13Human Development and Health Academic Unit, Faculty of Medicine, University of Southampton, Southampton SO16 6YD UK 12Institute of Genetics and Animal Breeding, Polish Academy of Science, Jastrzębiec, Postępu 36, 05-552 Magdalenka, Poland 13Human Development and Health Academic Unit, Faculty of Medicine, University of Southampton, Southampton 13Human Development and Health Academic Unit, Faculty of Medicine, University of Southampton, Southampton SO16 6YD, UK 14National Agricultural Research Foundation (NAGREF), Veterinary Research Institute of Thessaloniki, 57001 Thermi, Thessaloniki, Greece cational Training School, Gumushane University, 29600 Kelkit, Gumushane, Turkey 15Kelkit Aydin Vocational Training School, Gumushane University, 29600 Kelkit, Gumushane, Turkey 16Food Production and Quality Division, School of Agriculture, Policy and Development, Centre for Dairy Research, University of Reading, PO Box 237, Earley Gate, Reading RG6 6AR, UK of Reading, PO Box 237, Earley Gate, Reading RG6 6AR, UK (Submitted 12 January 2015 – Final revision received 13 November 2015 – Accepted 18 November 2015) (Submitted 12 January 2015 – Final revision received 13 November 2015 – Accepted 18 November 2015) Abbreviations: ALA, α-linolenic acid; DMI, DM intake; DPA, docosapentaenoic acid; EU, European Union; FA, fatty acids; LA, linoleic acid; MPD, mean percentage difference; SMD, standardised mean difference; UM, unweighted meta-analysis; VLC, very long-chain FA; WM, weighted meta-analysis. * C di h P f C L if f 44 1661 831 006 il l l if @ l k * Corresponding author: Professor C. Leifert, fax +44 1661 831 006, email carlo.leifert@newcastle.ac.uk Composition differences between organic and conventional meat: a systematic literature review and meta-analysis Dominika Średnicka-Tober1,7, Marcin Barański1, Chris Seal2, Roy Sanderson3, Charles Benbrook4, Håvard Steinshamn5, Joanna Gromadzka-Ostrowska6, Ewa Rembiałkowska7, Krystyna Skwarło-Sońta8, Mick Eyre1, Giulio Cozzi9, Mette Krogh Larsen10, Teresa Jordon1, Urs Niggli11, Tomasz Sakowski12, Philip C. Calder13, Graham C. Abbreviations: ALA, α-linolenic acid; DMI, DM intake; DPA, docosapentaenoic acid; EU, European Union; FA, fatty a percentage difference; SMD, standardised mean difference; UM, unweighted meta-analysis; VLC, very long-chain FA; WM Key words: Organic foods: Animal products: Meat: Iron: Meat fat composition: n-3 PUFA: n-6 PUFA Where possible, additional meta-analyses were carried out; these included some individual FA, the thrombogenicity and atherogenicity indices (which might be used to compare the overall CVD risk associated with different meat FA proﬁles(19,26,27)) and a range of other composition parameters (e.g. total protein, minerals, toxic metals), but for many of these only a small number of data pairs (n 3–5) were available. We were therefore unable to carry out meaningful meta-analyses for nutritionally relevant minerals, antioxidants and vitamins found in meat. The SFA in meat, in particular lauric (12 : 0), myristic (14 : 0) and palmitic (16 : 0) acids, are widely considered to have negative effects on human health, as they are linked to an increased risk of CVD in humans(10), although this is not universally accepted(11–13). In contrast, a range of PUFA found in meat are thought to reduce the risk of CVD(14). This includes linoleic acid (LA; the main n-6 PUFA found in meat), α-linolenic acid (ALA, the main n-3 PUFA found in meat) and, in particular, the very long-chain (VLC, ≥C20) n-3 PUFA EPA, docosapentaenoic acid (DPA) and DHA. Both LA and ALA are known to reduce LDL production and to enhance its clearance(14), whereas VLC n-3 PUFA are also shown to reduce arrhythmias, blood pressure, platelet sensitivity, inﬂammation and serum TAG concentrations(15,16). There is also evidence of other health beneﬁts from increasing VLC n-3 PUFA (especially DHA) intakes, including improved fetal brain development, delayed decline in cognitive function in elderly men and reduced risk of dementia (especially Alzheimer’s disease)(17). Previous meta-analyses of composition differences between organic and conventional foods (i.e. for crops, and milk and dairy products) have used variable inclusion criteria, data extraction and synthesis methods(4,5,8,9,25). In the present study, sensitivity analyses designed to identify the effect of using different inclusion criteria, extraction and analysis methods were therefore performed to assess the consistency of ﬁndings. Results are discussed in the context of known information on (1) the effects of livestock management practices (especially feeding regimens) and breed choice on meat composition and (2) potential health impacts of composition differences between organic and non-organic meat. Although LA may reduce CVD risk, intakes associated with typical Western diets are thought to be too high(18). This is mainly because LA is the precursor of the pro-inﬂammatory n-6 PUFA arachidonic acid (AA). In contrast, n-3 FA are considered to have an anti-inﬂammatory effect(15,16,19,20). Key words: Organic foods: Animal products: Meat: Iron: Meat fat composition: n-3 PUFA: n-6 PUFA children(23). LA may also reduce the rate of conversion of ALA to VLC n-3 PUFA in humans, because ALA and LA compete for Δ6 desaturase enzyme activity(24). The demand for organic meat products has increased steadily over the last 20 years(1). A major driver for this increase has been consumer perception that organic livestock products typically contain higher concentrations of nutritionally desirable compounds, therefore making them ‘healthier’(2,3). However, there is still considerable scientiﬁc uncertainty over whether, and to what extent, organic production standards result in sig- niﬁcant and nutritionally relevant changes in food quality(3–6). Systematic literature reviews and meta-analyses of compara- tive composition data for (1) crops, (2) milk and (3) milk, eggs and meat together have been published(4,5,8,9,25), but there are Systematic literature reviews and meta-analyses of compara- tive composition data for (1) crops, (2) milk and (3) milk, eggs and meat together have been published(4,5,8,9,25), but there are no published meta-analyses in which the composition of organic and non-organic meat is compared. In this study, we report the results of a systematic review of the literature published before March 2014 and meta-analyses of data designed to quantify nutritionally relevant composition para- meters in organic and conventional meat products. no published meta-analyses in which the composition of organic and non-organic meat is compared. In this study, we report the results of a systematic review of the literature published before March 2014 and meta-analyses of data designed to quantify nutritionally relevant composition para- meters in organic and conventional meat products. In Western European diets, meat is an important source of protein, essential fatty acids (FA), minerals (e.g. Fe, Zn, Se, Cu) and vitamins (e.g. vitamin A, vitamin B1, B6 and B12, riboﬂavin, folate, niacin, pantothenic acid)(7). Over the last 20 years, an increasing number of scientiﬁc studies have compared con- centrations of nutritionally relevant compounds in meat from organic and conventional livestock production systems. Most comparative studies have reported data on meat fat composi- tion, whereas there are limited published data on mineral and vitamin concentrations(4,8,9). For meta-analyses and interpreting the overall strength of evidence, total PUFA and n-3 PUFA concentrations were con- sidered the primary outcome, because they are considered to be most closely linked to potential human health outcomes (see above). A range of other nutritionally relevant meat fat parameters were considered secondary outcomes. Key words: Organic foods: Animal products: Meat: Iron: Meat fat composition: n-3 PUFA: n-6 PUFA In addition, high dietary n-6 PUFA intakes have been linked to an increased risk of other chronic diseases including certain cancers, inﬂamma- tory, autoimmune and CVD(16,21) as well as shown to stimulate adipogenesis (and thereby the risk of obesity) to a greater extent compared with n-3 FA(22). Excessive LA intakes during pregnancy and in the ﬁrst few years of life have been linked to a range of neurodevelopmental deﬁcits and abnormalities in Abstract Demand for organic meat is partially driven by consumer perceptions that organic foods are more nutritious than non-organic foods. However, there have been no systematic reviews comparing speciﬁcally the nutrient content of organic and conventionally produced meat. In this study, we report results of a meta-analysis based on sixty-seven published studies comparing the composition of organic and non-organic meat products. For many nutritionally relevant compounds (e.g. minerals, antioxidants and most individual fatty acids (FA)), the evidence base was too weak for meaningful meta-analyses. However, signiﬁcant differences in FA proﬁles were detected when data from all livestock species were pooled. Concentrations of SFA and MUFA were similar or slightly lower, respectively, in organic compared with conventional meat. Larger differences were detected for total PUFA and n-3 PUFA, which were an estimated 23 (95 % CI 11, 35) % and 47 (95 % CI 10, 84) % higher in organic meat, respectively. However, for these and many other composition parameters, for which meta-analyses found signiﬁcant Composition of organic meat products 995 differences, heterogeneity was high, and this could be explained by differences between animal species/meat types. Evidence from controlled experimental studies indicates that the high grazing/forage-based diets prescribed under organic farming standards may be the main reason for differences in FA proﬁles. Further studies are required to enable meta-analyses for a wider range of parameters (e.g. antioxidant, vitamin and mineral concentrations) and to improve both precision and consistency of results for FA proﬁles for all species. Potential impacts of composition differences on human health are discussed. Data acquisition: literature search strategy and inclusion criteria Data acquisition: literature search strategy and inclusion criteria The systematic review methods are described in a previously published meta-analysis by Barański et al.(25) focused on identifying composition differences between organic and con- ventional crops. The methods were based on a more detailed D. Średnicka-Tober et al. 996 publications were found by studying lists of references or directly contacting authors of published papers and reviews identiﬁed in the initial literature search (Fig. 1). protocol for systematic reviews of composition differences published by Brandt et al.(28). However, the protocols used in this study and by Barański et al.(25) differed from the detailed protocol published by Brandt et al.(28), notably in the emphasis on weighted meta-analysis (WM) rather than unweighted meta-analysis (UM), which had previously been recommended by Brandt et al.(5,28) and Dangour et al.(4). The abstracts of all publications were then examined to determine whether they contained original data obtained by comparing composition parameters in organic and conven- tional beef, lamb or goat meat, pork, poultry or rabbit meat. This identiﬁed seventy-ﬁve suitable publications; of these, eight were subsequently rejected, because they did not report suitable data sets or contained the same data as other papers. Relevant publications were identiﬁed through an initial search of the literature in the Web of Knowledge, Scopus, Ovid and EBSCO, Elton B. Stephens Company (EBSCO) databases using the following search terms: ‘organic* or ecologic* or biodynamic’, ‘conventional or integrated’ and ‘livestock or meat or pork or beef or poultry or chicken or turkey or lamb or goat or rabbit’ (Fig. 1). Papers in all languages, published in peer-reviewed and non- peer-reviewed journals, and reporting data on both desirable and undesirable compositional parameters, were considered relevant for inclusion in the meta-analyses. The search was restricted to the period between 1992 (the year when legally binding organic farming regulations were ﬁrst introduced in the European Union (EU)) and the end of the project in March 2014 and provided 707 references. An additional seventeen Relevant publications were identiﬁed through an initial search of the literature in the Web of Knowledge, Scopus, Ovid and EBSCO, Elton B. Stephens Company (EBSCO) databases using the following search terms: ‘organic* or ecologic* or biodynamic’, ‘conventional or integrated’ and ‘livestock or meat or pork or beef or poultry or chicken or turkey or lamb or goat or rabbit’ (Fig. 1). Data acquisition: literature search strategy and inclusion criteria Data sets were deemed suitable if data for at least one meat composition parameter were reported. As a result, sixty-seven publications (sixty-three peer-reviewed) were selected for data extraction (sixteen on beef, sixteen on lamb and goat meat, fourteen on pork, seventeen on chicken meat, three on rabbit meat and one on non-speciﬁed meats). Papers in all languages, published in peer-reviewed and non- peer-reviewed journals, and reporting data on both desirable and undesirable compositional parameters, were considered relevant for inclusion in the meta-analyses. The search was restricted to the period between 1992 (the year when legally binding organic farming regulations were ﬁrst introduced in the European Union (EU)) and the end of the project in March 2014 and provided 707 references. An additional seventeen Data from forty-eight publications (forty-seven peer- reviewed) fulﬁlled the criteria for inclusion in the random effects WM and UM. The additional nineteen publications (sixteen peer-reviewed) fulﬁlled the criteria for inclusion in the UM only. Initial search* (n 724) Web of Knowledge database (years 1992–2014) Scopus database (years 1992–2014) Ovid database (years 1992–2014) EBSCO database (years 1992–2014) Lists of references and direct contact with the authors (years 1977–2014) (n 611) (n 19) (n 73) (n 4) (n 17) Excluded (n 649) Publications did not present data on meat (appropriate population) or comparison between organic and conventional system (appropriate comparators) Suitable publications reviewed† (n 75) Excluded (n 8) Contained the same data as other studies Report not suitable data type Replication number was lower than 3 (n 3) (n 1) (n 4) Paper did meet inclusion criteria (n 67) Peer-reviewed journals Non-peer-reviewed journals (n 63) (n 4) Weighted meta-analysis Papers did provide information about number of replicates and SD or SE (n 48) CF (n 5) BS (n 12) EX (n 31) Unweighted meta-analysis Not all papers did provide information about number of replicates and SD or SE (n 67) CF (n 5) BS (n 20) EX (n 42) Fig. 1. Summary of the search and selection protocols used to identify papers included in the meta-analyses. EBSCO, Elton B. Stephens Company; CF, comparison of matched farms; BS, basket studies; EX, controlled experiments. * Review carried out by one reviewer. † Data extraction carried out by two reviewers. Suitable publications reviewed† (n 75) Fig. 1. Summary of the search and selection protocols used to identify papers included in the meta-analyses. EBSCO, Elton B. Data acquisition: literature search strategy and inclusion criteria Stephens Company; CF, comparison of matched farms; BS, basket studies; EX, controlled experiments. * Review carried out by one reviewer. † Data extraction carried out by two reviewers. Composition of organic meat products 997 (3) multiple time points within the same sampling year were averaged before use in the meta-analysis. This represents a signiﬁcantly greater evidence base compared with a previous systematic review of comparative studies by Dangour et al.(4) that (1) was based on eleven publications reporting meat composition data, (2) pooled meat, egg and milk/dairy product composition data and (3) used unweighted, under-powered analytical methods only. All pub- lications included in this previous review were also used in the random effects WM reported in this study. Two independent reviewers assessed publications for eligibility and extracted data. Discrepancies were detected for approximately 4 % of the data, and in these cases extraction was repeated following discussion. Study characteristics, summaries of methods used for sensi- tivity analyses and ancillary information are given in the online Supplementary Table S2–S7. They include information on (1) the number of papers from different countries and pub- lication years used in the meta-analyses (see online Supple- mentary Fig. S1 and S2), (2) study type, location, meat product, animal group and information regarding FA analysis methods used in different studies (online Supplementary Table S2), (3) production system information for studies with more than two systems (online Supplementary Table S3), (4) the type of information extracted from papers (online Supplementary Table S4), (5) data handling and inclusion criteria and meta- analysis methods used in sensitivity analyses (online Supple- mentary Table S5), (6) the list of composition parameters included in the meta-analyses (online Supplementary Table S6) and (7) the list of composition parameters for which meta- analyses were not possible (n < 3) (online Supplementary Table S7). A Preferred Reporting Items for Systematic Reviews and Meta-Analyses ﬂow diagram illustrates the search and study inclusion strategies (Fig. 1). Eligibility assessment was performed by two independent reviewers, with discrepancies resolved by consensus and reference to a third reviewer when necessary. Data extraction Data were extracted from three types of studies: (1) compar- isons of matched farms (CF), farm surveys in which meat was obtained from organic and conventional farms in the same country or region; (2) basket studies (BS), retail product surveys in which organic and conventional meats were obtained from retail outlets; and (3) controlled experiments (EX) in which meat was obtained from experimental animals reared according to organic or conventional farming standards/protocols. Data from the three study types were deemed relevant for meta- analysis if the authors stated that (1) organic farms included in farm surveys were using organic farming methods, (2) organic products collected in retail surveys were labelled as organic and (3) animals from organically reared herds used in controlled experiments were managed according to organic farming standards, even if animals and land used for ‘organic treatments’ in experiments were not organically certiﬁed. The online Supplementary Table S8 summarises the basic statistics on the number of studies, individual comparisons, organic and conventional samples sizes and comparisons showing statistically or numerically higher concentrations in organic or conventional meat for the composition parameters included in Fig. 2–4. Meta-analyses Several studies compared more than one organic or conven- tional system or treatment – for example, additional conventional systems were described as ‘intensive’ or ‘free range’. In such cases, a pragmatic choice was made to compare the organic with the standard conventional (non-organic) comparator. Standard systems were identiﬁed as closest to the typical, contemporary organic/conventional farming system, as recommended by Brandt et al.(5). Full references of the publications and summary descriptions of studies included in the meta-analyses are given in the online Supplementary Tables S1–S3. In total, six analyses were undertaken (online Supplementary Table S5). The standard WM and UM sensitivity analysis 1 compared data only from pragmatically chosen standard organic and conventional systems. Fig. 2–4 show the pooled effects obtained using standard random-effects meta-analysis weighted by inverse variance and a common random-effects variance component and UM of differences in means. The standard WM protocol is the primary analysis, but it is useful to augment the results with UM (particularly to explore the impact of including data from the studies that do not report measures of variance, and thus a wider range of studies). Information and data were extracted from all selected publications and compiled in a Microsoft Access database. The database is freely available on the Newcastle University website (http://research.ncl.ac.uk/nefg/QOF) for use and scrutiny by others. A list of the information extracted from publications and recorded in the database is given in the online Supplementary Table S4. Four additional sensitivity analyses were carried out. Two analyses (sensitivity analysis 2 and 3) were designed to identify whether exclusion of data for comparisons with non-standard organic or conventional systems would affect the results of the meta-analyses; in these analyses, comparative data for all organic and conventional production systems reported by authors were included (see online Supplementary Table S3). In sensitivity analysis 4, we explored the effect of excluding the 20 % of studies with the least precise treatment effects from the WM. Data reported as numerical values in the text or tables were copied directly into the database. Results only published in graphical form were enlarged, printed, measured (using a ruler) and then entered into the database as previously described(5). Data reported in the same publication for different animal species, products, study types, countries and outcomes were treated as independent effects. Meta-analyses n, number of data points included in meta-analyses; VLC n-3 PUFA, very long-chain n-3 PUFA; DPA, docosapentaenoic acid; OA, oleic acid; ALA, α-linolenic acid; LA, linoleic acid; AA, arachidonic acid; SMD, standardised mean difference; ○, MPD calculated using data included in standard unweighted meta-analyses; ▷, MPD calculated using data include in standard weighted meta-analysis; ◆, SMD with 95 % CI represented by horizontal bars. Fig. 2. Results of the standard weighted meta-analysis and sensitivity analysis 1 for fat composition of meat (data for all animal groups included in the same analysis). * Numerical values for mean percentage difference (MPD) and 95 % CI are given in the online Supplementary Table S9. † Ln ratio = ln (ORG/CONV × 100 %). ‡ P value <0·05 indicates a significant difference between organic samples (ORG) and conventional samples (CONV). § Heterogeneity and the I 2 statistic. \|\| Outlying data points (where the MPD between ORG and CONV was more than fifty times greater than the mean value including the outliers) were removed. ¶ Calculated based on published fatty acids (FA) composition data. n, number of data points included in meta-analyses; VLC n-3 PUFA, very long-chain n-3 PUFA; DPA, docosapentaenoic acid; OA, oleic acid; ALA, α-linolenic acid; LA, linoleic acid; AA, arachidonic acid; SMD, standardised mean difference; ○, MPD calculated using data included in standard unweighted meta-analyses; ▷, MPD calculated using data include in standard weighted meta-analysis; ◆, SMD with 95 % CI represented by horizontal bars. A positive SMD value indicates that mean concentrations of the observed compound were greater in the organic meat samples, whereas a negative SMD indicates that mean con- centrations were higher in conventional (non-organic) samples. The statistical signiﬁcance of a reported effect size (i.e. SMDtot) and CI were estimated based on standard methods(36) using ‘metafor’(32). The inﬂuence of potential moderators, particularly (1) meat type (beef, lamb and goat, pork, rabbit or chicken meat) and (2) study type (CF, EX, BS), were additionally tested using mixed-effect models(37) and subgroup analyses (Fig. 3 and 4, and online Supplementary Fig. S3–S5). GC-based protocols and described methods in sufﬁcient detail. Seven studies may be classiﬁed as being of lower quality, which included two studies that used an near IR-spectroscopy method calibrated with GC data (ID209 and ID355) and ﬁve studies that provided only brief descriptions of the methods used (ID159, ID407, ID560, ID570 and ID606). Meta-analyses However, data extracted from the same publication for (1) different years, (2) different regions, retail outlets or brands in the same country or Data reported as numerical values in the text or tables were copied directly into the database. Results only published in graphical form were enlarged, printed, measured (using a ruler) and then entered into the database as previously described(5). The suitability of analytical methods used in studies contributing data for WM and UM of FA proﬁles was assessed, and for most studies it was considered to be scientiﬁcally sound for comparison of relative differences between organic and conventional meat samples. Most studies used established Data reported in the same publication for different animal species, products, study types, countries and outcomes were treated as independent effects. However, data extracted from the same publication for (1) different years, (2) different regions, retail outlets or brands in the same country or D. Średnicka-Tober et al. 998 –80 –60 –40 –20 0 20 40 60 80 MPD* % higher in ORG % higher in CONV Standard meta-analysis Sensitivity meta-analysis 1 n Ln ratio† P‡ P‡ n Heterogeneity§ Primary outcome PUFA n-3 FA Secondary outcome 12 : 0 (lauric acid) 14 : 0 (myristic acid) 16 : 0 (palmitic acid) 23 <0.001 Yes (95%) 35 4.75 <0.001 <0.001 4.85 31 Yes (98%) 21 0.026 11 23 24 0.974 0.049 0.044 Yes (84%) Yes (98%) Yes (91%) 15 27 30 15 4.66 4.47 4.55 4.79 0.270 0.003 0.043 0.017 – – – VLC n-3 PUFA (EPA+DPA+DHA)¶ Exploratory outcome Fat Intramuscular fat SFA MUFA OA (cis-9-18 : 1) ALA (cis-9,12,15-18 : 3) EPA (cis-5, 8,11,14,17-20 : 5)\|\| DPA (cis-7,10,13,16,19-22 : 5) DHA (cis-4,7,10,13,16,19-22 : 6) n-6 FA LA (cis-9,12-18 : 2) AA (cis-5,8,11,14-20 : 4)\|\| LA:ALA ratio¶ n-6:n-3 ratio –5.0 –2.5 0.0 2.5 5.0 SMD 22 0.125 Yes (89%) 34 4.45 0.012 7 0.331 Yes (79%) 9 4.44 0.065 26 0.127 Yes (92%) 38 4.59 0.103 24 <0.001 Yes (94%) 36 4.55 <0.001 22 22 13 11 14 19 23 13 – – – 17 0.133 Yes (97%) 32 4.42 0.004 0.079 Yes (80%) 28 4.46 0.026 19 4.61 0.461 0.077 Yes (95%) 30 4.68 0.097 0.138 Yes (94%) 27 4.56 0.016 0.008 0.403 0.007 0.246 0.010 4.80 4.58 4.82 4.68 4.70 32 20 15 22 29 Yes (97%) Yes (95%) Yes (92%) Yes (75%) Yes (96%) 0.169 0.966 0.304 0.404 0.020 Parameters Fig. 2. Meta-analyses Results of the standard weighted meta-analysis and sensitivity analysis 1 for fat composition of meat (data for all animal groups included in the same analysis). * Numerical values for mean percentage difference (MPD) and 95 % CI are given in the online Supplementary Table S9. † Ln ratio = ln (ORG/CONV × 100 %). ‡ P value <0·05 indicates a significant difference between organic samples (ORG) and conventional samples (CONV). § Heterogeneity and the I 2 statistic. \|\| Outlying data points (where the MPD between ORG and CONV was more than fifty times greater than the mean value including the outliers) were removed. ¶ Calculated based on published fatty acids (FA) composition data. n, number of data points included in meta-analyses; VLC n-3 PUFA, very long-chain n-3 PUFA; DPA, docosapentaenoic acid; OA, oleic acid; ALA, α-linolenic acid; LA, linoleic acid; AA, arachidonic acid; SMD, standardised mean difference; ○, MPD calculated using data included in standard unweighted meta-analyses; ▷, MPD calculated using data include in standard weighted meta-analysis; ◆, SMD with 95 % CI represented by horizontal bars. Meta-analyses –80 –60 –40 –20 0 20 40 60 80 MPD* % higher in ORG % higher in CONV Standard meta-analysis Sensitivity meta-analysis 1 n Ln ratio† P‡ P‡ n Heterogeneity§ Primary outcome PUFA n-3 FA Secondary outcome 12 : 0 (lauric acid) 14 : 0 (myristic acid) 16 : 0 (palmitic acid) 23 <0.001 Yes (95%) 35 4.75 <0.001 <0.001 4.85 31 Yes (98%) 21 0.026 11 23 24 0.974 0.049 0.044 Yes (84%) Yes (98%) Yes (91%) 15 27 30 15 4.66 4.47 4.55 4.79 0.270 0.003 0.043 0.017 – – – VLC n-3 PUFA (EPA+DPA+DHA)¶ Exploratory outcome Fat Intramuscular fat SFA MUFA OA (cis-9-18 : 1) ALA (cis-9,12,15-18 : 3) EPA (cis-5, 8,11,14,17-20 : 5)\|\| DPA (cis-7,10,13,16,19-22 : 5) DHA (cis-4,7,10,13,16,19-22 : 6) n-6 FA LA (cis-9,12-18 : 2) AA (cis-5,8,11,14-20 : 4)\|\| LA:ALA ratio¶ n-6:n-3 ratio –5.0 –2.5 0.0 2.5 5.0 SMD 22 0.125 Yes (89%) 34 4.45 0.012 7 0.331 Yes (79%) 9 4.44 0.065 26 0.127 Yes (92%) 38 4.59 0.103 24 <0.001 Yes (94%) 36 4.55 <0.001 22 22 13 11 14 19 23 13 – – – 17 0.133 Yes (97%) 32 4.42 0.004 0.079 Yes (80%) 28 4.46 0.026 19 4.61 0.461 0.077 Yes (95%) 30 4.68 0.097 0.138 Yes (94%) 27 4.56 0.016 0.008 0.403 0.007 0.246 0.010 4.80 4.58 4.82 4.68 4.70 32 20 15 22 29 Yes (97%) Yes (95%) Yes (92%) Yes (75%) Yes (96%) 0.169 0.966 0.304 0.404 0.020 Parameters Fig. 2. Results of the standard weighted meta-analysis and sensitivity analysis 1 for fat composition of meat (data for all animal groups included in the same analysis). * Numerical values for mean percentage difference (MPD) and 95 % CI are given in the online Supplementary Table S9. † Ln ratio = ln (ORG/CONV × 100 %). ‡ P value <0·05 indicates a significant difference between organic samples (ORG) and conventional samples (CONV). § Heterogeneity and the I 2 statistic. \|\| Outlying data points (where the MPD between ORG and CONV was more than fifty times greater than the mean value including the outliers) were removed. ¶ Calculated based on published fatty acids (FA) composition data. Meta-analyses Composition of organic meat products 999 MPD* –80 –60 –40 –20 0 20 40 60 80 Animal groups† % higher in CONV % higher in ORG Standard meta-analysis Sensitivity meta-analysis 1 n P‡ n Ln ratio § P‡ –5.0 –2.5 0.0 SMD 2.5 5.0 Fat Beef Lamb and goat meat Pork Poultry Intramuscular fat Pork SFA Beef Lamb and goat meat Pork Poultry 12 : 0 (lauric acid) Beef Lamb and goat meat 14 : 0 (myristic acid) Beef Lamb and goat meat Pork Poultry 16 : 0 (palmitic acid) Beef Lamb and goat meat Pork Poultry MUFA Beef Lamb and goat meat Pork Poultry OA (cis-9-18 : 1) Beef Lamb and goat meat Pork Poultry PUFA Beef Lamb and goat meat Pork Poultry n-3 FA Beef Lamb and goat meat Poultry 6 7 – 4 4 5 9 4 5 – 7 5 9 4 4 5 9 4 5 4 8 4 5 5 9 4 – 4 7 4 5 – 8 6 0.076 0.600 – 0.158 0.879 0.835 0.870 0.224 0.046 – 0.674 0.081 0.828 0.713 <0.001 0.266 0.480 0.465 0.116 0.097 0.914 0.028 <0.001 0.981 0.928 0.327 – 0.277 0.577 0.025 <0.001 – 0.878 0.027 7 11 5 9 5 8 14 6 7 4 9 6 11 4 5 7 11 5 6 7 13 6 7 6 11 5 4 7 12 6 7 7 11 8 4.35 4.58 4.57 4.35 4.55 4.57 4.61 4.56 4.60 4.49 4.70 4.45 4.61 4.46 4.14 4.58 4.61 4.57 4.40 4.53 4.60 4.58 4.45 4.62 4.59 4.57 4.38 4.76 4.73 4.69 4.85 5.01 4.70 4.83 0.080 0.400 0.432 0.074 0.281 0.231 0.357 0.045 0.433 0.125 0.192 0.046 0.442 0.189 0.066 0.202 0.491 0.063 0.190 0.016 0.383 0.208 0.007 0.349 0.156 0.189 0.060 0.006 0.019 0.270 0.009 0.008 0.070 0.162 Fig. 3. Results of the standard weighted meta-analysis and sensitivity analysis 1 for different animal groups for fat composition in meat. * Numerical values for mean percentage difference (MPD) and 95 % CI are given in the online Supplementary Table S10. † For parameters for which n ≤3 for specific animal group, results obtained in the meta-analyses are not shown. ‡ Ln ratio = ln (ORG/CONV × 100 %). § P value <0·05 indicates a significant difference between organic samples (ORG) and conventional samples (CONV). Meta-analyses When these studies were excluded from the meta-analyses (sensitivity analysis 5), broadly similar results were obtained. However, as the laboratories that carried out these ﬁve studies were reputable institutions and to minimise publication bias, we included data from all studies in the standard WM reported here. The results of sensitivity analyses 2–5 are available in the Appendix on the Newcastle University website (http://research.ncl.ac.uk/ nefg/QOF). GC-based protocols and described methods in sufﬁcient detail. Seven studies may be classiﬁed as being of lower quality, which included two studies that used an near IR-spectroscopy method calibrated with GC data (ID209 and ID355) and ﬁve studies that provided only brief descriptions of the methods used (ID159, ID407, ID560, ID570 and ID606). When these studies were excluded from the meta-analyses (sensitivity analysis 5), broadly similar results were obtained. However, as the laboratories that carried out these ﬁve studies were reputable institutions and to minimise publication bias, we included data from all studies in the standard WM reported here. The results of sensitivity analyses 2–5 are available in the Appendix on the Newcastle University website (http://research.ncl.ac.uk/ nefg/QOF). We carried out tests of homogeneity (Q statistics and I 2 statistics) on all summary effect sizes. Homogeneity was indicated when I 2 was <25 % and the P value for the Q statistics was >0·010. Funnel plots, Egger’s tests of funnel plot asymmetry and fail-safe number tests were used to assess publication bias(38) (see online Supplementary Table S13 for further information). Effect sizes for all WM were based on standardised mean differences (SMD) as recommended for studies that include data obtained by measuring the same parameters on different scales(29,30). In the UM, the signiﬁcance and magnitude of differences in contents of the compounds were calculated using a resampling method, where the ratio of organic means/conventional means (X̅O/X̅C) expressed as a percentage was ln-transformed and values used to determine if the arithmetic average of the ln-transformed ratios was signiﬁcantly greater than ln(100)(39). Reported P values were derived from Fisher’s one-sample randomisation test(40), and a P< 0·05 was considered to be statistically signiﬁcant. Both WM and UM were carried out using the R statistical programming environment(31). WM, with the SMD as the basic response variable, were carried out using standard methods and the open-source ‘metafor’ statistical package(32–35). A detailed description of the methods and calculations is provided in the ‘Additional Methods and Results’ in the Supplementary Information available online. Meta-analyses n, number of data points included in the meta-analyses; OA, oleic acid; FA, fatty acids; SMD, standardised mean difference; ○, MPD calculated using data included in standard unweighted meta-analyses; ▷, MPD calculated using data include in standard weighted meta-analysis; ◆, SMD with 95 % CI represented by horizontal bars. Composition of organic meat products 999 MPD* –80 –60 –40 –20 0 20 40 60 80 Animal groups† % higher in CONV % higher in ORG Standard meta-analysis Sensitivity meta-analysis 1 n P‡ n Ln ratio § P‡ –5.0 –2.5 0.0 SMD 2.5 5.0 Fat Beef Lamb and goat meat Pork Poultry Intramuscular fat Pork SFA Beef Lamb and goat meat Pork Poultry 12 : 0 (lauric acid) Beef Lamb and goat meat 14 : 0 (myristic acid) Beef Lamb and goat meat Pork Poultry 16 : 0 (palmitic acid) Beef Lamb and goat meat Pork Poultry MUFA Beef Lamb and goat meat Pork Poultry OA (cis-9-18 : 1) Beef Lamb and goat meat Pork Poultry PUFA Beef Lamb and goat meat Pork Poultry n-3 FA Beef Lamb and goat meat Poultry 6 7 – 4 4 5 9 4 5 – 7 5 9 4 4 5 9 4 5 4 8 4 5 5 9 4 – 4 7 4 5 – 8 6 0.076 0.600 – 0.158 0.879 0.835 0.870 0.224 0.046 – 0.674 0.081 0.828 0.713 <0.001 0.266 0.480 0.465 0.116 0.097 0.914 0.028 <0.001 0.981 0.928 0.327 – 0.277 0.577 0.025 <0.001 – 0.878 0.027 7 11 5 9 5 8 14 6 7 4 9 6 11 4 5 7 11 5 6 7 13 6 7 6 11 5 4 7 12 6 7 7 11 8 4.35 4.58 4.57 4.35 4.55 4.57 4.61 4.56 4.60 4.49 4.70 4.45 4.61 4.46 4.14 4.58 4.61 4.57 4.40 4.53 4.60 4.58 4.45 4.62 4.59 4.57 4.38 4.76 4.73 4.69 4.85 5.01 4.70 4.83 0.080 0.400 0.432 0.074 0.281 0.231 0.357 0.045 0.433 0.125 0.192 0.046 0.442 0.189 0.066 0.202 0.491 0.063 0.190 0.016 0.383 0.208 0.007 0.349 0.156 0.189 0.060 0.006 0.019 0.270 0.009 0.008 0.070 0.162 Fig. 3. Results of the standard weighted meta-analysis and sensitivity analysis 1 for different animal groups for fat composition in meat. * Numerical values for mean percentage difference (MPD) and 95 % CI are given in the online Supplementary Table S10. Meta-analyses † For parameters for which n ≤3 for specific animal group, results obtained in the meta-analyses are not shown. ‡ Ln ratio = ln (ORG/CONV × 100 %). § P value <0·05 indicates a significant difference between organic samples (ORG) and conventional samples (CONV). n, number of data points included in the meta-analyses; OA, oleic acid; FA, fatty acids; SMD, standardised mean difference; ○, MPD calculated using data included in standard unweighted meta-analyses; ▷, MPD calculated using data include in standard weighted meta-analysis; ◆, SMD with 95 % CI represented by horizontal bars. Fig. 3. Results of the standard weighted meta-analysis and sensitivity analysis 1 for different animal groups for fat composition in meat. * Numerical values for mean percentage difference (MPD) and 95 % CI are given in the online Supplementary Table S10. † For parameters for which n ≤3 for specific animal group, results obtained in the meta-analyses are not shown. ‡ Ln ratio = ln (ORG/CONV × 100 %). § P value <0·05 indicates a significant difference between organic samples (ORG) and conventional samples (CONV). n, number of data points included in the meta-analyses; OA, oleic acid; FA, fatty acids; SMD, standardised mean difference; ○, MPD calculated using data included in standard unweighted meta-analyses; ▷, MPD calculated using data include in standard weighted meta-analysis; ◆, SMD with 95 % CI represented by horizontal bars. D. Średnicka-Tober et al. 1000 Th l f bli i h MPD* –80 –60 –40 –20 0 20 40 60 8 % higher in CONV % higher in ORG –5.0 –2.5 0.0 SMD 2.5 5 Fig. 4. Results of the standard weighted meta-analysis and sen percentage difference (MPD) and 95 % CI are given in the online in the meta-analyses are not shown. ‡ Ln ratio = ln (ORG/CON conventional samples (CONV). \|\| Outlying data points (where the outliers) were removed. ¶ Calculated based on published FA co docosapentaenoic acid; VLC n-3 PUFA, very long-chain n-3 P ○, MPD calculated using data included in standard unweighted with 95 % CI represented by horizontal bars. Meta-analyses There are currently very few publications that report com- parative data for thrombogenicity and/or atherogenicity indices, and all provide information on lamb and goat meat only However, a much larger number of publications covering a range of meat types reported sufﬁcient data for individual FA/groups of FA to calculate the two indices On the basis of MPD* –80 –60 –40 –20 0 20 40 60 80 % higher in CONV % higher in ORG Animal groups† ALA (cis-9,12,15-18 : 3) Beef Lamb and goat meat Pork Poultry EPA (cis-5,8,11,14,17-20 : 5)\|\| Beef Lamb and goat meat Poultry DPA (cis-7,10,13,16,19-22 : 5) Beef Lamb and goat meat DHA (cis-4,7,10,13,16,19-22 : 6) Beef Lamb and goat meat Poultry VLC n-3 PUFA (EPA+DPA+DHA)¶ Beef Lamb and goat meat n-6 FA Beef Lamb and goat meat Poultry LA (cis-9,12-18 : 2) Beef Lamb and goat meat Pork Poultry AA (cis-5,8,11,14-20 : 4)\|\| Beef Lamb and goat meat Poultry LA:ALA ratio¶ Beef Lamb and goat meat Pork Poultry n-6:n-3 ratio Beef Lamb and goat meat Poultry –5.0 –2.5 0.0 SMD 2.5 5.0 n P‡ n Ln ratio § P‡ 4 8 4 5 7 – 7 6 5 8 4 5 8 4 5 6 5 6 0.109 0.926 0.783 0.074 0.307 0.719 0.393 0.653 0.844 <0.001 0.696 0.862 0.366 0.025 0.601 0.093 0.656 8 12 5 6 5 10 4 5 8 5 10 6 5 8 7 11 6 8 10 5 6 4 8 4 7 10 5 5 9 13 5 4.69 4.46 4.18 4.59 4.68 4.38 4.28 4.80 4.55 4.63 4.70 4.68 4.70 4.65 4.88 4.62 4.61 4.70 5.00 4.44 4.75 4.87 4.77 5.01 4.45 4.44 5.03 4.66 4.61 4.90 4.96 0.003 0.017 0.504 0.433 0.029 0.135 0.312 0.029 0.088 0.063 0.103 0.298 0.030 0.197 0.488 0.372 0.015 0.354 0.120 0.307 0.312 0.438 0.245 0.188 0.025 0.058 0.291 0.471 0.002 0.058 0.370 – – – – – – – – – – – – – – – – – – – – – – – – – – – Standard meta-analysis Sensitivity meta-analysis 1 Fig. 4. Results of the standard weighted meta-analysis and sensitivity analysis 1 for different animal groups for fat composition in meat. * Numerical values for mean percentage difference (MPD) and 95 % CI are given in the online Supplementary Table S10. Meta-analyses † For parameters for which n ≤3 for specific animal group, results obtained in the meta-analyses are not shown. ‡ Ln ratio = ln (ORG/CONV × 100 %). § P value <0·05 indicates a significant difference between organic samples (ORG) and conventional samples (CONV). \|\| Outlying data points (where the MPD between ORG and CONV was more than fifty times greater than the mean value including the outliers) were removed. ¶ Calculated based on published FA composition data. n, number of data points included in the meta-analyses; ALA, α-linolenic acid; DPA, docosapentaenoic acid; VLC n-3 PUFA, very long-chain n-3 PUFA; FA, fatty acids; LA, linoleic acid; AA, arachidonic acid; SMD, standardised mean difference; ○, MPD calculated using data included in standard unweighted meta-analyses; ▷, MPD calculated using data include in standard weighted meta-analysis; ◆, SMD with 95 % CI represented by horizontal bars. Meta-analyses There are currently very few publications that report com- parative data for thrombogenicity and/or atherogenicity indices However, a much larger number of publications covering a range of meat types reported sufﬁcient data for individual MPD* –80 –60 –40 –20 0 20 40 60 80 % higher in CONV % higher in ORG Animal groups† ALA (cis-9,12,15-18 : 3) Beef Lamb and goat meat Pork Poultry EPA (cis-5,8,11,14,17-20 : 5)\|\| Beef Lamb and goat meat Poultry DPA (cis-7,10,13,16,19-22 : 5) Beef Lamb and goat meat DHA (cis-4,7,10,13,16,19-22 : 6) Beef Lamb and goat meat Poultry VLC n-3 PUFA (EPA+DPA+DHA)¶ Beef Lamb and goat meat n-6 FA Beef Lamb and goat meat Poultry LA (cis-9,12-18 : 2) Beef Lamb and goat meat Pork Poultry AA (cis-5,8,11,14-20 : 4)\|\| Beef Lamb and goat meat Poultry LA:ALA ratio¶ Beef Lamb and goat meat Pork Poultry n-6:n-3 ratio Beef Lamb and goat meat Poultry –5.0 –2.5 0.0 SMD 2.5 5.0 n P‡ n Ln ratio § P‡ 4 8 4 5 7 – 7 6 5 8 4 5 8 4 5 6 5 6 0.109 0.926 0.783 0.074 0.307 0.719 0.393 0.653 0.844 <0.001 0.696 0.862 0.366 0.025 0.601 0.093 0.656 8 12 5 6 5 10 4 5 8 5 10 6 5 8 7 11 6 8 10 5 6 4 8 4 7 10 5 5 9 13 5 4.69 4.46 4.18 4.59 4.68 4.38 4.28 4.80 4.55 4.63 4.70 4.68 4.70 4.65 4.88 4.62 4.61 4.70 5.00 4.44 4.75 4.87 4.77 5.01 4.45 4.44 5.03 4.66 4.61 4.90 4.96 0.003 0.017 0.504 0.433 0.029 0.135 0.312 0.029 0.088 0.063 0.103 0.298 0.030 0.197 0.488 0.372 0.015 0.354 0.120 0.307 0.312 0.438 0.245 0.188 0.025 0.058 0.291 0.471 0.002 0.058 0.370 – – – – – – – – – – – – – – – – – – – – – – – – – – – Standard meta-analysis Sensitivity meta-analysis 1 Fig. 4. Results of the standard weighted meta-analysis and sensitivity analysis 1 for different animal groups for fat composition in meat. * Numerical values for mean percentage difference (MPD) and 95 % CI are given in the online Supplementary Table S10. † For parameters for which n ≤3 for specific animal group, results obtained in the meta-analyses are not shown. ‡ Ln ratio = ln (ORG/CONV × 100 %). Estimation of fatty acid intakes Estimation of fatty acid intakes Estimation of fatty acid intakes Forest plots were constructed to show pooled SMD and corresponding 95 % CI for all compositional parameters inves- tigated. Additional forest plots were presented for selected Intakes were estimated for FA parameters for which WM based on pooled data from all meat types had detected signiﬁcant Table 1. Grading of Recommendations Assessments, Development and Evaluation (GRADE) assessment of the strength of evidence for standard weighted meta-analysis for parameters shown in Fig. 2 (Standardised mean difference (SMD) values and 95 % confidence intervals) weighted meta-analysis for parameters shown in Fig. 2 (Standardised mean difference (SMD) values and 95 % confidence intervals) Parameters SMD 95 % CI Effect magnitude* Inconsistency† Precision‡ Publication bias§ Overall reliability\|\| Fat composition Fat −0·35 −0·80, 0·10 Small Low Poor Medium Low Intramuscular fat −0·25 −0·74, 0·25 Small Low Moderate Strong Low SFA −0·35 −0·79, 0·10 Small Medium Poor No Moderate 12 : 0 (lauric acid) −0·01 −0·55, 0·53 Small Low High Medium Moderate 14 : 0 (myristic acid) −1·02 −2·09, 0·04 Moderate High Poor Strong Very low 16 : 0 (palmitic acid) −0·47 −0·96, 0·02 Small Low Poor Strong Very low MUFA −1·01 −1·57, −0·45 Moderate High Moderate Medium Moderate OA (cis-9-18 : 1) −0·48 −1·12, 0·16 Small Low Poor Medium Low PUFA 1·15 0·51, 1·80 Moderate High Moderate Medium Moderate n-3 FA 1·31 0·16, 2·45 Moderate Medium Poor Strong Low ALA (cis-9,12,15-18 : 3) 0·73 −0·27, 1·73 Small High Poor Strong Very low EPA (cis-5,8,11,14,17-20 : 5)¶ 0·02 −0·85, 0·90 Small High Moderate Strong Very low DPA (cis-7,10,13,16,19-22 : 5) 0·40 −0·36, 1·17 Small Low Moderate Strong Low DHA (cis-4,7,10,13,16,19-22 : 6) 0·22 −0·17, 0·61 Small Medium High Strong Low VLC n-3 PUFA (EPA + DPA + DHA) – – – – – – – n-6 FA 0·97 0·15, 1·78 Moderate High Moderate Strong Low LA (cis-9,12-18 : 2) 0·65 −0·01, 1·30 Small Medium Poor Medium Low AA (cis-5,8,11,14-20 : 4)¶ 0·45 −0·05, 0·94 Small Medium Poor Medium Low LA:ALA ratio – – – – – – – n-6:n-3 ratio −0·75 −1·72, 0·23 Moderate High Poor Medium Low OA, oleic acid; FA, fatty acids; ALA, α-linolenic acid; DPA, docosapentaenoic acid; VLC n-3 PUFA, very long-chain n-3 PUFA; LA, linoleic acid; AA, arachidonic acid. * Study quality was considered low because of high risks of bias and potential for confounding. Meta-analyses § P value <0·05 indicates a significant difference between organic samples (ORG) and conventional samples (CONV). \|\| Outlying data points (where the MPD between ORG and CONV was more than fifty times greater than the mean value including the outliers) were removed. ¶ Calculated based on published FA composition data. n, number of data points included in the meta-analyses; ALA, α-linolenic acid; DPA, docosapentaenoic acid; VLC n-3 PUFA, very long-chain n-3 PUFA; FA, fatty acids; LA, linoleic acid; AA, arachidonic acid; SMD, standardised mean difference; ○, MPD calculated using data included in standard unweighted meta-analyses; ▷, MPD calculated using data include in standard weighted meta-analysis; ◆, SMD with 95 % CI represented by horizontal bars. Fig. 4. Results of the standard weighted meta-analysis and sensitivity analysis 1 for different animal groups for fat composition in meat. * Numerical values for mean percentage difference (MPD) and 95 % CI are given in the online Supplementary Table S10. † For parameters for which n ≤3 for specific animal group, results obtained in the meta-analyses are not shown. ‡ Ln ratio = ln (ORG/CONV × 100 %). § P value <0·05 indicates a significant difference between organic samples (ORG) and conventional samples (CONV). \|\| Outlying data points (where the MPD between ORG and CONV was more than fifty times greater than the mean value including the outliers) were removed. ¶ Calculated based on published FA composition data. n, number of data points included in the meta-analyses; ALA, α-linolenic acid; DPA, docosapentaenoic acid; VLC n-3 PUFA, very long-chain n-3 PUFA; FA, fatty acids; LA, linoleic acid; AA, arachidonic acid; SMD, standardised mean difference; ○, MPD calculated using data included in standard unweighted meta-analyses; ▷, MPD calculated using data include in standard weighted meta-analysis; ◆, SMD with 95 % CI represented by horizontal bars. However, a much larger number of publications covering a range of meat types reported sufﬁcient data for individual FA/groups of FA to calculate the two indices. On the basis of There are currently very few publications that report com- parative data for thrombogenicity and/or atherogenicity indices, and all provide information on lamb and goat meat only. Composition of organic meat products 1001 those reported data, we calculated values of the thrombogeni- city and atherogenicity indices as follows: results to illustrate heterogeneity between subgroups based on types of meat (see online Supplementary Fig. S6–S35). Meta-analyses The mean percentage difference (MPD) was calculated for all parameters for which statistically signiﬁcant effects were detected by either WM or UM. This was carried out to facilitate value judgements regarding the biological importance of the relative effect magnitudes using the calculations described by Barański et al.(25). Thrombogenicity index ¼ 14 : 0 + 16 : 0 + 18 : 0 ð05 ´ MUFAÞ + ð05 ´ n-6 PUFAÞ + ð3 ´ n-3 PUFAÞ + ðn-3:n-6 PUFAÞ ; Atherogenicity index ¼ 12 : 0 + ð4 ´ 14 : 0Þ + 16 : 0 MUFA + PUFA : Thrombogenicity index ¼ 14 : 0 + 16 : 0 + 18 : 0 ð05 ´ MUFAÞ + ð05 ´ n-6 PUFAÞ + ð3 ´ n-3 PUFAÞ + ðn-3:n-6 PUFAÞ ; Atherogenicity index ¼ 12 : 0 + ð4 ´ 14 : 0Þ + 16 : 0 MUFA + PUFA : We calculated MPD for data pairs included in both the WM and the UM in order to estimate the impact of excluding data for which no measures of variance were reported on the magnitude of dif- ference. As the MPD can be expressed as ‘% higher’ in conven- tional or organic meat, they provide estimates for the magnitude of composition differences that are easier to relate to existing infor- mation on potential health impacts of changing dietary intakes for individual or groups of compounds than the SMD values. The 95 % CI for MPD were estimated using a standard method(36). For the thrombogenicity index ﬁfteen data points (three for beef, seven for lamb and goat meat, two for pork and three for chicken meat) and for the atherogenicity index thirteen data points (three for beef, eight for lamb and goat meat, one for pork and one for rabbit meat) were available. We carried out separate meta-analyses for the published and calculated estimates of the two indices (Fig. 2 and 4; online Supplementary Tables S9–S11 and Fig. S5). For all parameters (thrombogenicity index, atherogenicity index, total VLC n-3 PUFA, LA:ALA ratio) that were calculated based on published information it was only possible to carry out UM (Fig. 2–4), as measures of variance were not available. An overall assessment of the strength of evidence was made using an adaptation of the Grading of Recommendations Assess- ment, Development and Evaluation (GRADE)(41) system (Table 1). , sistency was based on the measure of heterogeneity and consistency of effect direction sensu GRADE. Estimation of fatty acid intakes These values were then used to calculate mean FA concentrations in different meat types. These means were then used to calculate total FA intakes from organic and conventional meats using (1) published data on fat consumption from different meat types in the EU(42) and (2) for mean concentrations of total FA esters in organic and conventional meats (Fig. 3 and 4). MPD in FA intakes between organic and conventional meats was then calculated (see Table 2). It should be pointed out that the European fat consumption data were based on means from all EU countries, whereas means for FA concentrations in organic and conven- tional meats were based on published data from eight EU countries (Germany, Denmark, Spain, France, UK, Italy, Poland, Sweden; contributing approximately 70 % of data) and seven countries from outside the EU (Switzerland, Brazil, Republic of Korea, Turkey, Taiwan, Province of China, USA, Uruguay). Estimates of FA intakes for speciﬁc countries were not possible owing to a lack of published data (comparative studies for all different meat types were not available for any one country). individual studies were available for WM of SFA contents in chicken meat and that results differed between studies and/or countries/regions. Three studies (from the UK and Italy) reported no signiﬁcant difference, whereas two others (from the Republic of Korea and the USA) reported signiﬁcantly lower SFA concentra- tions in organic chicken meat (online Supplementary Table S9). For MUFA, WM detected signiﬁcantly lower concentrations for pork and chicken only (Fig. 3 and online Supplementary Fig. S14). However, it should be noted that only three and ﬁve individual studies were available for WM of MUFA contents in pork and chicken meat, respectively. For pork, results differed between studies and/or countries/regions; one study (from Poland) reported no signiﬁcant difference, and two (from the Republic of Korea and Sweden) studies reported signiﬁcantly lower MUFA concentrations in organic meat (online Supplementary Table S14). For chicken meat, all ﬁve studies (from the UK, Italy, Republic of Korea and the USA) reported signiﬁcantly lower MUFA concentrations in organic chicken meat (online Supplementary Table S14). For PUFA, signiﬁcantly higher concentrations were detected for pork and chicken meat, but not for beef and lamb and goat meat (Fig. 3 and online Supplementary Fig. S19). Estimation of fatty acid intakes However, we considered large effects to mitigate this sensu GRADE; large effects were defined as >20 %, moderate effects 10–20 and small <10 %. † Inconsistency was based on the measure of heterogeneity and consistency of effect direction sensu GRADE. ‡ Precision was based on the width of the pooled effect CI and the extent of overlap in substantive interpretation of effect magnitude sensu GRADE. § Publication bias was assessed using visual inspection of funnel plots, the Egger’s test, two-tests of fail-safe N and trim and fill (see online Supplementary Table S13). Overall publication bias was considered high when indicated by two or more methods, moderate when indicated by one method and low when no methods suggested publication bias. \|\| Overall quality of evidence was then assessed across domains as in standard GRADE appraisal; high when there was very high confidence that the true effect lies close to that of the estimate, moderate when there was moderately confidence in effect estimate and the true effect is likely to be close to the estimate but there is a possibility that it is substantially different, low when the confidence in the effect estimate was limited and the true effect may be substantially different from the estimate, very low when there was very little confidence in the effect estimate and the true effect is likely to be substantially different from the estimate. ¶ Outlying data pairs (where the mean percentage difference between organic and conventional meat samples was over fifty times greater than the mean value including outliers) were removed. ** Calculated based on published FA composition data. weighted meta-analysis for parameters shown in Fig. 2 (Standardised mean difference (SMD) values and 95 % confidence intervals) SMD 95 % CI Effect magnitude* Inconsistency† Precision‡ Publication bias§ Overall reliability\|\| OA, oleic acid; FA, fatty acids; ALA, α-linolenic acid; DPA, docosapentaenoic acid; VLC n-3 PUFA, very long-chain n-3 PUFA; LA, linoleic acid; AA, arachidonic acid. * Study quality was considered low because of high risks of bias and potential for confounding. However, we considered large effects to mitigate this sensu GRADE; large effects were defined as >20 % moderate effects 10 20 and small <10 % D. Średnicka-Tober et al. 1002 differences between organic and conventional meat. All FA data extracted from the original publications were converted into a common unit (g/100 g total FA esters). Characteristics of studies and data included in the meta-analyses The WM and UM were based on data from sixty-three peer- reviewed papers and four non-peer-reviewed studies, including publications reporting farm surveys (ﬁve papers), controlled experiments (forty-two papers) and BS (twenty papers). Most of the eligible studies were from Europe, mainly from Spain, UK, Italy, Sweden, Poland and Germany, with most of the others coming from the USA and Brazil (online Supple- mentary Table S2 and Fig. S2). Publications reported data on 373 different composition parameters, but the majority of studies (thirty-nine papers) focused on meat fat composition parameters (online Supplementary Tables S6 and S7). In contrast, relatively few studies (thirteen papers) reported data on mineral nutrients, toxic metals and/or other composition parameters. Meta-analyses were carried out on 122 meat-quality parameters (online Supplementary Tables S6 and S7). When data for all meat types were analysed together, WM identiﬁed signiﬁcantly lower concentrations of the SFA myristic acid (14 : 0) and palmitic acid (16 : 0) in organic compared with conventional meat. The MPD were −18 (95 % CI −32, −5) % for myristic acid and −11 (95 % CI −28, 5) % for palmitic acid (Fig. 2). When data for different meat types were analysed separately, WM detected signiﬁcantly lower 14 : 0 concentrations for organic chicken meat only (Fig. 3 and online Supplementary Fig. S11). However, it should be noted that only four studies were available for WM of PUFA in chicken meat and that results dif- fered between studies and/or countries/regions; two studies (both from the UK) reported no signiﬁcant difference, whereas two others studies (from the UK and Republic of Korea) reported signiﬁcantly lower 14 : 0 concentrations in organic chicken meat (online Supplementary Fig. S11). Estimation of fatty acid intakes However, it should be noted that only four and ﬁve individual studies were available for WM of PUFA contents in pork and chicken meat, respectively, and for both pork and chicken meat results differed between studies and/or countries/regions (online Supplementary Table S19). For pork, one study (from Sweden) reported no signiﬁcant differences and two studies (from the Republic of Korea and Poland) reported signiﬁcantly higher PUFA concentrations in organic meat. For chicken meat, two studies (from the UK and Italy) reported no signiﬁcant differences, whereas three studies (from the UK, Republic of Korea and the USA) reported signiﬁcantly higher PUFA in organic chicken meat (online Supplementary Table S19). Composition of organic and conventional meat products Estimated fatty acids (mg/person per d) intake from organic (ORG) and conventional (CONV) meat based on FAO’s fat supply quantity data(42) for bovine meat, pig meat, sheep and goat meat and poultry meat in the European Union, calculated using the data included in the unweighted meta-analysis shown in Fig. 2 Consumption associated with Beef* Lamb and goat meat† Pork‡ Chicken meat§ Total meat Parameters ORG CONV MPD ORG CONV MPD ORG CONV MPD ORG CONV MPD ORG CONV MPD SFA 1518 1507 1 527 528 0 6648 6868 −3 1408 1419 −1 10 100 10 322 −2 14 : 0 (myristic acid) 59 66 −12 60 61 −2 217 252 −16 27 41 −50 363 420 −16 16 : 0 (palmitic acid) 709 715 −1 252 254 −1 4238 4368 −3 993 999 −1 6191 6337 −2 MUFA 1307 1395 −7 406 414 −2 8229 8417 −2 1587 1858 −17 11 528 12 083 −5 PUFA 525 455 15 142 132 8 2930 2561 14 1482 1200 24 5080 4348 17 n-3 PUFA 128 78 64 41 40 2 419 360 16 161 136 19 748 613 22 n-6 PUFA 290 277 5 94 95 −1 4400 3637 21 1396 1100 27 6180 5110 21 MPD, mean percentage difference. * Calculated assuming an average fat consumption from bovine meat of 3·5 g/person per d. † Calculated assuming an average fat consumption from sheep and goat meat of 1·2 g/person per d. ‡ Calculated assuming an average fat consumption from pig meat of 19·1 g/person per d. § Calculated assuming an average fat consumption from poultry meat of 4·7 g/person per d. studies were available for WM of n-3 PUFA in chicken meat and that results differed between studies and/or countries/regions; two studies (both from the UK) reported no signiﬁcant difference, whereas four other studies (from the UK, Italy, Republic of Korea and the USA) reported signiﬁcantly higher n-3 PUFA in organic chicken meat (online Supplementary Fig. S11). WM detected no signiﬁcant differences for CLA, EPA, DPA and DHA, a range of other SFA, MUFA and PUFA and the n-6:n-3 ratio (Fig. 2 and online Supplementary Table S12). UM were carried out as ‘sensitivity analyses’ to estimate the extent to which an increase in the evidence base (inclusion of publications in which no measures of variance were reported) would identify additional composition differences. Composition of organic and conventional meat products When data for different meat types were pooled, UM results were similar to those obtained by WM for total SFA, MUFA and PUFA and for n-3 PUFA, n-6 PUFA, 14 :0 and 16 :0 (Fig. 2). However, different to the WM, the UM-based sensitivity analyses also detected sig- niﬁcant differences for a range of other fat composition para- meters. Speciﬁcally, UM detected (1) lower total fat and oleic acid concentrations, (2) higher ALA, DPA and total VLC n-3 PUFA (EPA+DPA+DHA) concentrations, (3) a lower n-6:n-3 PUFA ratio and (4) a lower thrombogenicity index in organic meat (Fig. 2; online Supplementary Table S9). For individual meat types, UM (sensitivity analysis 1) allowed comparisons for a wider range of composition parameters for all meat types and detected additional differences between organic and conventional meats (Fig. 3). This included (1) lower 14 :0 and MUFA but higher PUFA, n-3 PUFA, EPA, DPA and total VLC n-3 PUFA concentrations in beef, (2) higher PUFA and ALA concentrations in lamb and goat meat and (3) lower SFA concentrations in organic pork (Fig. 3). MPD, mean percentage difference. * Calculated assuming an average fat consumption from bovine meat of 3·5 g/person per d. † Calculated assuming an average fat consumption from sheep and goat meat of 1·2 g/person per d. ‡ Calculated assuming an average fat consumption from pig meat of 19·1 g/person per d. § Calculated assuming an average fat consumption from poultry meat of 4·7 g/person per d. Estimation of fatty acid intakes from organic and conven- tional meats. Accurate comparisons of FA intakes between organic and conventional meats are currently not possible, due to (a) the contrasting pattern of total meat and types of meat (e.g. beef, lamb, pork, chicken meat) consumed in different countries and (b) lack of sufﬁcient comparative data sets to estimate FA composition difference for speciﬁc countries. This makes it impossible to carry out country-speciﬁc intake esti- mates. Estimates of FA intakes were therefore calculated using published meat fat consumption data for the EU and mean FA composition data obtained from the systematic literature review. Moreover, intake estimates were only carried out for FA parameters for which relatively large data sets (n > 20) were available and for which the WM had detected signiﬁcant dif- ferences between organic and conventional meat (Table 2). Composition of organic and conventional meat products Fat composition. When data for all meat types were analysed together, WM detected signiﬁcant differences in FA proﬁles between organic and conventional meat (Fig. 2). Organic meat had similar SFA, lower MUFA and higher PUFA concentrations compared with conventional meat. The MPD (calculated based on data used for the WM) were −8 (95 % CI −13, −4) % for MUFA and 23 (95 % CI 11, 35) % for PUFA, respectively (Fig. 2 and online Supplementary Table S9). For 16 : 0, WM detected no signiﬁcant difference for all indi- vidual meat types (Fig. 3 and online Supplementary Fig. S12). When data for all meat types were analysed together, WM detected signiﬁcantly higher n-3 and n-6 concentrations in organic compared with conventional meat (Fig. 2). The MPD (calculated based on the data used for the WM) were 47 (95% CI 10, 84) % for n-3 PUFA and 16 (95 % CI 2, 31)% for n-6 PUFA, respectively. When data for all meat types were analysed together, WM detected signiﬁcantly higher n-3 and n-6 concentrations in organic compared with conventional meat (Fig. 2). The MPD (calculated based on the data used for the WM) were 47 (95% CI 10, 84) % for n-3 PUFA and 16 (95 % CI 2, 31)% for n-6 PUFA, respectively. When data for different meat types were analysed separately, WM detected signiﬁcantly higher concentrations of total n-3 PUFA in organic chicken meat only (Fig. 3 and online Supple- mentary Fig. S20). However, it should be noted that only six When data for different meat types were analysed separately, no differences in SFA were detected for beef, lamb and goat meat and pork, but WM detected slightly but signiﬁcantly lower SFA concentrations in organic chicken meat (Fig. 3 and online Sup- plementary Fig. S9). However, it should be noted that only ﬁve When data for different meat types were analysed separately, WM detected signiﬁcantly higher concentrations of total n-3 PUFA in organic chicken meat only (Fig. 3 and online Supple- mentary Fig. S20). However, it should be noted that only six 1003 Composition of organic meat products Table 2. Discussion Heterogeneity was high (I 2 > 75 %) for nearly all composition parameters, with I 2 ranging from 79 % for fat content to 98 % for 14 : 0 and n-3 PUFA concentrations (Fig. 2). Results of the meta-analyses reported in this study indicate for the ﬁrst time that there are signiﬁcant and nutritionally mean- ingful composition differences between organic and non-organic meat. This contradicts the results of a previous literature review by Dangour et al.(4), which pooled comparative data for meat, eggs, milk and dairy products in their analyses and concluded that overall there are no signiﬁcant composition differences between organic and conventional livestock products (meat, dairy products and eggs). However, results for speciﬁc para- meters reported in this study were variable, and both previous reviews(4,9) covering livestock products and the present study acknowledge serious deﬁciencies in the evidence, which result in considerable uncertainty. Plausible mechanistic explanations for the ﬁndings in this study are discussed below. When meta-analysis results obtained from different study types (BS, CF and EX) were compared, broadly similar results were obtained for most of the composition parameters included in Fig. 2 (see online Supplementary Fig. S3–S5). However, there was considerable variation between results for different meat types or studies carried out in different countries (see Fig. 3 and online Supplementary Fig. S6–S35). Non-weighted MPD were calculated to aid the biological interpretation of effect size magnitude where either the weighted or UM had identiﬁed statistically signiﬁcant differ- ences. For many parameters, MPD based on all the available data produced values very similar to those calculated using only data for which measures of variance were reported (those used for the WM; Fig. 2). However, for some parameters (n-3 PUFA, ALA), inclusion criteria had a moderate effect on the MPD. Meta-analysis results suggesting that certain organic meats (beef, lamb and pork) have higher concentrations of PUFA and n-3 PUFA are broadly consistent with results from controlled animal experiments that studied the effect of grazing or high- forage diets and the use of legume-rich forages (both of which are typically used in organic production) on meat quality(43–45). Strength of evidence The overall assessment of the strength of evidence based on WM using an adapted GRADE(41) approach highlighted strong uncertainties, with the overall strength of evidence being very low or low for most composition parameters, and moderate overall reliability was found only for 12 : 0, SFA, MUFA and PUFA concentrations (Table 1). Minerals, toxic metals and other composition parameters. Compared with fat composition parameters, relatively few comparative data sets were available for meta-analyses of minerals (e.g. Fe, Se, Zn), toxic metals (e.g. As, Pb, Cd) and other composition parameters (including protein, vitamins and pesticides) in meat (online Supplementary Tables S6, S7 and S12). The meta-analyses detected some signiﬁcant effects (e.g. for Cu), but these are not presented in detail in this study, because of the high level of uncertainty associated with meta-analysis results based on data from a very few studies. In general, there were substantial issues with study quality and reporting measures of variance, which were not generally mitigated by large effects. Inconsistency was high and precision was low. Strong or medium funnel plot asymmetry consistent with publication biases was also apparent for many parameters (see online Supplementary Table S13). However, it is not possible to deﬁnitely attribute discrepancies between large precise studies and small imprecise studies to publication bias, which remains strongly suspected rather than detected where asymmetry is severe. Composition of organic and conventional meat products Intakes of total SFA and palmitic acid had similar numerical values, whereas values for myristic acid (14 : 0) were lower with organic meat consumption (Table 2). Larger differences in numerical values were found for beef (−12 %), pork (−16 %) and chicken (−50 %), and overall the intake of myristic acid was estimated to be 16 % lower based on average meat consump- tion pattern in the EU (Table 2). Intakes of total MUFA with meat were estimated to be similar (−5 %) based on average meat consumption pattern in the EU (Table 2). D. Średnicka-Tober et al. 1004 total n-3, for which non-signiﬁcant differences were detected in some of the sensitivity analyses (see http://research.ncl.ac.uk/ nefg/QOF for detailed results of the sensitivity analyses). Larger numerical differences in intakes were calculated for total PUFA, n-3 PUFA and n-6 PUFA, which were all higher (by 17, 22 and 21 %, respectively) with organic meat consumption based on average meat consumption pattern in the EU (Table 2). However, there was considerable variation in the MPD calcu- lated for intakes for different meat types (Table 2). Owing to the more limited data available, comparisons of intakes with organic and conventional meat are currently not possible for other FA parameters including VLC FA (EPA + DPA + DHA). Discussion However, it should be pointed out that (a) the evidence base for individual meat/types/livestock species was very small (usually between two and seven studies), (2) the meta-analyses did not detect signiﬁcant differences for all meat types/livestock species and (3) that results for PUFA and n-3 PUFA varied between individual studies and studies carried out in different countries/ regions. Other composition differences (e.g. the lower con- centrations of 14 : 0 and 16 : 0 and higher concentrations of total n-6 PUFA in organic chicken meat) detected by meta-analyses may also be explained by differences in management practices between organic and conventional production systems(46–48). In addition, when the calculated MPD were superimposed onto SMD results (with 95 % CI) at an appropriate scale (−80 to +80 for MPD and −3 to +3 for SMD), a reasonable match was observed, with MPD for most compounds being present within the 95 % CI for SMD (Fig. 2). However, for some para- meters (fat, intramuscular fat, PUFA, n-3 PUFA, DPA and DHA), MPD were outside the 95 % CI of SMD, and therefore these should be seen as less reliable. However, it should be pointed out that (a) the evidence base for individual meat/types/livestock species was very small (usually between two and seven studies), (2) the meta-analyses did not detect signiﬁcant differences for all meat types/livestock species and (3) that results for PUFA and n-3 PUFA varied between individual studies and studies carried out in different countries/ regions. Other composition differences (e.g. the lower con- centrations of 14 : 0 and 16 : 0 and higher concentrations of total n-6 PUFA in organic chicken meat) detected by meta-analyses may also be explained by differences in management practices between organic and conventional production systems(46–48). Sensitivity analyses designed to identify the effect of using different inclusion criteria and data-handling methods yielded results broadly similar to those of the standard weighted and UM for the composition parameters included in Fig. 2. Links between livestock management and meat composition/quality Organic livestock production standards prescribe that livestock are to be reared outdoors for a part of the year, although the length of outdoor periods differs among regions and livestock species(49–51). EU organic standards prescribe that (1) ruminants receive at least 60 % of total DM intake (DMI) from forage (from grazing, cut fresh forage or conserved forage such as silage or hay) and (2) pigs and poultry are provided with access to forage but intake levels are not speciﬁed(49–51). For ruminants, organic regulations also prescribe that fresh forage intake is from grazing ‘when conditions allow’, and as a result the duration of grazing and the ratio of fresh:conserved forage in organic diets vary signiﬁcantly between European regions, mainly due to differ- ences in pedo-climatic and agronomic conditions(48,52). Where organic pigs and poultry have access to grassland, this may also result in signiﬁcant fresh forage intake, but in many regions organic pigs and poultry are fed conserved forage only(46,47). For poultry, there are limited data from controlled experi- mental studies that could potentially explain impacts of feeding regimens used in organic farming systems on meat quality, but access to forage may also at least partially explain the differences detected. For pigs and poultry, differences in the type of concentrate (and in particular protein supplements) may also contribute to composition differences between organic and conventional meat, especially FA proﬁles – for example, although conventional pig and poultry production relies on chemically extracted soya meal (which has low levels of residual fat) to supply high-quality protein, organic standards only allow cold-pressed soya and other oil seed meals (which have a higher oil content). Moreover, on- farm-produced grain legumes (peas and beans) are more widely used as protein supplements in organic production, mainly because there is a need for a proportion of feed to be produced on farm because of the limited availability, high cost and ethical concerns about imported feeds(46,47,55). The higher intake of soya oil (which has a high LA content) with cold-pressed soya meal may therefore explain the higher LA and n-6 concentrations detected by meta-analyses for organic chicken meat(46,47). Discussion The sensitivity analyses, designed to identify the effect of removing data from the 20 % of studies with least precise treatment effects also yielded broadly similar results, except for 14 : 0 and 16 : 0 and We therefore discuss below (1) current knowledge about the effects of management practices (especially feeding regimens) Composition of organic meat products 1005 fat in the longissimus muscle of beef, although it had no sig- niﬁcant effect on total n-6 PUFA or LA concentrations(44). In lamb production, a switch from grain- to grass-based ﬁnishing diets signiﬁcant increased ALA (>2-fold), EPA (>2-fold), DPA (88 %) and DHA (100 %) in the intramuscular fat of pelvic limb muscle meat and decreased concentrations of LA (30 %) and AA (21 %)(43). Although forage intakes in monogastric livestock are much lower than that in ruminants, free-range rearing of pigs with access to pasture grazing had signiﬁcantly increased con- centrations of PUFA, n-3 PUFA and ALA in the intramuscular fat when compared with meat from pigs reared indoors on standard concentrate-based diets(45). However, the relative differences were smaller (<50 %) than those detected in studies with beef and lamb(43,44). This suggests that there is considerable potential for both conventional and organic production to increase n-3 PUFA (including VLC n-3 concentrations) concentrations in beef, lamb and pork meat by further increasing grazing and the proportion of forage in livestock diets. that may explain composition differences between organic and conventional meat, (2) the strength of evidence and potential reasons for the heterogeneity of the available data/evidence, (3) potential nutritional/health impacts of meat from organic and other grazing or high-forage livestock production systems, (4) the need for expanding the current evidence base available for meta-analysis and (5) the requirement for dietary interven- tion and/or cohort studies to quantify potential health impacts of organic meat consumption. Links between livestock management and meat composition/quality clover, lucerne) or grass- legume mixtures are typically used in organic farming systems (where standards demand a speciﬁc proportion of fertility- building legume crops in the rotation). In contrast, pure grass or swards with a high proportion of grasses are more widely used in conventional/non-organic production systems, because the permitted use of mineral NPK fertilisers allows for higher DM yields per hectare compared with legume-grass mixtures. Evidence from studies comparing milk fat composition in extensive (grazing only) organic and non-organic dairy production systems (which use similar cross-breeds and grazing DMI) showed that organic milk (from cows grazing swards with a higher clover content) had signiﬁcantly more n-3 PUFA, but lower CLA concentrations compared with milk from non-organic farms(56,57). Similar impacts of legumes have also been reported for meat quality(58): longissimus dorsi muscle from lambs grazing lucerne or red clover swards (more widely used in organic production systems) had signiﬁcantly greater PUFA:SFA ratios and higher concentrations of both LA and ALA compared with lambs grazing grass swards. However, despite these uncertainties, there is a substantial body of evidence indicating that overall organic meat may have a more desirable FA proﬁle than non-organic comparators. The consistency of association directions across the multiple outcomes and analyses mitigates some of the uncertainty associated with individual parameters from a decision-analytical perspective, but the currently available evidence requires cautious interpretation. A major reason for the heterogeneity of the available data is likely to be the considerable variation in the intensity of both conventional and organic meat production systems. Non- organic production may range from intensive indoor produc- tion systems with high concentrate-based diets (>90 % of total DMI for pigs and poultry) to extensive outdoor grazing-based systems with high fresh and/or conserved forage (up to 100 % of total DMI) diets(53–55). Although limited by the restrictions of organic farming regulations, there is also variation in production intensity within organic systems – for example, concentrate intakes may vary between 0 and 40 % of DMI for organic ruminant diets(48,52). Links between livestock management and meat composition/quality In addition, although organic ruminant diets are thought to be based on higher fresh forage from grazing and lower concentrate intakes in most European countries/regions, lower grazing-based DMI in organic, com- pared with extensive non-organic, have been documented for some ruminant livestock species in some regions of Europe – for example, dairy cattle in Southern Wales(52,56) and dairy sheep and lamb meat production systems in Crete (Smaro Sotiraki, personal communication). This could explain why some studies showed a different trend (e.g. lower PUFA and n-3 FA in organic meat) to the overall results obtained by meta-analyses of pooled data or data for individual livestock species/meat types. Mineral supply and supplementation. Although some trends towards differences in mineral composition were detected by meta-analyses, these were based on a very limited evidence base and cannot be used to draw conclusions. However, they demonstrate the importance to carry out additional well-designed comparative studies, as organic and conventional livestock systems differ in a range of management practices that may affect the mineral composition of meat – for example, (1) conventional forage and grain crops often receive high inputs of mineral P fertilisers, a practice that has been linked to higher Cd concentrations in crops(25,59), and (2) conventional livestock feeding regimens often use higher levels of mineral supplementation (e.g. more widespread use of Cu supplements in conventional pig production). In addition, Fe concentrations in meat may be increased by access to the outside or higher proportions of forage in the diet (as recommended by organic farming standards), as forages contain higher Fe concentrations than concentrate feeds, and it is well recognised that piglets with access to soil in their environment do not need Fe injections, routinely used in housed production systems(60). In contrast, Cu deﬁciency in organically reared calves was linked to high forage and low concentrate intakes in one recent study(61), and this may have been due to low Cu contents in soils used for forage production and/or the mineral supplements in the concentrate feed used for rearing calves in conventional systems. Other potential sources of heterogeneity are the range of dif- ferent livestock species, meat types and countries and/or variable study designs and methodologies used in the studies from which data were extracted. Links between livestock management and meat composition/quality In contrast, in conventional beef, pork and poultry (and in some regions also lamb and goat) production, there has been a trend towards (1) reduced outdoor grazing or all-year-round housing and (2) reductions in both fresh and conserved forage intakes, but (3) increased use of concentrate feeds based on maize, other cereals, soya, other grain legumes and by-products from the food processing industry(53–55). Feeding regimens. A range of controlled animal experiments showed that high grazing/forage-based diets (similar to those prescribed under organic farming standards) reduce the total fat and/or nutritionally undesirable SFA (12 : 0, 14 : 0 and/or 16 : 0) content, while increasing concentrations of total PUFA, n-3 PUFA and VLC n-3 PUFA in meat, compared with concentrate- based diets (typical for intensive conventional farming systems)(43–45). These results suggest that the relative diver- gence in feeding practices between the organic and conven- tional livestock sectors is a major driver for both the differences in meat FA composition between systems and the variability of results between countries/regions and individual studies detected in this study by meta-analyses. Breed choice. The use of traditional and robust breeds/geno- types is often recommended by organic sector bodies and advisors. However, there is limited information on the relative differences in breed choice/breeding regimens between organic and conventional beef cattle, lamb, goat, pig and poultry production systems, and the papers used for meta- analyses provided no or insufﬁcient data on the breeds used in the organic and conventional systems they compared. It was therefore not possible to determine whether breed choice contributed signiﬁcantly to the composition differences reported in this study. However, controlled experimental studies have demonstrated that breed choice does affect FA proﬁles of meat(43–45). Differences in meat composition (e.g. for n-3 PUFA) reported by controlled experimental studies are greater than the differ- ences detected in this study between organic and conventional meat by meta-analysis, especially for ruminant livestock – for example, in beef production, a switch from grain- to grass- based ﬁnishing diets produced signiﬁcant increases in total PUFA (45 %), total n-3 PUFA (>3-fold), ALA (>3-fold), EPA (>5-fold), DPA (>2-fold) and DHA (129 %) in the intramuscular Grassland/forage composition. The composition of grazing swards and conserved forages may also partially explain the differences between organic and conventional meat. Most D. Średnicka-Tober et al. 1006 to facilitate inclusion in WM and (3) the establishment of registers of primary research(29). importantly, forage-legume (e.g. Links between livestock management and meat composition/quality In addition, data used in the meta-analyses were collected over a >20-year period and agronomic practices in both organic and conventional systems may have changed over time; this may also have contributed to heterogeneity. As described in previous reviews focused on composition differences between organic and conventional crop-based foods(5,25), pooling diverse information was necessary, because for most composition parameters the number of published studies available was insufﬁcient to carry out separate meta-analyses for speciﬁc countries/regions, livestock species/meat types or study types. Consequently heterogeneity was high, although only PUFA appeared to be sensitive to variable inclusion criteria. Potential nutritional impacts of composition differences The 47 % higher total n-3 PUFA concentration detected by WM and estimated 17 % higher n-3 PUFA intake with organic meat could therefore be potentially beneﬁcial, especially if intakes of VLC n-3 PUFA were increased. However, it is currently unclear whether there are systematic differences in VLC n-3 PUFA concentrations between organic and conventional meat, because there is currently insufﬁcient data to carry out WM comparing VLC n-3 PUFA concentration in most individual meat types. UM were possible for a larger number of meat types and detected higher concentrations of VLC n-3 PUFA in beef, but not other meat types for which sufﬁcient data were available. Meat fat is an important source for VLC n-3 PUFA. Average consumption levels of meat have been estimated to be 240 and 340 g/d per person, with red meat at 184 and 270 g/d per person in Europe and the USA, respectively(62). For the majority of North American and European consumers, meat is therefore the main dietary source for VLC n-3 PUFA, supplying up to an estimated 50 % of the recommended adequate intake. A priority for future studies should therefore be to substantially expand the evidence base for VLC n-3 PUFA for all meat types to allow accurate estimates of composition differences and dietary intakes with organic and conventional meat. The currently very high level of meat, particularly red meat, consumption is thought to be nutritionally undesirable, as it has been linked to obesity, CVD, type 2 diabetes and a range of cancers(77). Current dietary recommendations in the USA and Europe are to reduce red meat intakes to <70 g/d(78,79). Com- pliance with these guidelines will substantially reduce total fat and VLC n-3 intakes. The need to identify alternative approaches to increase VLC n-3 PUFA intake is discussed in the supplementary data (see online additional discussion section). Although UM of pooled data for all meat types and beef indicated that organic production may reduce the LA:ALA and n-6:n-3 ratio, this cannot currently be conﬁrmed by WM. These ratios may be nutritionally relevant, as additional VLC n-3 PUFA may be generated from dietary ALA, because humans can elongate ALA to produce longer-chain n-3 PUFA(17,24,63–75). However, ALA to EPA conversion rates are thought to be low in humans and synthesis of DHA is very low, especially in men(71). Potential nutritional impacts of composition differences The high inconsistency and low precision of meta-analyses for many meat composition parameters may reﬂect both the paucity of information and variability associated with agri- production systems and especially livestock diets (see detailed description below). This highlights the need for (1) further well-designed studies delivering substantial additional primary data sets, (2) reporting of measures of variance in publications Fat composition. The lower thrombogenicity index detected by UM for organic meat fat was due to both (1) lower concentra- tions of undesirable 14 : 0 and 16 : 0 (linked to an increased risk of CVD) and (2) higher concentrations of n-3 PUFA (linked to a decreased risk of CVD) found in organic meat. However, it should be pointed out that the thrombogenicity index as a Composition of organic meat products 1007 predictor for CVD risk(19) has not so far been validated in human dietary intervention or cohort studies. It is therefore currently not possible to estimate to what extent the changes in FA proﬁles and intakes may affect CVD risk (see also discussion below). Overall, results of the meta-analyses indicate that the relative impact of using organic production methods on meat FA proﬁles differs between livestock species. The impact of switching to organic meat consumption therefore not only depends on the amount but also on the type of meat consumed. However, there are large differences in the relative amounts of beef, lamb, pork and chicken meat fat consumed between countries/regions in the EU and elsewhere(42). In addition, calculations of estimated FA intakes assumed that (1) fat concentrations in organic and conventional meats are similar and (2) there is no difference in the relative proportion of different types of meat consumed by organic and conventional consumers, whereas there is insufﬁ- cient published information to conﬁrm that these assumptions are correct. However, it is well documented that (1) meat intakes vary considerably between individuals, (2) the FA composition of intramuscular fat may differ signiﬁcantly from that of subcutaneous/storage(48) and (3) meat processing and con- sumption methods (e.g. amount of fat being removed) may greatly affect both total fat and FA intakes. Estimates of total daily FA intakes calculated using data on current average EU meat fat consumption therefore have to be interpreted with caution. Increasing n-3 (especially VLC n-3) PUFA intake in human diets has been linked to a range of other health beneﬁts in humans(16,17,21–23). Potential nutritional impacts of composition differences The proportion of ALA (the main n-3 in the human diet) converted to longer-chain n-3 FA in humans is thought to increase with decreasing LA:ALA ratios in the diet, as ALA and LA compete for Δ6 desaturase activity(24). In addition, the nutritional impact of switching consumption from conventional to organic meat (or that from other high-forage systems) relating to higher n-3 PUFA intakes (and conversion of ALA to VLC n-3 PUFA) will depend on a range of other dietary factors including total fat intake, the proportion of dairy products, meat and vegetable fat in total fat intake, the type of vegetable fats in the diet and the relative capacity of individuals to convert/elongate ALA into longer-chain n-3 PUFA(17,24,63–75). Minerals. Owing to the very limited evidence base, it is not currently possible to estimate differences in mineral composi- tion and potential impacts on human health. The need to investigate the potential effects of organic and conventional production protocols on the mineral composition of meat is discussed in the supplementary data (see online additional discussion section). Deﬁciencies in the evidence base Meat composition data. Compared with the large amount of comparative composition data now available for crop-based foods(25), the data sets available for the meta-analyses of meat composition parameters reported in this study were limited. Results showed low statistical power for many parameters and limited ability to understand between-study heterogeneity, and these are the major reasons for the very low or low overall reliability for many of the outcomes. However, for a range of composition parameters for which signiﬁcant differences were detected, the method of synthesis did not have large effects, in terms of either statistical signiﬁcance or effect magnitude. Additional data from further, well-designed studies would alleviate the current uncertainties in the evidence and may allow exploration of between-study covariates. Future studies should be registered to eliminate potential publication biases. Apart from FA proﬁles, a particular emphasis should be placed on comparing nutritionally important meat composition A recent dietary intervention study showed that concentrations of VLC n-3 PUFA in both plasma and platelets were signiﬁcantly higher in individuals consuming pasture-ﬁnished compared with concentrate-ﬁnished beef and lamb(76). This indicates that consumption of meat from grazing/forage-based systems (such as organic meat) may raise VLC n-3 concentrations in the human body, although it is currently unclear to what extent this is due to (1) higher VLC n-3 intakes or (2) higher ALA to VLC n-3 conversion associated with the low LA:ALA ratio in meat from grazing-based systems. D. Średnicka-Tober et al. 1008 parameters for which there are currently no or too few studies to carry out meta-analyses, especially antioxidants/vitamins (e.g. vitamin A, vitamin B1, B6 and B12, riboﬂavin, folate, niacin, pantothenic acid) and minerals (e.g. Fe, Zn, Se) for which meat is a major dietary source. Overall, the present study indicates that organic livestock production may change the FA proﬁles, and possibly other composition parameters, and that some of these changes (e.g. higher n-3 PUFA) may be nutritionally desirable. It is therefore important to carry out additional studies to address the limita- tions in the current evidence base. If nutritionally relevant composition differences can be conﬁrmed and/or linked to speciﬁc agronomic practices (e.g. high forage diets), this would then justify dietary intervention or cohort studies designed to identify the impact of consuming meat with contrasting com- position generated by switching to organic production or spe- ciﬁc agronomic practices. Effect of speciﬁc agronomic practices. Acknowledgements Support from Lord Peter Melchett (Policy Director, Soil Asso- ciation, Bristol, UK) and Bruno Martin (Centre Clermont- Ferrand-Theix, Institut National de la Recerche Agronomique, INRA, Saint Genès Champanelle, France) for the critical review/ editing of the manuscript is gratefully acknowledged. The authors are grateful for funding from the European Community ﬁnancial participation under the Sixth Framework Programme for Research, Technological Development and Demonstration Activities for the Integrated Project QUALITY- LOWINPUTFOOD, FP6-FOOD-CT-2003-506358. The authors also gratefully acknowledge ﬁnancial and technical support from the Sheepdrove Trust for ‘The meta-analyses of data on composition of organic and conventional foods’. The Sheep- drove Trust supports independent R&D underpinning the development of organic and sustainable farming and food systems. Financial support by the Trust was without conditions, and the Trust had no inﬂuence on the design and management of the research project and the preparation of publications from the project. Dietary intervention and cohort studies. Potential impacts of composition differences in meat composition on human health (e.g. risk of CVD) currently have to be extrapolated from existing information about the effects of compounds such as 12 : 0, 14 : 0 and 16 : 0 SFA, LA and n-3 (especially VLC n-3) PUFA on human health, as there are a few studies that have assessed impacts of organic food consumption on animal or human health or health-related biomarkers. If the signiﬁcant differences in nutritionally relevant compounds identiﬁed in this study are conﬁrmed, this would highlight the need to carry out human dietary intervention and cohort studies designed to quantify the potential health impacts of switching to organic food production. Experimental studies comparing meat from non-organic forage and concentrate-based production systems suggest that other grazing-based livestock production systems deliver similar improvements in FA proﬁles(43–45) and poten- tially other meat-quality parameters. This should be considered in the design of future dietary intervention/cohort studies. p j D. Ś.-T. is a nutritionist who carried out a major part of the literature search and extraction and contributed to writing the manuscript. M. B. is an animal and food scientist who designed the database, carried out most of the meta-analyses and con- tributed to writing the manuscript. C. S. is a human nutritionist who contributed to the design of the study, discussion of potential health impacts of composition differences and the critical review of the manuscript. R. S. Deﬁciencies in the evidence base Current knowledge on the effect of feeding regimens on meat quality and the results of the meta-analyses reported in this study suggest that increasing the requirements for grazing and applying further restrictions on the use of concentrate feeds (especially during the ﬁnishing period) under organic and other extensive (e.g. pasture-reared) production standards will further improve the nutritional quality of meat and the differential in quality compared with meat products from intensive indoor meat production systems(48). However, additional well-designed comparative studies are needed to increase the sensitivity of meta-analyses and to quantify more speciﬁcally which production system parameters (e.g. speciﬁc feed composition components, especially during the ﬁnishing period, breed choice/breeding systems, veterinary interventions) are the most signiﬁcant drivers for nutritionally relevant composition differences for different livestock species. Acknowledgements is an animal scientist, who supported the literature search, critical review of the manuscript and the discussion relating to interactions between feeding regimens and meat quality. M. K. L. is a biochemist/nutritionist and provided data sets and supported the literature review and critical review of the manuscript. T. J. is the Nafferton Ecological Farming Group ofﬁce manager and supported the literature search and data extraction. U. N. is head of FiBL, Europe’s largest organic farming institute, and supported the literature review (especially with respect to studies linking feeding regimens and meat quality parameters) and critical review of the manuscript. T. S. is an animal physiologist and supported the literature review and critical revision of the manuscript, especially with respect to studies from Eastern European countries. P. C. C. is a nutritionist who supported the preparation (in particular introduction and discussion sections describing potential health impacts of changes in fatty acid proﬁles in meat) and critical review of the manuscript. G. C. B. is a nutritionist who supported the pre- paration (in particular introduction and discussion sections describing potential health impacts of changes in fatty acid proﬁles in meat) and critical review of the manuscript. S. S. is a veterinarian and animal scientist who supported the literature review and critical review of sections of the discussion. A. S. is a veterinarian and animal scientist who supported the literature review and critical review of sections of the introduction and discussion. H. Y. is forage-production agronomist who sup- ported the literature review and preparation of the discussion sections dealing with associations between forage-based feeding regimens and meat composition. S. S. is an animal scientist who supported the literature review and prepared sections of the discussion. G. B. is an animal nutritionist/scientist who sup- ported the literature review and critical review of the manuscript. G. S. is a lecturer in Evidence Synthesis who provided advice on the conduct and interpretation of the meta-analysis and critical review of the manuscript. C. L. is an agronomist specialising on agricultural production systems design/improvement and the study of interactions between agronomic practices and food quality and safety. He led the design of the study, management of research project and the preparation of the manuscript. 3. Oughton E & Ritson C (2007) Food consumers and organic agriculture. In Handbook of Organic Food Quality and Safety, pp. 74–94 [J Cooper, U Niggli and C Leifert, editors]. Cambridge: Woodhouse Publishing Ltd. 1. Willer H & Kilcher L (2011) The World of Organic Agriculture. Statistics and Emerging Trends 2011. FiBL-IFOAM Report. Rheinbreitbach, Germany: IFOAM, Bonn and FiBL, Frick. Acknowledgements Ulbricht TL & Southgate DA (1991) Coronary heart disease: seven dietary factors. Lancet 338, 985–992. The senior author of the paper, C. L., owns farmland in Germany that is managed by conventional farming standards and a smallholding in Greece that is managed by organic farming standards. 20. Calder PC (2015) Marine omega-3 fatty acids and inﬂamma- tory processes: effects, mechanisms and clinical relevance. Biochim Biophys Acta 1851, 469–484. 21. European Food Safety Authority (2010) Scientiﬁc opinion on dietary reference values for fats, including saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, trans fatty acids, and cholesterol. EFSA J 8, 1461. Acknowledgements 4. Dangour AD, Dodhia SK, Hayter A, et al. (2009) Nutritional quality of organic foods: a systematic review. Am J Clin Nutr 90, 680–685. 5. Brandt K, Leifert C, Sanderson R, et al. (2011) Agroecosystem management and nutritional quality of plant foods: the case of organic fruits and vegetables. Crit Rev Plant Sci 30, 177–197. 6. Cooper J, Niggli U & Leifert C (2007) Handbook of Organic Food Safety and Quality. Cambridge: CRC Press. Food Safety and Quality. Cambridge: CRC Press. 7. Williams P (2007) Nutritional composition of red meat. Nutr Diet 64, S113–S119. 8. Palupi E, Jayanegara A, Ploeger A, et al. (2012) Comparison of nutritional quality between conventional and organic dairy products: a meta-analysis. 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Wijendran V & Hayes KC (2004) Dietary n-6 and n-3 fatty acid balance and cardiovascular health. Annu Rev Nutr 24, 597–615. 16. Simopoulos AP & Cleland LG (editors) (2003) Omega-6/ omega-3 Essential Fatty Acid Ratio: The Scientiﬁc Evidence. Basel, Switzerland: Karger. 17. Givens DI & Gibbs RA (2008) Current intakes of EPA and DHA in European populations and the potential of animal-derived foods to increase them. Proc Nutr Soc 67, 273–280. 18. Blasbalg TL, Hibbeln JR, Ramsden CE, et al. (2011) Changes in consumption of omega-3 and omega-6 fatty acids in the United States during the 20th century. Am J Clin Nutr 93, 950–962. 19. Acknowledgements is an environmental modeller and data analyser, who helped design the literature search and database storage, helped to design the study and provided guidance the meta-analyses used. C. B. is an agronomist specialising on organic production systems, who supported the literature review (especially with respect to stu- dies in North and South America) and the preparation/review of the manuscript. H. S. is an animal nutritionist, who supported the literature review and critical revision of the manuscript, especially with respect to studies from Scandinavian countries. J. G.-O. is a human nutritionist, who supported the literature review and the discussion of potential health impacts of composition differences identiﬁed in the meta-analyses. E. R. is a human nutritionist, who supported the literature review and critical revision of the manuscript, especially with respect to human intervention studies focused on health impacts of organic food consumption. K. S.-S. is an animal nutritionist/ The potential of carrying out dietary intervention/cohort studies was demonstrated by a recent investigation into the effect of organic milk consumption on eczema in children younger than 2 years of age in the Netherlands (a country with relatively high milk consumption)(64). It reported that eczema was signiﬁcantly reduced in children from families consuming organic rather than non-organic milk. This may have been caused by the higher n-3 PUFA concentrations and lower n-6:n-3 PUFA ratio in organic milk, as there is increasing evi- dence for anti-allergic effects of n-3 FA(65) – for example, a recent animal study showed that increasing dietary VLC n-3 PUFA intake prevents allergic sensitisation to cows’ milk protein in mice(66). However, it is important to point out that there are so far no cohort studies showing a link between organic meat consumption and reduced incidence in eczema and other positive health outcomes. Composition of organic meat products 1009 physiologist who supported the literature review and critical revision of the manuscript, especially with respect to animal dietary intervention studies focused on physiological and health impacts of organic feed consumption. M. E. is an ecologist and statistician who advised and supported the statistical analyses. G. C. is an animal scientist, who supported the literature search, critical review of the manuscript and the discussion relating to interactions between feeding regimens and meat quality. M. K. L. is a biochemist/nutritionist and provided data sets and supported the literature review and critical review of the manuscript. T. J. Supplementary material For supplementary material/s referred to in this article, please visit http://dx.doi.org/doi:10.1017/S0007114515005073 22. Massiera F, Barbry P, Guesnet P, et al. (2010) A western-like fat diet is sufﬁcient to induce a gradual enhancement in fat mass over generations. J Lipid Res 51, 2352–2361. Acknowledgements is the Nafferton Ecological Farming Group ofﬁce manager and supported the literature search and data extraction. U. N. is head of FiBL, Europe’s largest organic farming institute, and supported the literature review (especially with respect to studies linking feeding regimens and meat quality parameters) and critical review of the manuscript. T. S. is an animal physiologist and supported the literature review and critical revision of the manuscript, especially with respect to studies from Eastern European countries. P. C. C. is a nutritionist who supported the preparation (in particular introduction and discussion sections describing potential health impacts of changes in fatty acid proﬁles in meat) and critical review of the manuscript. G. C. B. is a nutritionist who supported the pre- paration (in particular introduction and discussion sections describing potential health impacts of changes in fatty acid proﬁles in meat) and critical review of the manuscript. S. S. is a veterinarian and animal scientist who supported the literature review and critical review of sections of the discussion. A. S. is a veterinarian and animal scientist who supported the literature review and critical review of sections of the introduction and discussion. H. Y. is forage-production agronomist who sup- ported the literature review and preparation of the discussion sections dealing with associations between forage-based feeding regimens and meat composition. S. S. is an animal scientist who supported the literature review and prepared sections of the discussion. G. B. is an animal nutritionist/scientist who sup- ported the literature review and critical review of the manuscript. G. 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https://openalex.org/W2051359566	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0049552&type=printable	English	null	Phylogenetic Patterns of Geographical and Ecological Diversification in the Subgenus Drosophila	PloS one	2,012	cc-by	12,860	Abstract Colonisation of new geographic regions and/or of new ecological resources can result in rapid species diversification into the new ecological niches available. Members of the subgenus Drosophila are distributed across the globe and show a large diversity of ecological niches. Furthermore, taxonomic classification of Drosophila includes the rank radiation, which refers to closely related species groups. Nevertheless, it has never been tested if these taxonomic radiations correspond to evolutionary radiations. Here we present a study of the patterns of diversification of Drosophila to test for increased diversification rates in relation to the geographic and ecological diversification processes. For this, we have estimated and dated a phylogeny of 218 species belonging to the major species groups of the subgenus. The obtained phylogenies are largely consistent with previous studies and indicate that the major groups appeared during the Oligocene/Miocene transition or early Miocene, characterized by a trend of climate warming with brief periods of glaciation. Ancestral reconstruction of geographic ranges and ecological resource use suggest at least two dispersals to the Neotropics from the ancestral Asiatic tropical disribution, and several transitions to specialized ecological resource use (mycophagous and cactophilic). Colonisation of new geographic regions and/or of new ecological resources can result in rapid species diversification into the new ecological niches available. However, diversification analyses show no significant support for adaptive radiations as a result of geographic dispersal or ecological resource shift. Also, cactophily has not resulted in an increase in the diversification rate of the repleta and related groups. It is thus concluded that the taxonomic radiations do not correspond to adaptive radiations. Citation: Morales-Hojas R, Vieira J (2012) Phylogenetic Patterns of Geographical and Ecological Diversification in the Subgenus Drosophila. PLoS ONE 7(11): e49552. doi:10.1371/journal.pone.0049552 Editor: Nadia Singh, North Carolina State University, United States of America Received May 15, 2012; Accepted October 15, 2012; Published November 12, 2012 Received May 15, 2012; Accepted October 15, 2012; Published November 12, 2012 ales-Hojas, Vieira. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2012 Morales-Hojas, Vieira. This is an open-access article distributed under the terms of the Creative Commons Attribu unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction and, while the phylogeny, ecology and distribution for some species are not well characterized, there is broad information for most of the species groups [12,13]. There is some difficulty in resolving the phylogenetic relationships between the main groups of Drosophila, which has been attributed to the rapid divergence of these lineages as the descendants adapted to a variety of ecological resources [12]. This pattern, if confirmed, could indicate that the Drosophila lineage is the result of an adaptive radiation. The large diversity of life forms that we see today is the result of different biological processes, one of which is adaptive radiation [1,2,3]. Adaptive radiations refer to lineages that exhibit a di- versification of species intodifferent ecologicalniches.This process is generally triggered by ecological opportunity, in which an under- utilized environment becomes available to an ancestral species allowing a rapid species diversification into the new ecological niches available. The new environment can be colonized either by the evolutionofakeyinnovation,thedispersalintoanewgeographicarea or by the extinction of antagonist species [4,5]. The study of the rates of diversification across phylogenetic lineages can be used to identify adaptive radiations and provides valuable information about the processesthat underlietheoriginofbiologicaldiversity[3].Apattern that is generally considered to be the result of an adaptive radiation is when there is a rapid origin of species that adapt to a diversity of ecological niches followed by a slow down of the diversification rate through time as the new niches become occupied [2,6]. This is a common pattern observed in many taxonomic groups (e.g. [7,8,9,10]). p g p The genus Drosophila is paraphyletic as several other genera are included within the phylogeny of the Drosophila [12]. Ten subgenera are presently recognized within the genus Drosophila, of which the Sophophora and Drosophila are the major ones [14]; these are further taxonomically subdivided into radiations and species groups. It should be noted that the term radiation refers to a taxonomic rank that comprises several closely related species groups, and should not be confused with an adaptive radiation. Furthermore, it has never been tested whether these taxonomic radiations correspond to adaptive radiations. Of the two main subgenera, Drosophila is the largest one. It has a wide distribution and some of its members show interesting ecological niches such as fungi and cacti. Abstract Funding: This work was funded by the Fundac¸a˜o para a Cieˆncia e Tecnologia (FCT) through research projects PTDC/BIA-BDE/66765/2006 and PTDC/BIA-BEC/ 099933/2008, funded by Programa Operacional para a Cieˆncia e Inovac¸a˜o (POCI-2010), co-funded by Fundo Europeu para o Desenvolvimento Regional (FEDER) and Programa Operacional para a Promoc¸a˜o da competividade (COMPETE). Ramiro Morales-Hojas is supported by a Cieˆncia 2007 contract. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rmhojas@ibmc.up.pt Ramiro Morales-Hojas, Jorge Vieira Molecular Evolution Lab, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Molecular Evolution Lab, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Phylogenetic Patterns of Geographical and Ecological Diversification in the Subgenus Drosophila Ramiro Morales-Hojas, Jorge Vieira November 2012 \| Volume 7 \| Issue 11 \| e49552 PLOS ONE \| www.plosone.org Phylogenetic Analyses Phylogenetic reconstruction was performed using Bayesian Inference (BI) and Maximum Likelihood (ML) as optimality criteria. For the BI approach, phylogenetic relationships between the species were estimated at the same time as the estimation of the divergence times with BEAST version 1.7.2 [32] run in CIPRES Science Gateway [33]. The divergence times were estimated under the uncorrelated relaxed-clock tree model [34]. Runs were performed allowing for different substitution rates and clocks at nuclear and mitochondrial genes. The model of evolution used for each data partition was the GTR+I+G. Runs were performed with two different matrices: 1) all 218 species, which included non- overlapping characters; and, 2) alignment of 153 species with overlapping sequences (2–3 genes). Also, two calibration schemes were used in these analyses. First, the calibration of the tree was done using dates of divergence obtained from the literature for 9 nodes: 1) antopocerus-modified tarsi species groups, mean = 9 Mya61.0 (standard deviation) [35]; 2) haleakalae species group, mean = 10 Mya61.0 (stdev) [35]; 3) modified mouthparts group, mean = 16 Mya61.0 (stdev) [35]; 4) picturewing-nudidrosophila groups, mean = 15 Mya61.0 (stdev) [35]; 5) D. picticornis - planitibia group, mean = 4.6 Mya60.3 (stdev) [36,37]; 6) virilis group, mean = 9.4 Mya60.6 (stdev) [29]; 7) virilis subgroup, mean = 4.05 Mya60.6 (stdev) [29]; 8) montana subgroup, mean = 4.9 Mya60.5 (stdev) [29]; and 9) D. mojavensis - D. arizonae, mean = 2.4 Mya60.3 (stdev) [38]. Calibrating times were introduced in the analysis as priors with a normal distribution; the standard deviation specified for each calibration point was chosen to include the confidence limits reported in the respective studies. As some of these calibration points are approximate estimates and had no confidence limits associated to them, analyses were also run using only 5 calibration points based on geological and climatic data and that had associated intervals: 1) D. picticornis - planitibia group, mean = 4.6 Mya60.3 (stdev) [36,37]; 2) virilis group, mean = 9.4 Mya60.6 (stdev) [29]; 3) virilis subgroup, mean = 4.05 Mya60.6 (stdev) [29]; 4) montana subgroup, mean = 4.9 Mya60.5 (stdev) [29]; and 5) D. mojavensis - D. arizonae, mean = 2.4 Mya60.3 (stdev) [38]. The divergence between D. picticornis and other species from the planitibia subgroup has been estimated to have occurred 5.1 mya based on the oldest surface rock of the island of Kauai [36,37]. Drosophila Diversification Patterns Drosophila Diversification Patterns excludes the genus Hirtodrosophila in contrast to the immigrans- Hirtodrosophila radiation of Throckmorton [11]. The phylogenetic position of the funebris group is not well resolved, and some studies suggest that it is part of the immigrans-tripunctata radiation [15,16]. It should be noted that the subgenus Drosophila is paraphyletic and includes the Hawaiian Drosophilidae (Hawaiian Drosophila + genus Scaptomyza), which form a monophyletic, sister group to the virilis- repleta radiation [15,17] or to the virilis, robusta, melanica clade within the virilis-repleta radiation [18]. This Hawaiian lineage comprises approximately 1000 species and it forms an adaptive radiation of its own, with a large diversity of forms and ecological niches [19]. some of the taxa had data for just one gene, it has been shown that highly incomplete taxa can still be accurately placed in the phylogeny if there are overlapping characters [30]. As the DNA alignment included non-overlapping characters, analyses were also run with 153 species for which there was sequence data for 2–3 genes. This controlled for any potential bias introduced in the phylogenies by non-overlapping characters. Alignment of gene sequences was done with Clustal X v2.0 [31] with minor adjustments by eye based on the amino-acid trans- lation of the cds to avoid alignment of non-orthologous nucleotides and distortion of the ORF. Alignment editing and coding sequence translation was done in Se-Al v2.0a11 [Rambaut (1996) http://tree.bio.ed.ac.uk/software/seal/]. , g y g [ ] Based on biogeographic data, the origin of the virilis-repleta radiation has been placed in the Old World tropics, most likely in the Asiatic tropical regions [11]; from this ancestor two lineages evolved leading to the Old World tropics (e.g. the polychaeta group) and temperate species groups (e.g. virilis, robusta and melanica species groups). A Neotropical radiation, which comprises the repleta, canalinea, mesophragmatica, dreyfusi, annulimana and nannoptera species groups, evolved from a third lineage of the Asiatic tropical ancestor. The origin of the immigrans-tripunctata radiation has also been placed in the Old World tropics, from where two lineages arose, the Asiatic immigrans group and the New World tripunctata radiation that comprises the tripunctata and closely related species group [11]. From an ecological point of view, the members of the subgenus Drosophila occupy a wide variety of niches, from sap feeders (e.g. robusta, melanica and virilis) to cactophilic species (e.g. repleta), mycophagous (e.g. quinaria) and flower feeders (e.g. Phylogenetic Analyses The mean value and associated standard deviation introduced as normal prior was chosen to incorporate the 1 my time span (5.1–4.1 mya) described to elapse between the pre-shield and shield stages according to the models of volcano growth. As outgroups we used D. melanogaster, D. yakuba, D. ananassae, D. pseudoobscura and D. willistoni. The analyses with 218 spp. and 9 calibration points were run for 2006106 generations, with sampling every 10000th generations. The first 5000 trees were discarded as burn-in and the remaining 15001 phylogenies were summarized using maximum clade credibility. The other three Given the widespread geographic distribution of the different species groups and the variety of the ecological resources they exploit, the subgenus Drosophila could represent an adaptive radiation. In order to test for this hypothesis we have estimated a time-calibrated phylogeny of 218 species representing the main species groups of the subgenus Drosophila. This phylogenetic analysis is the first attempt to date the divergence events of the main lineages of this group of organisms using a relaxed molecular clock approach. Therefore, the results of the present study will be relevant for many research areas because the subgenus Drosophila includes some of the best-studied model organisms in ecological and evolutionary research [22,23,24,25,26,27,28]. Based on the obtained phylogeny, and taking into account topological and dating uncertainties, we have reconstructed the ancestral states for the species’ geographic distribution and ecological resource use. We have also investigated the patterns of diversification of those lineages that dispersed into the Neotropic and acquired the capacity to exploit new resources. Drosophila Diversification Patterns tripunctata) [13,14], although many of the species are generalists and can exploit different resources. Cactophily is not observed in any species outside the lineage including the repleta, nannoptera and mesophragmatica species groups. As the ability to exploit cacti as an ecological resource implies acquiring the capacity to degrade an array of toxic compounds that are produced in rotting cacti material [20,21], cactophily can be considered an apomorphy of this Neotropical lineage. Introduction It comprises three major lineages: 1) the funebris species group; 2) the virilis-repleta radiation; and 3) the immigrans- tripunctata radiation, which following Remsen and O’Grady [15] The genus Drosophila is a large, diverse and widely distributed group of organisms [11]. Its taxonomy is relatively well established November 2012 \| Volume 7 \| Issue 11 \| e49552 PLOS ONE \| www.plosone.org 1 Ancestral State Reconstruction Two different approaches were used for the ancestral character state reconstruction of the geographic distribution and ecological resource use. First it was used the likelihood method implemented in Mesquite [42]. Character mapping was done on the summa- rized BI chronogram (218 spp. +9 calibration points) with the Hawaiian Drosophilidae clade pruned. Being a monophyletic, derived lineage its removal will not affect the assignment of ancestral states for the main lineages of the study. Furthermore, this lineage has been comprehensively investigated in a recent study [19]. In order to account for phylogenetic uncertainty, the Bayesian ancestral state reconstruction implemented in SIMMAP v1.5 [43,44] was used. This method estimates the marginal posterior probabilities of each possible character state at the internal nodes of a sample of phylogenies. To account for topological uncertainty we used a random sub-sample of 100 trees from the posterior distribution of phylogenies obtained with BEAST (218 spp, 9 calibrations). The parameters for the prior distributions of the models implemented in the analyses were estimated using an MCMC approach as described in the SIMMAP webpage. The temporal method BDL implemented in LASER was used to test for departure from constant rate and to detect rate shifts in the presence of extinction [48]. Rate-constant diversification models (RC) fitted to the data were pureBirth and birth-death models. The rate-variable (RV) models tested were the density- dependent models with exponential and logistic variants (DDX and DDL) and the yule2rate and yule3rate models, which allow for two and three different rates of speciation across the phylogeny, respectively. The models were fitted to the branching times of the maximum clade credibility (MCC) trees obtained with BEAST and shifts were only allowed at the branching times. The significance of the change in the Akaike Information Criterion (DAIC) scores between the RC and RV models was tested fitting the models to the branching times of a simulated sample of 5000 trees. In order to account for incomplete sampling, the trees were simulated to have the total number of species as the lineages analysed and were then pruned to contain the same number of tips as our phylogeny. The number of categories introduced in the ancestral re- construction was restricted by the limitation of SIMMAP 1.5, which accepts a maximum of 7 character states. Drosophila Diversification Patterns Drosophila Diversification Patterns analyses (218 spp.-5 calibration points, 153 spp.-9 and -5 calibration points) were run for 1006106 generations, sampling every 10000th generations. The burn-in was set to 10%. In order to confirm that the MCMC had run long enough to get valid estimates and establish the burn-in level, results were analysed with TRACER [39] and the effective sample size (ESS) confirmed to be greater than 200. The constant-rate (CR) test [45] was used to examine the departure of the lineage accumulation from the null hypothesis of a constant rate of diversification. The CR test evaluates the relative position of nodes in the phylogeny against the expected under a CR model of diversification using the c statistic, calculated with LASER [46] in R. Negative values of c indicate that the nodes are closer to the root than expected, signifying a deceleration in the rate of diversification; positive values indicate a bias towards the tips of the tree and denote acceleration in the diversification rate towards the present. The c-statistic was computed for 1000 posterior distribution trees from each of the two BEAST analyses to control for the uncertainty in topology and branching times. Incomplete taxon sampling can bias the CR test and, in order to correct for this effect we adjusted the critical value using the MCCR test [45] implemented in LASER. The total number of species in the lineages analysed was obtained from Markow and O’Grady [14] and the TaxoDros database (Table 1). Number of Monte Carlo simulations run was 5000. To test the significance of the empirical c value distribution estimated for the 1000 posterior trees, the average and median values of c from the 1000 posterior trees were compared to the critical c value obtained from the null distribution. A second source of bias in the c-statistic comes from the violation of the random sampling assumption. It has been recently shown that non-random taxonomic sampling inflates the type-I error of the CR and MCCR tests [47]. The degree of bias introduced in the analysis by non-random sampling is here evaluated using the scaling parameter a as described by Brock et al. [47]. Maximum Likelihood (ML) analyses were run using RAxML 7.2.8 [40,41] in CIPRES Science Gateway [33]. The analysis was performed with a partitioned dataset (one model for each gene Adh, ND2 and COI) and 1000 rapid bootstrap inferences were executed with a thorough ML search thereafter. Ancestral State Reconstruction The bio- geographic categories used in the analyses were based on the regions of TaxoDros (www.taxodros.uzh.ch): 0 - cosmopolitan (when one species is found in more than one region); 1 - European; 2 - African; 3 - North American; 4 - Neotropical; 5 - Asian; and 6 - Australia + Oceania. We are aware that the records for some species from the database can be dubious, nevertheless, as we are assessing general trends for a rather large lineage we are confident that incorrect records for a small proportion of species will not bias the results or their interpretation. The ecological resources included were those natural substrates from where Drosophila adults and larvae have been collected. Following Throckmorton [11] and Markow and O’Grady [13] with additional information obtained from the literature (table S2 and references therein), the following categories were used: 0 - generalist (species that use more than one type of substrate except cacti); 1 - mycophagous; 2 - frugivorous; 3 - sap feeders; 4 - cactophilic; and 5 - generalist + cactophilic (those species that can use any part of any plant including cacti). In order to detect exceptionally radiating lineages within the subgenus Drosophila we used MEDUSA [49] implemented in Geiger [50]. MEDUSA tests among-clade variation in rates of speciation (and not rate variation through time as the tests above) by combining phylogenetic information about the timing of splits with taxonomic richness data. The advantage of this method is that it accommodates incomplete sampling by using taxonomic richness information. MEDUSA was run with different backbone phylogenies containing 13 tips corresponding to the main monophyletic lineages of the phylogeny and combined with a species richness table (Table 1). The backbone phylogenies used were obtained by pruning the MCC trees from the 218 spp. with 9 calibrations and 5 calibrations analyses, to test for possible biases introduced by the different topologies and divergence dates. Those species groups that were paraphyletic, were combined into a single tip thus, the robusta clade was considered to have 44 spp. including the robusta, melanica, quadrisetata and clefta species groups, and the tripunctata clade (170 spp.) included the tripunctata, sticta, pallidipennis, Drosophila Diversification Patterns To estimate clade support, 350 bootstrap replicates were performed. Samples and Gene Sequences The phylogeny of the subgenus Drosophila was reconstructed using sequences for the nuclear Adh and the mitochondrial ND2 and COI for 218 species representing all the major lineages of the subgenus (table S1; sequences were obtained from GenBank with exception of the Adh sequences of some species of the virilis group, obtained by us [29]). More than 70% of the species included in the analyses have sequence data for at least two of the genes. While November 2012 \| Volume 7 \| Issue 11 \| e49552 PLOS ONE \| www.plosone.org 2 Phylogeny of the Subgenus Drosophila The BI analyses performed with 218 and 153 spp. to control for the bias of non-overlapping characters, and using 9 or 5 calibration points to control for less robust times of divergence, resulted in phylogenies that were consistent except for the placement of the polychaeta and angor groups (Fig. 1–3). The phylogeny obtained with the ML approach is also consistent in topology with the 218 (5 calibration points) and 153 spp. (Fig. 1). These results show that the presence of non-overlapping sequences is not a significant source of bias in the present study. However, the times of divergence estimated with 5 calibration points were significantly younger than those resulting from the analyses with 9 points (Fig. 1–3), which is likely the result of the removal of those at deeper nodes. The crown age of the subgenus Drosophila is placed in the Eocene/Oligocene transition around 34.33 Mya (30.24–38.30 Mya 95% HPD) when all 9 calibration points are used, while it is placed in the Oligocene/Miocene transition, 23.79 Mya (19.24– 28.83 Mya 95% HPD), when the calibration points used are reduced to 5 (Fig. 1 and 3). The phylogeny of the subgenus includes two clades corresponding to the immigrans-tripunctata [98% and 100% Bayesian Posterior Probability (BPP) in the BI with 9 and 5 calibrations, respectively; 60% bootstrap in the ML analysis] and the virilis-repleta (98% and 100% BPP; 55% bootstrap) radiations. The analysis using 9 calibration points places the crown ages of these lineages during the early Oligocene around 31 Mya (27.23–35.46 and 27.29–35.13 for the immigrans-tripunctata and virilis-repleta, respectively). When 5 calibration points were used the crown ages of these lineages were placed at 20 Mya (15.84– 24.38) and 22.86 Mya (18.67–27.76) for the immigrans-tripunctata and virilis-repleta lineages, respectively. p g , p y The first lineage to diverge within the virilis-repleta radiation in the BI phylogeny with 218 spp. and 9 calibration points is the polychaeta species group, with a crown age of 26.63 Mya (22.12– 30.93). However, in the ML and BI (218 spp. and 5 calibration points; 153 spp.) trees this lineage is placed as the sister group to the clade comprising the annulimana, nannoptera, dreyfusi, canalinea, mesophragmatica and repleta species groups, with bootstrap support ,50% and 99% BPP. cardini, guarani, testacea, calloptera, funebris, quinaria and macroptera. Species richness was taken from TaxoDros and Markow and O’Grady [14]. cardini, guarani, testacea, calloptera, funebris, quinaria and macroptera. Species richness was taken from TaxoDros and Markow and O’Grady [14]. Analyses of Diversification Analyses of diversification were run with the phylogenetic results obtained with 218 spp. and 9 calibration points, and with 153 spp. and 5 calibration nodes to test for potential bias as result of the different divergence times obtained with the different calibration points. November 2012 \| Volume 7 \| Issue 11 \| e49552 November 2012 \| Volume 7 \| Issue 11 \| e49552 PLOS ONE \| www.plosone.org 3 Drosophila Diversification Patterns Table 1. Summary data for the species groups of the Drosophila subgenus. Species group Number spp. Missing spp. angor 7 0 annulimana 16 14 calloptera 8 5 canalinea 14 12 cardini 16 9 clefta 3 0 dreyfusi 9 8 funebris 7 6 guarani 16 11 immigrans 104 84 macroptera 5 4 melanica 13 4 mesophragamatica 13 7 nannoptera 5 2 pallidipennis 1 0 polychaeta 10 3 quadrisetata 12 2 quinaria 33 25 repleta 101 57 robusta 16 2 sticta 1 0 testacea 4 3 tripunctata 79 57 virilis 13 1 Hawaiian Drosophila 1000 972 Number spp. is the extant number of species, missing spp. is the number of species listed in TaxoDros and Markow and O’Grady [14] not represented in the phylogeny. doi:10.1371/journal.pone.0049552.t001 Results Table 1. Summary data for the species groups of the Drosophila subgenus. Species group Number spp. Missing spp. angor 7 0 annulimana 16 14 calloptera 8 5 canalinea 14 12 cardini 16 9 clefta 3 0 dreyfusi 9 8 funebris 7 6 guarani 16 11 immigrans 104 84 macroptera 5 4 melanica 13 4 mesophragamatica 13 7 nannoptera 5 2 pallidipennis 1 0 polychaeta 10 3 quadrisetata 12 2 quinaria 33 25 repleta 101 57 robusta 16 2 sticta 1 0 testacea 4 3 tripunctata 79 57 virilis 13 1 Hawaiian Drosophila 1000 972 Number spp. is the extant number of species, missing spp. is the number of species listed in TaxoDros and Markow and O’Grady [14] not represented in the phylogeny. doi:10.1371/journal.pone.0049552.t001 Phylogeny of the Subgenus Drosophila The crown age of the polychaeta species group estimated in this case was 18.42 Mya (14.56–22.81) The monophyly of the repleta radiation, which includes the repleta, mesophragmatica and canalinea species groups, is recovered although it is only well supported in the BI trees (85% to 91% BPP). Its sister clade is formed by the nannoptera and annulimana species groups. D. camargoi, member of the dreyfusi species group, is placed as sister species to the nannoptera, although this is not well supported by BPP or bootstrap. The crown age of the repleta radiation is estimated to be 24.36 Mya (21.04–27.78) or 18.31 Mya (14.61–22.36) with 9 or 5 calibration points, respectively, and it shared a last common ancestor with its sister species groups (nannoptera and annulimana) 27.76 Mya (24.34–31.50, 218 spp., 9 calibration points) or 19.78 Mya (15.83–24.12, 218 spp., 5 calibration points). The clade comprising the virilis, Hawaiian Drosophilidae, robusta, melanica, quadrisetata and angor species groups is recovered with different support (Fig. 1 and 3). This clade has an estimated crown age of 29.71 Mya (26.37–33.32) and 22.02 Mya with 9 and 5 calibration points, respectively. The first group to diverge in this clade is the angor group, with an estimated crown age of 24.91 Mya (21.23– 28.48), but surprisingly it is not recovered as monophyletic in the BI analysis with 5 calibration points. Also, the angor species group is placed as the sister clade of the polychaeta-repleta lineage in the BI analysis with 153 spp. and 9 calibration points (Fig. 2). Monophyly Number spp. is the extant number of species, missing spp. is the number of species listed in TaxoDros and Markow and O’Grady [14] not represented in the phylogeny. doi:10.1371/journal.pone.0049552.t001 Number spp. is the extant number of species, missing spp. is the number of species listed in TaxoDros and Markow and O’Grady [14] not represented in the phylogeny. doi:10 1371/journal pone 0049552 t001 Test of Macroevolutionary Hypothesis Bursts of diversification can be the result of the invasion of previously unoccupied ecological niches. Whether adaptation to cacti in the repleta and closely related species groups has allowed an increased rate of diversification in this group has been tested with BiSSE [51], implemented in Mesquite [42]. BiSSE uses a likelihood-based approach to test the association of a discrete character, in the present case being cactophilic or not, with the rate of diversification of different lineages of a phylogeny. The likelihoods of our empirical data (summarized chronogram of the Drosophila subgenus and character states) were estimated under two models, unconstrained and constrained, using BiSSE. The unconstrained model had all parameters (l, m and q) free to vary while the constrained model was forced to have the same speciation rate for both character states (l0 = l1). The statistical significance of the log-likelihoods difference was tested with a likelihood ratio test assuming a x2 distribution with one degree of freedom. PLOS ONE \| www.plosone.org November 2012 \| Volume 7 \| Issue 11 \| e49552 4 Drosophila Diversification Patterns November 2012 \| Volume 7 \| Issue 11 \| e49552 November 2012 \| Volume 7 \| Issue 11 \| e49552 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org Drosophila Diversification Patterns Figure 1. Summarized chronogram for the subgenus Drosophila obtained with BEAST using 218 species and 9 calibration points. Numbers on nodes indicate ages for the corresponding nodes; asterisks on branches indicate BPP $ 90% and numbers are bootstrap support values. Bars represent the 95% highest posterior density (HPD) interval for the divergence times. doi:10.1371/journal.pone.0049552.g001 as cosmopolitan, which is in agreement with the holarctic origin inferred in a recent analysis of the systematics of this group [29]. The ancestral species of the melanica species group has an equivocal distribution being inferred as Asiatic by the ML approach (PL = 0.81) and as North American with the Bayesian method (North America PP = 0.63 vs. Asia PP = 0.32). The Neotropical origin of the repleta radiation and closely related species groups is supported by both approaches (PL = 0.68 and PP = 0.75). Simi- larly, the ancestral species of the annulimana, nannoptera and D. camargoi lineage and the repleta, mesophragmatica and canalinea are also estimated to be Neotropical. Nevertheless, the repleta species group has an equivocal origin, as the ML analysis supports a Neotropical origin (PL = 0.69 vs. Test of Macroevolutionary Hypothesis PL = 0.27 for a distribution across more than one region, namely the Neotropics and North America) and the Bayesian method inferred an ancestral distribution across more than one region (PP = 0.97 Neotropics and North American). A more detailed analysis of the biogeographic history of the repleta species group would need a better resolution of the phylogenetic relationships of the species it comprises. of the Hawaiian Drosophilidae, virilis, robusta, melanica and quadrisetata species groups is well supported in the BI trees (100% and 89% BPP with 9 and 5 calibration nodes, respectively) although the bootstrap support is low in the ML analysis (56%). The crown age of this clade is 27.95 (24.68–31.41) Mya or 21.15 (16.79–25.46) Mya with 9 or 5 calibration nodes, respectively. The position of the Hawaiian Drosophila differs in the phylogenies, being monophyletic with virilis in the BI analysis of 218 spp. and 9 calibration points, but being recovered as the sister clade to the virilis, robusta, melanica, quadrisetata and clefta species groups in the other analyses (Fig. 2–3). The member of the immigrans clade D. annulipes is placed as sister species to the Hawaiian Drosophila with high support (Fig. 1 and 3). The robusta species group is recovered as polyphyletic in the present analysis and very closely related to the melanica and quadrisetata species groups. The first species of the immigrans-tripunctata radiation to diverge is D. quadrilineata, a member of the immigrans group. The sister group is subdivided in two clades, one corresponding to the immigrans group and the second clade including the tripunctata, pallidipennis, quinaria, cardini, guarani, testacea, macroptera, calloptera and funebris species groups (Fig. 1–3). These two clades have crown ages corresponding to the Oligocene/Miocene transition or mid- Miocene depending on the analysis. Thus, the tripunctata and closely related groups have a crown age of 24.84 (21.69–28.04) Mya with 9 calibration points, or 17.48 (14.05–21.33) Mya with 5 calibration points. The immigrans species group (excluding D. quadrilineata) have crown ages of 22.95 (19.02–26.83) Mya (9 calibration points) or 15.57 (11.91–19.57) Mya (5 calibration points). The tripunctata is recovered as polyphyletic. The species D. funebris is also recovered as closely related to the quinaria and macroptera species groups, although its placement differs between the BI and ML trees. Evolution of Ecological Resource Use g Results of the ancestral reconstruction of the ecological resource use are shown in Fig. 4B and Table 3. Results indicate that the ancestor of the Drosophila subgenus was a generalist, although the support for this state is not strong in either of the two approaches (PL = 0.57 and PP = 0.55). The alternatives, however, show also low probabilities (the second most supported state is frugivore with a PL = 0.20 and PP = 0.27). The inferred ancestral state of the immigrans species group is also equivocal; the ML method results indicate that the ancestral species of the group was a generalist (PL = 0.51) or frugivorous (PL = 0.48), the Bayesian approach supports the frugivorous ancestral state with a posterior probability of 0.88 (the generalist state has a PP of 0.11). The majority of the species of this group included in the analyses are frugivorous, nevertheless, six species (D. albomicans, D. oritisa, D. ruberrima, D. signata, D. immigrans and D. curviceps) show a more generalist ecological usage having been caught in tree sap, fungi and fruit (table S2 and references included). This indicates that at least some species retained the ancestral character state of the immigrans- tripunctata lineage. Within the immigrans-tripunctata lineage the quinaria and macroptera groups are specialized in using fungi as resource [although some of the species of this group may be generalist such as D. quinaria [13]]. In our analyses, it is equivocal when these groups became mycophagous. While the ML reconstruction approach inferred that the ancestor of the funebris, quinaria and macroptera was a generalist with a proportional likelihood of 0.89, the Bayesian method resulted in a posterior probability of being mycophagous of 0.55 (0.44 for the generalist state). Although the species of the funebris group use fungi as resource, they can also use other resources [13]. The ML and Bayesian methods indicate that the ancestral species of the quinaria and macroptera was mycophagous (PL = 0.98 and PP = 0.99). It should be noted that some of the generalist species of the immigrans group also use fungi as ecological resource; therefore, we interpret this as evidence that the mycophagous state is not an apomorphy but that instead, species have specialized in fungi probably without losing the capacity to use other ecological resources. November 2012 \| Volume 7 \| Issue 11 \| e49552 Diversification Analysis This indicates that a type-I error is likely with a little degree of sampling bias and The estimated mean c value for the posterior distribution of trees of the Drosophila subgenus is 25.184201 (24. 105418 to 26.363904), indicative of a deceleration of the speciation rate. However, results of the MCCR test (total number of species 1506, missing species 1288) indicate that the negative value obtained from our distribution of trees is not significant when compared with the null distribution (critical c value 29.019494, P = 1). The BDL analysis resulted in a better fit of the rate-variable (RV) models than the rate-constant (RC) models, and found the yule3rate model as being the best RC model (Table 4A). Nevertheless, the improvement in AIC (DAIC) of the RV models was not significant when compared to that observed in a null sample of 5000 simulated trees (P = 0.9634). This result is congruent with the CR test and both indicate that a constant mode of evolution of the Drosophila subgenus cannot be rejected. In order to analyse the effect that the dispersal to the New World may have had in the diversification pattern of particular lineages of the subgenus Drosophila, we analysed the clade including the tripunctata, pallidipennis, cardini, guarani, testacea, calloptera, funebris, quinaria, sticta and macroptera species groups. These species groups have a last common ancestor with an inferred Neotropical distribution. The estimated mean c value for the posterior distribution of 1000 trees is 25.359199 (24.558396 to 26.304164). The MCCR test (assuming a total number of species of 170 and 120 missing) indicated that the value obtained for the posterior distribution trees is significant (critical c value 23.789056, P,0.0006). Furthermore, the BDL analysis found the RV models to fit better the data than the RC. The logarithmic density-dependent model (DDL) model showed the lowest AIC value (Table 4B). This DAIC of the RV models was significant when compared to a null sample obtained from 5000 simulated trees (P = 0). Nevertheless, the c value loses its significance at low levels of non-random sampling (a = 0.55; P.0.05). This indicates that a type-I error is likely with a little degree of sampling bias and Figure 3. Summarized chronogram for the subgenus Drosophila obtained with BEAST using 218 species and 5 calibration points. Numbers on nodes indicate ages for the corresponding nodes; numbers on branches indicate BPP values. Diversification Analysis Results of the tests obtained with the phylogenies from both BI inferences were consistent (Table 4 and Table S3). Thus, henceforth it is reported only the results from the analyses run with the 218 spp. and 9 calibration points. with the 218 spp. and 9 calibration points. The estimated mean c value for the posterior distribution of trees of the Drosophila subgenus is 25.184201 (24. 105418 to 26.363904), indicative of a deceleration of the speciation rate. However, results of the MCCR test (total number of species 1506, missing species 1288) indicate that the negative value obtained from our distribution of trees is not significant when compared with the null distribution (critical c value 29.019494, P = 1). The BDL analysis resulted in a better fit of the rate-variable (RV) models than the rate-constant (RC) models, and found the yule3rate model as being the best RC model (Table 4A). Nevertheless, the improvement in AIC (DAIC) of the RV models was not significant when compared to that observed in a null sample of 5000 simulated trees (P = 0.9634). This result is congruent with the CR test and both indicate that a constant mode of evolution of the Drosophila subgenus cannot be rejected. In order to analyse the effect that the dispersal to the New World may have had in the diversification pattern of particular lineages of the subgenus Drosophila, we analysed the clade including the tripunctata, pallidipennis, cardini, guarani, testacea, calloptera, funebris, quinaria, sticta and macroptera species groups. These species groups have a last common ancestor with an inferred Neotropical distribution. The estimated mean c value for the posterior distribution of 1000 trees is 25.359199 (24.558396 to 26.304164). The MCCR test (assuming a total number of species of 170 and 120 missing) indicated that the value obtained for the posterior distribution trees is significant (critical c value 23.789056, P,0.0006). Furthermore, the BDL analysis found the RV models to fit better the data than the RC. The logarithmic density-dependent model (DDL) model showed the lowest AIC value (Table 4B). This DAIC of the RV models was significant when compared to a null sample obtained from 5000 simulated trees (P = 0). Nevertheless, the c value loses its significance at low levels of non-random sampling (a = 0.55; P.0.05). Patterns of Geographical Dispersal Results obtained with the ML and Bayesian approaches are consistent (Fig. 4A and Table 2). Analyses placed the root of the Drosophila subgenus in Asia. The immigrans-tripunctata clade and the immigrans species group were inferred to have an Asiatic origin, while the lineage comprising the tripunctata, pallidipennis, quinaria, cardini, guarani, testacea, macroptera, calloptera and funebris species groups had a last common ancestor in the Neotropics. Within this clade, the last common ancestor of the cosmopolitan D. funebris, the North American quinaria species group (although it includes the Asiatic D. brachynephros and the cosmopolitan D. kuntzei) and D. macroptera was inferred to have a North American distribution, although this is better supported in the Bayesian analysis than in the ML analysis [marginal posterior probability (PP) = 0.95, proportional likelihood (PL) = 0.55]. The remaining species groups of this clade (tripunctata, guarani, pallidipennis, calloptera, cardini and testacea) all have a last common ancestor with an unequivocal Neotropical range. The virilis-repleta radiation is estimated to have an Asiatic origin. This is further supported by the fact that the first lineage to diverge, the polychaeta, includes species that are mainly Asiatic. Of the two main clades of the virilis-repleta radiation, the one comprising the virilis, robusta, melanica, quadrisetata and angor species groups had a last common ancestor in Asia. Within this Asiatic clade, two species groups have dispersed to other geographic regions. The ancestral range of the virilis species group is estimated The ecological resource of the last common ancestor of the virilis-repleta radiation is equivocal. The ML approach recon- November 2012 \| Volume 7 \| Issue 11 \| e49552 PLOS ONE \| www.plosone.org November 2012 \| Volume 7 \| Issue 11 \| e49552 6 Drosophila Diversification Patterns PLOS ONE \| www.plosone.org 7 November 2012 \| Volume 7 \| Issue November 2012 \| Volume 7 \| Issue 11 \| e49552 November 2012 \| Volume 7 \| Issue 11 \| e49552 PLOS ONE \| www.plosone.org 7 PLOS ONE \| www.plosone.org Figure 2. Summarized chronogram for the subgenus Drosophila obtained with BEAST using 153 species and 9 calibration points. Numbers on nodes indicate ages for the corresponding nodes; numbers on branches indicate BPP values. Bars represent the 95% highest posterior density (HPD) interval for the divergence times. doi:10.1371/journal.pone.0049552.g002 Drosophila Diversification Patterns Drosophila Diversification Patterns Figure 2. Summarized chronogram for the subgenus Drosophila obtained with BEAST using 153 species and 9 calibration points. Patterns of Geographical Dispersal Numbers on nodes indicate ages for the corresponding nodes; numbers on branches indicate BPP values. Bars represent the 95% highest posterior density (HPD) interval for the divergence times. doi:10.1371/journal.pone.0049552.g002 structed the node as generalist (PL = 0.38; the alternative states frugivore and sap feeder had PL = 0.24), while the Bayesian method estimated the ancestor to be frugivorous with a posterior probability of 0.47 (sap feeder PP = 0.25, cactophilic PP = 0.16 and generalist PP = 0.10). The polychaeta group includes frugivorous species, and the ancestral state of this lineage is here inferred to be frugivorous by both methods. The remaining lineages of the virilis- repleta radiation can be classified depending on the ecological resource they use. The sap feeder species groups, the virilis, robusta, melanica and quadrisetata forms one of the two monophyletic lineages of the radiation; the second clade includes the repleta radiation and closely related species groups, which are predominantly cactophi- lic. Nevertheless, within this second clade there are species that are frugivorous such as the annulimana species group, or that are able to utilize other plant parts besides cacti, such as some species of the repleta group ([52], and references therein). However, it is inferred that the ability to use cacti as an ecological resource first appeared in the last common ancestor of these groups (PL = 0.55 and PP = 0.85). Figure 3. Summarized chronogram for the subgenus Drosophila obtained with BEAST using 218 species and 5 calibration points. Numbers on nodes indicate ages for the corresponding nodes; numbers on branches indicate BPP values. Bars represent the 95% highest posterior density (HPD) interval for the divergence times. doi:10.1371/journal.pone.0049552.g003 Phylogenenetic Inferences in the Subgenus Drosophila Phylogenenetic Inferences in the Subgenus Drosophila The phylogenies obtained in the present analysis are in general agreement with previous studies including some of the groups here analysed [12,15,17,18,54]. Thus, two major clades are identified, one corresponding to the immigrans-tripunctata radiation and the other one corresponding to the virilis-repleta radiation, that also includes the Hawaiian Drosophilidae [15,17,18]. Also in agree- ment with the previous studies is the paraphyly of the tripunctata, immigrans, guarani, calloptera and robusta species groups [54,55,56,57]. Within the virilis-repleta radiation, the position of the polychaeta group is not well resolved. In the BI tree (218 spp. and 9 calibration points) the polychaeta lineage is the sister group to all other species groups, a topology consistent with previous studies [15,54,58]. On the other hand, the remaining BI and ML analyses place the polychaeta as the sister group to the clade comprising the repleta, annulimana, nannoptera, dreyfusi, canalinea and mesophragmatica, a relationship observed previously with mitochondrial markers [54]. The sister relationship between the Hawaiian Drosophila and the virilis group recovered in the BI analysis (218 spp., 9 calibration points) is novel, nevertheless this is not supported by the BPP. On the other hand, the other BI and ML analyses placed the Hawaiian Drosophila as the sister group of the virilis-robusta-melanica- quadrisetata clade, a relationship that has been observed in other studies [18]. Another alternative hypothesis place the Hawaiian Drosophilidae as the sister lineage of the virilis-repleta [15,17] and The effect that adaptation to cacti may have had in the speciation rate of the clade including the repleta and closely related groups has been also tested using BiSSE. Results show no statistically significant difference in the speciation rates between the cactophilic clades (l1 = 0.0856) and the non-cactophilic (l0 = 0.0846) (P.0.05). The comparative method MEDUSA was used to detect clades with increased rates of diversification within the subgenus. Given that poorly resolved lineages can bias the results of this method [53], two backbone trees were used to test for uncertainty bias in the topology and divergence times (Fig. 5). Results are similar and indicate that the net rate of diversification is significantly greater in the Hawaiian Drosophilidae (r = 0.20) than in the other groups of the subgenus (r = 0.14), but among the non-Hawaiian lineages phic reconstructions obtained with Likelihood (ML) and Bayesian approaches. ble 2. Ancestral biogeographic reconstructions obtained with Likelihood (ML) and Bayesian approaches Table 2. Diversification Analysis Bars represent the 95% highest posterior density (HPD) interval for the divergence times. doi:10.1371/journal.pone.0049552.g003 November 2012 \| Volume 7 \| Issue 11 \| e49552 PLOS ONE \| www.plosone.org 8 Drosophila Diversification Patterns November 2012 \| Volume 7 \| Issue 11 \| e49552 Drosophila Diversification Patterns Figure 4. Ancestral reconstruction of geographic distribution and ecological niche. Trees showing the main dispersal events (A) and ecological shifts (B) inferred to have occurred during the evolution of the Drosophila subgenus. Clades involved are collapsed and the shift indicated with symbols above the branch. doi:10.1371/journal.pone.0049552.g004 Figure 4. Ancestral reconstruction of geographic distribution and ecological niche. Trees showing the main dispersal events (A) and ecological shifts (B) inferred to have occurred during the evolution of the Drosophila subgenus. Clades involved are collapsed and the shift indicated with symbols above the branch. doi:10.1371/journal.pone.0049552.g004 there are no differences in the rate of diversification. This method assumes a constant rate of diversification through time, which is met, as the analyses above indicate no departure from the CR models. therefore, the significance of the signal of a deceleration in the rate of diversification with time could be the result of a bias due to non- random sampling. Similarly to the tripunctata and closely related species groups, the repleta, annulimana, nannoptera, dreyfusi, canalinea and mesophragmatica species groups had a last common ancestor with a New World distribution. Furthermore, the ability to use cacti as ecological resource was estimated to have evolved in this last common ancestor. The mean c-statistic value estimated for 1000 trees of the posterior distribution obtained with BEAST is 22.0340890 (interval from 20.897719 to 23.039011), indicating a decrease in the lineage accumulation as time proceeds. Nevertheless, this value is not significant as indicated by the MCCR test (total number of species 158, missing 100) (critical c value = 23.505972; P = 0.5087). Furthermore, although the BDL analysis found a RV model (DDX) to have the best fit to our data (Table 4C), this improvement was not significant when compared to a null sample of simulated trees (P = 0.2951). Phylogenenetic Inferences in the Subgenus Drosophila Few fossils belonging to Drosophilids have been described to date from samples of Dominican Republic amber, estimated to have been deposited during the early Miocene (,23 Mya) [63]. One of the few is a member of the genus Scaptomyza [63], which is the sister group of the Hawaiian Drosophila and thus within the virilis-repleta lineage. Other two extinct species from Dominican Republic amber, D. poinari and D. succini, have been described samples as belonging to the genus Drosophila (and sharing some morphological features with members of the subgenus Drosophila) [63]. These fossils suggest that by the Oligocene/ Miocene transition some of the lineages of the subgenus Drosophila were already diverging. This is more compatible with the crown age of the subgenus Drosophila being 34.33 Mya estimated with 9 calibration points than the 23.79 Mya estimated with 5 calibration points. cannot be ruled out. The placement in the present analysis of D. annulipes, a member of the immigrans species group, as the sister species to the Hawaiian Drosophila is similar to that obtained by Katoh et al. [57]. Within the immigrans-tripunctata lineage, two clades are recovered in the present study, one corresponding to the immigrans species group and a second one comprising the tripunctata, pallidipennis, quinaria, cardini, guarani, testacea, macroptera, calloptera and funebris. This is consistent with previous studies [12,17,18,55,56]. The sister species to these two clades is a member of the immigrans species group, D. quadrilineata, rendering this group paraphyletic; however, this position has been reported in a previous study [57]. The polyphyly of the tripunctata species group here observed is in agreement with other recent study of the group [55]. The D. funebris is placed in the present study together with the quinaria and macroptera species groups. Although the funebris species group was considered by Throckmorton [11] to be the sister group of all other groups of the subgenus Drosophila, previous molecular analyses have also placed this group within the immigrans-tripunctata radiation [15,16,56]. Few previous studies have attempted to date the phylogeny of the subgenus Drosophila. Throckmorton [11] based on biogeo- graphic information proposed an evolutionary history for the Drosophilidae, although the divergence times of lineages are only vaguely specified. Other studies have dated the divergence of some of the groups using immunological and DNA sequence data [37,59,60]. Phylogenenetic Inferences in the Subgenus Drosophila Ancestral biogeographic reconstructions obtained with Likelihood (ML) and Bayesian approaches. ML approach Bayesian approach Node region PL region MPP Drosophila subgenus Asia 0.92 Asia 0.90 immigrans-tripunctata Asia 0.85 Asia 0.80 immigransa Asia 0.93 Asia 0.92 (trip,pall,quin,card,gua,test,macrop,call,fun) Neotropics 0.98 Neotropics 0.99 quinaria, funebris, macroptera North America 0.55 North America 0.95 virilis-repleta Asia 0.96 Asia 0.99 polychaeta + D. fluvialis Asia 0.98 Asia 0.97 (vir, rob, mel, quad, ang) Asia 0.99 Asia 0.99 Virilis .1 region 0.85 .1 region 0.83 robusta, melanica, quadrisetata Asia 0.99 Asia 0.99 Melanica Asia 0.81 North America 0.63 Quadrisetata Asia 0.99 Asia 0.99 rpl, ann, nan, drey, cana, meso Neotropics 0.68 Neotropics 0.75 repleta radiation Neotropics 0.72 Neotropics 0.67 repleta Neotropics 0.69 .1 region 0.97 aexcluding D. quadrilineata. doi:10.1371/journal.pone.0049552.t002 PLOS ONE \| www.plosone.org 10 November 2012 \| Volume 7 \| Issue 11 \| e49552 November 2012 \| Volume 7 \| Issue 11 \| e49552 10 PLOS ONE \| www.plosone.org Drosophila Diversification Patterns Table 3. Ancestral reconstruction of the ecological resource used obtained with Likelihood (ML) and Bayesian approaches. Table 3. Ancestral reconstruction of the ecological resource used obtained with Likelihood (ML) and Bayesian approaches. ML approach Bayesian approach Node resource PL resource MPP Drosophila subgenus unspecific 0.57 unspecific 0.55 immigrans-tripunctata unspecific 0.80 unspecific 0.98 immigransa unspecific 0.51 frugivorous 0.88 (trip,pall,quin,card,gua,test,macrop,call,fun) unspecific 0.99 unspecific 0.99 quinaria, funebris, macroptera unspecific 0.89 mycophagous 0.55 quinaria, macroptera mycophagous 0.98 mycophagous 0.99 virilis-repleta unspecific 0.38 frugivorous 0.47 polychaetab frugivorous 0.95 frugivorous 0.99 (vir, rob, mel, quad) sap feeders 0.90 sap feeders 0.99 virilis sap feeders 0.99 sap feeders 0.99 robusta, melanica, quadrisetata sap feeders 0.99 sap feeders 0.99 melanica sap feeders 0.99 sap feeders 0.99 (rpl, ann, nan, drey, cana, meso) cactophilic 0.55 cactophilic 0.85 repleta radiation cactophilic 0.70 cactophilic 0.68 repleta cactophilic 0.91 cactophilic/unspecific+cactophilic 0.49/0.49 aexcluding D. quadrilineata. bnot including the unclassified species D. fluvialis. doi:10.1371/journal.pone.0049552.t003 aexcluding D. quadrilineata. bnot including the unclassified species D. fluvialis. doi:10.1371/journal.pone.0049552.t003 [62]. The use of reliable points of calibration will be of relevance in obtaining good estimates of species divergence. It is also expected that the more points are used, the more reliable the estimation will be. The present study is the first one to estimate times of divergence using a relaxed molecular clock and a large number of species. The scarcity of fossil data poses a challenge towards estimating the origin and evolutionary history of this group of organisms. Phylogenenetic Inferences in the Subgenus Drosophila Furthermore, two studies have attempted to calibrate the molecular clock of Drosophila using mutation rates as an approximation of substitution rates [61,62]. All these studies have used few species and have resulted in contrasting times of speciation. These differing times of divergence observed among studies reflect the uncertainties of the assumptions of the different methods used. For example, different models of emergence and colonisation of the Hawaiian islands result in contrasting times of speciation, or the use of mutation rates to estimate times of divergence also rely on the use of appropriate generation times According to the results from the analysis including more calibrations, the divergence between the Drosophila and the Sophophora (outgroup) subgenera occurred around 36 Mya, which is a much younger estimate than the 61–65 Mya estimated using immunological distances [59] and synonymous mutation- based molecular clock [37], but is similar to the 39 Mya estimated using the Adh gene [60] and the 32 (25–40) Mya estimated using the mutation rate as a proxy for substitution rate [62]. The crown age for the Drosophila subgenus (and, therefore, the divergence of the two major lineages, the immigrans-tripunctata and virilis-repleta), is in the present study placed in the late Eocene, approximately 34 Mya, which is similar to the ,33 Mya divergence estimate between the D. immigrans and D. repleta groups by Russo et al. [60]. This is also 11 November 2012 \| Volume 7 \| Issue 11 \| e49552 PLOS ONE \| www.plosone.org Drosophila Diversification Patterns Table 4. Results of fitting diversification models to the Drosophila subgenus (A), the tripunctata and closely related species groups (B), and the repleta and closely related species groups (C). Patterns of Evolution in the Drosophila Subgenus A commonly observed pattern of diversification is that lineage diversification rates decline through time (e.g. [7,66]). This density-dependent trend can be explained by an early greater opportunity for occupying new ecological niches where the competition pressure is reduced and allows for a rapid di- versification rate [5,6,7]. This is followed by a decrease in the speciation rate as the niche becomes saturated and the competition for ecological space increases. Some authors have suggested that the diversification of the main lineages of the Drosophila subgenus occurred rapidly early in the evolution of the group, remaining stable for a long time until the present [12,67]. Furthermore, the subgenus contains a large number of species that show a consider- able diversity in geographic distribution and use a significant variety of ecological resources. These features are suggestive of an adaptive radiation. However, results do not support the hypothesis that the Drosophila subgenus is an adaptive radiation. p g p Dispersal into new areas and evolution of characters that allow the use of new resources, often result in an ecological opportunity that leads to adaptive radiations [2,5]. Dispersal of lineages of the Drosophila subgenus to the New World has occurred at least twice independently and at similar times; both, the ancestors of the tripunctata and closely related species groups, and the repleta and closely related species groups had a Neotropical distribution. These results support the Neotropical origin of these lineages proposed by Throckmorton [11]. In contrast with the origin of repleta and closely related species groups, our analyses have not been able to resolve the ancestral distribution of the repleta species group. Similarly, a recent analysis of the ancestral geographic distribution of the repleta species group was not able to place the origin of this group in either North America or South America, and the authors suggest that the biogeographic history of this group is marked by a repeated exchange of fauna between these subcontinents [64]. Our results would support the hypothesis of a close relationship between the repleta subgroups of North and South America. Also, several resource shifts are here inferred to have occurred from the generalist ancestral state of the subgenus. Phylogenenetic Inferences in the Subgenus Drosophila A) pureBirth BD DDL DDX yule2rateyule3rate Parameters r1 = 0.087 r1 = 0.087 a = 0 r1 = 0.126 k = 436.00 r1 = 0.630 x = 0.430 r1 = 0.161 r2 = 0.068 st = 13.34 r1 = 0.161 r2 = 0.089 r3 = 0.057 st1 = 13.34 st2 = 7.42 Ln(L) 211.705 211.705 223.905 227.668 229.101 232.127 AIC 2421.410 2419.410 2443.810 2451.337 2452.203 2454.254 DAIC 32.844 34.844 10.444 2.916 2.051 0 P = 0.9634 B) pureBirth BD DDL DDX yule2rateyule3rate Parameters r1 = 0.069 r1 = 0.069 a = 0 r1 = 0.255 k = 51.083 r1 = 1.796 x = 0.975 r1 = 0.181 r2 = 0.034 st = 12.44 r1 = 0.325 r2 = 0.110 r3 = 0.030 st1 = 17.61 st2 = 10.85 Ln(L) 231.142 231.142 212.964 216.367 215.299 211.293 AIC 64.285 66.285 29.927 36.734 36.599 32.585 DAIC 34.358 36.358 0 6.807 6.672 2.658 P = 0.0000 C) pureBirth BD DDL DDX yule2rateyule3rate Parameters r1 = 0.090 r1 = 0.090 a = 0 r1 = 0.158 k = 82.055 r1 = 0.434 x = 0.475 r1 = 0.149 r2 = 0.065 st = 10.38 r1 = 0.149 r2 = 0.058 r3 = 0.141 st1 = 10.38 st2 = 0.63 Ln(L) 214.003 214.003 210.824 210.251 29.269 27.943 AIC 30.006 32.006 25.647 24.501 24.538 25.886 DAIC 5.504 7.504 1.146 0 0.037 1.385 P = 0.2951 The phylogeny used was that obtained using the 218 species and 9 calibration points. P indicates the significance of the DAIC between the rate-constant and rate-variable models. (BD – Birth-Death model; DDL – Density-dependent logarithmic model; DDX – Density-dependent exponential model). doi:10.1371/journal.pone.0049552.t004 conditions are likely to have influenced speciation in Drosophila as well as in other biota. Patterns of Evolution in the Drosophila Subgenus Thus, the ancestor of the repleta and closely related species groups shifted to a cactophilic state, also observed in a recent study of this group [64], that of the immigrans and the polychaeta groups became frugivorous, and the quinaria and macroptera shared a common ancestor that was mycophagous. Within the repleta species group, shifts from Opuntia to columnar cacti species have occurred several times independently [64]. Despite these dispersal events and ecological shifts, we are not able to detect in any of the clades tested a pattern of speciation through time consistent with a density-dependent model, which would be indicative of an adaptive radiation [7,8]. Furthermore, apart from the Hawaiian Drosophilidae, there is no evidence for higher rates of di- versification among the other species groups of the subgenus, further supporting the lack of influence in the net diversification rate of the ecological shifts or geographic dispersals. The phylogeny used was that obtained using the 218 species and 9 calibration points. P indicates the significance of the DAIC between the rate-constant and rate-variable models. (BD – Birth-Death model; DDL – Density-dependent logarithmic model; DDX – Density-dependent exponential model). doi:10.1371/journal.pone.0049552.t004 consistent with the Oligocene date proposed by Throckmorton [11]. Beverly and Wilson [59] estimated the divergence of the repleta and robusta groups to be 35 Mya, an age that is consistent with the ,31 Mya our results indicate. The divergence between the mettleri and mulleri subgroups of the repleta were estimated by Russo et al. [60] to be of ,16 Mya, while the estimate in the present analysis is of ,21 Mya. Oliveira et al. [64], using as calibration points those divergence times of Russo et al. [60] have also estimated a crown age for the repleta species group of ,16 Mya, an estimate more similar to our results using 5 calibration points. Nevertheless, the phylogenetic resolution within some groups is not well supported. Results here presented indicate that the major lineages within the two radiations appeared between the late Oligocene and the first age of the early Miocene (28.5–20.5 Mya), and by the middle Miocene epoch most of the species groups had already diverged. This period of time corresponds with a long-term trend of climate warming that started from 26–27 Mya and lasted until the middle Miocene (15 Mya), with the exception of brief periods of glaciation approximately 23 Mya [65]. November 2012 \| Volume 7 \| Issue 11 \| e49552 Patterns of Evolution in the Drosophila Subgenus A) phylogeny backbone from analysis with 218 species and 9 calibration points; B) phylogeny backbone from analysis with 218 species and 5 calibration points. Tip names refer to the species groups (those polyphyletic were clustered into a single clade) and numbers in brackets refer to the species richness of the tip. Numbers on nodes indicate divergence times. doi:10.1371/journal.pone.0049552.g005 Figure 5. Backbone topologies used in the MEDUSA analyses. A) phylogeny backbone from analysis with 218 species and 9 calibration points; B) phylogeny backbone from analysis with 218 species and 5 calibration points. Tip names refer to the species groups (those polyphyletic were clustered into a single clade) and numbers in brackets refer to the species richness of the tip. Numbers on nodes indicate divergence times. doi:10.1371/journal.pone.0049552.g005 the propensity to radiate of some lineages [72]. In particular, this study finds that sexual dichromatism, a better proxy than species mating system for sexual selection intensity, in combi- nation with ecological opportunity (lake depth) influence the radiation pattern of cichlids [72]. Thus, the intensity of sexual selection in species of Drosophila could be a good candidate for future studies of diversification patterns. species [64]. In contrast we have specifically tested the hypothesis of an increased rate of speciation in this lineage as a result of becoming cactophilic, and were not able to reject the constant rate model. Thus, there is no support for a rapid radiation of the repleta species group. There is evidence for genetically differentiated host- races in some of the cactophilic species, indicating the relevance of cactus species use in the evolution of this group. However, this differentiation sometimes reflects geographic separation and races show no reproductive isolation [38,68,69,70,71]. Thus, adaptation to different cacti species does not necessarily have to be associated to an increase in the rate of speciation, even though it might be relevant for the evolution of the clade. Patterns of Evolution in the Drosophila Subgenus These climatic consistent with the Oligocene date proposed by Throckmorton [11]. Beverly and Wilson [59] estimated the divergence of the repleta and robusta groups to be 35 Mya, an age that is consistent with the ,31 Mya our results indicate. The divergence between the mettleri and mulleri subgroups of the repleta were estimated by Russo et al. [60] to be of ,16 Mya, while the estimate in the present analysis is of ,21 Mya. Oliveira et al. [64], using as calibration points those divergence times of Russo et al. [60] have also estimated a crown age for the repleta species group of ,16 Mya, an estimate more similar to our results using 5 calibration points. Nevertheless, the phylogenetic resolution within some groups is not well supported. Results here presented indicate that the major lineages within the two radiations appeared between the late Oligocene and the first age of the early Miocene (28.5–20.5 Mya), and by the middle Miocene epoch most of the species groups had already diverged. This period of time corresponds with a long-term trend of climate warming that started from 26–27 Mya and lasted until the middle Miocene (15 Mya), with the exception of brief periods of glaciation approximately 23 Mya [65]. These climatic Surprisingly, the lineage of cactophilic species groups does not show any departure from a constant rate of speciation. Despite colonising the Neotropics and acquiring the capacity to exploit cacti as ecological resource, no signature of adaptive radiation has been detected and there is no difference in the diversification rate between cactophilic and noncactophilic lineages. These results contrast with the recent suggestion of a rapid radiation of the repleta species group along its cacti hosts [64]. However, Oliveira et al. do not test for an increase in the rates of speciation in this group of November 2012 \| Volume 7 \| Issue 11 \| e49552 PLOS ONE \| www.plosone.org 12 Drosophila Diversification Patterns Drosophila Diversification Patterns Figure 5. Backbone topologies used in the MEDUSA analyses. A) phylogeny backbone from analysis with 218 species and 9 calibration points; B) phylogeny backbone from analysis with 218 species and 5 calibration points. Tip names refer to the species groups (those polyphyletic were clustered into a single clade) and numbers in brackets refer to the species richness of the tip. Numbers on nodes indicate divergence times. doi:10.1371/journal.pone.0049552.g005 Figure 5. Backbone topologies used in the MEDUSA analyses. November 2012 \| Volume 7 \| Issue 11 \| e49552 PLOS ONE \| www.plosone.org References 1. Losos JB (2010) Adaptive radiation, ecological opportunity, and evolutionary determinism. American Society of Naturalists E. O. Wilson award address. Am Nat 175: 623–639. 28. Perlman SJ, Spicer GS, Shoemaker DD, Jaenike J (2003) Associations between mycophagous Drosophila and their Howardula nematode parasites: a worldwide phylogenetic shuffle. Mol Ecol 12: 237–249. 29. Morales-Hojas R, Reis M, Vieira CP, Vieira J (2011) Resolving the phylogenetic relationships and evolutionary history of the Drosophila virilis group using multilocus data. Mol Phylogen Evol 60: 249–258. 2. 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References Bonacum J, OGrady PM, Kambysellis M, Desalle R (2005) Phylogeny and age of diversification of the planitibia species group of the Hawaiian Drosophila. Mol Phylogen Evol 37: 73–82. 9. Weir JT (2006) Divergent timing and patterns of species accumulation in lowland and highland neotropical birds. Evolution 60: 842–855. 10. Alfaro ME, Karns DR, Voris HK, Brock CD, Stuart BL (2008) Phylogeny, evolutionary history, and biogeography of Oriental-Australian rear-fanged water snakes (Colubroidea: Homalopsidae) inferred from mitochondrial and nuclear DNA sequences. Mol Phylogen Evol 46: 576–593. y g 37. Tamura K, Subramanian S, Kumar S (2004) Temporal patterns of fruit fly (Drosophila) evolution revealed by mutation clocks. Mol Biol Evol 21: 36–44. 38. Reed LK, Nyboer M, Markow TA (2007) Evolutionary relationships of Drosophila mojavensis geographic host races and their sister species Drosophila arizonae. Mol Ecol 16: 1007–1022. 11. 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Huelsenbeck JP, Bollback JP (2001) Empirical and hierarchical Bayesian estimation of ancestral states. Syst Biol 50: 351–366. 15. Remsen J, OGrady P (2002) Phylogeny of Drosophilinae (Diptera: Drosophi- lidae), with comments on combined analysis and character support. Mol Phylogen Evol 24: 249–264. y 44. Bollback JP (2006) SIMMAP: Stochastic character mapping of discrete traits on phylogenies. BMC Bioinformatics 7: 88. y g 16. Tatarenkov A, Zurovcova M, Ayala FJ (2001) Ddc and amd sequences resolve phylogenetic relationships of Drosophila. Mol Phylogen Evol 20: 321–325. p y g 45. Pybus OG, Harvey PH (2000) Testing macro-evolutionary models using incomplete molecular phylogenies. Proc R Soc Lond B Biol Sci 267: 2267–2272. 17. Conclusion Our results show that the proposed Drosophila taxonomic radiations do not correspond to adaptive radiations. Furthermore, none of the ecological resource shifts or the geographic dispersal events observed in the phylogeny of the Drosophila subgenus can be unequivocally linked to an adaptive radiation of the clade. In particular, the evolution of cactophily should not be invoked as a general explanation for the diversity of the repleta group. Results lend support towards the idea that in some groups, the pace of diversification can be more limited by the rate of speciation (the time it takes to achieve reproductive isolation) than by the evolution of new traits or colonisation of new regions, and Despite not having found evidence for adaptive radiation in the subgenus Drosophila in relation to ecological opportunity as a result of colonization of new geographic regions or new ecological resources, it is still possible that other intrinsic characteristics could have resulted in an increase in the speciation rate of other lineages than the ones explored here. Indeed, a recent study in cichlids shows that it is a combination of intrinsic characteristics and extrinsic factors that best explains November 2012 \| Volume 7 \| Issue 11 \| e49552 PLOS ONE \| www.plosone.org November 2012 \| Volume 7 \| Issue 11 \| e49552 13 Drosophila Diversification Patterns References van der Linde K, Houle D, Spicer GS, Steppan SJ (2010) A supermatrix-based molecular phylogeny of the family Drosophilidae. Genet Res (Camb) 92: 25–38. 46. Rabosky DL (2006) LASER: A Maximum Likelihood toolkit for detecting temporal shifts in diversification rates. Evol Bioinform Online 2006: 257–260. 18. OGrady P, DeSalle R (2008) Out of Hawaii: the origin and biogeography of the genus Scaptomyza (Diptera : Drosophilidae). Biol Lett 4: 195–199. 47. Brock CD, Harmon LJ, Alfaro ME (2011) Testing for temporal variation in diversification rates when sampling is incomplete and nonrandom. Syst Biol 60: 410–419. 19. O’Grady PM, Lapoint RT, Bonacum J, Lasola J, Owen E, et al. 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Starmer WT, Barker JS, Phaff HJ, Fogleman JC (1986) Adaptations of Drosophila and yeasts: their interactions with the volatile 2-propanol in the cactus- microorganism-Drosophila model system. Aust J Biol Sci 39: 69–77. 51. Maddison WP, Midford PE, Otto SP (2007) Estimating a binary character’s effect on speciation and extinction. Syst Biol 56: 701–710. 22. Barker JSF, Starmer WT (1982) Ecological Genetics and Evolution: The Cactus- Yeast-Drosophila Model System. New York: Academic Press. 22. Barker JSF, Starmer WT (1982) Ecological Genetics and Evolution 52. Silva-Bernardi ECC, Morales AC, Sene FM, Manfrin MH (2006) Phylogenetic relationships in the Drosophila fasciola species subgroup (Diptera, Drosophilidae) inferred from partial sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene. Genet Mol Biol 29: 566–571. Yeast-Drosophila Model System. New York: Academic Press. 23. Patterson JT, Stone WS (1952) Evolution in the genus Drosophila. New York: Macmillan. 24. Table S3 (DOCX) reproductive isolation may be a prior requisite for adaptive divergence to occur [6,73]. reproductive isolation may be a prior requisite for adaptive divergence to occur [6,73]. Table S1 (XLSX) Conceived and designed the experiments: RMH. Performed the experi- ments: RMH. Analyzed the data: RMH. Contributed reagents/materials/ analysis tools: JV RMH. Wrote the paper: RMH JV. Conceived and designed the experiments: RMH. Performed the experi- ments: RMH. Analyzed the data: RMH. Contributed reagents/materials/ analysis tools: JV RMH. Wrote the paper: RMH JV. Author Contributions Table S1 (XLSX) Table S2 (XLSX) Drosophila Diversification Patterns 56. Robe LJ, Valente VL, Budnik M, Loreto EL (2005) Molecular phylogeny of the subgenus Drosophila (Diptera, Drosophilidae) with an emphasis on Neotropical species and groups: a nuclear versus mitochondrial gene approach. Mol Phylogen Evol 36: 623–640. from a revised phylogeny of the Drosophila repleta species group. Mol Phylogen Evol 64: 533–544. from a revised phylogeny of the Drosophila repleta species group. Mol Phylogen Evol 64: 533–544. 65. Zachos J, Pagani M, Sloan L, Thomas E, Billups K (2001) Trends, rhythms, and aberrations in global climate 65 Ma to present. Science 292: 686–693. 66. Rabosky DL, Lovette IJ (2008) Explosive evolutionary radiations: decreasing speciation or increasing extinction through time? Evolution 62: 1866–1875. 57. Katoh T, Nakaya D, Tamura K, Aotsuka T (2007) Phylogeny of the Drosophila immigrans species group (Diptera : Drosophilidae) based on Adh and Gpdh sequences. Zool Sci 24: 913–921. 67. Powell J (1997) Progress and prospects in Evolutionary Biology: The Drosophila model. New York: Oxford University Press. 562 p. q 58. Tatarenkov A, Ayala FJ (2001) Phylogenetic relationships among species groups of the virilis-repleta radiation of Drosophila. Mol Phylogen Evol 21: 327–331. 68. Bono JM, Matzkin LM, Castrezana S, Markow TA (2008) Molecular evolution and population genetics of two Drosophila mettleri cytochrome P450 genes involved in host plant utilization. Mol Ecol 17: 3211–3221. 59. Beverley SM, Wilson AC (1984) Molecular Evolution in Drosophila and the Higher Diptera.2. A Time Scale for Fly Evolution. J Mol Evol 21: 1–13. 69. Hurtado LA, Erez T, Castrezana S, Markow TA (2004) Contrasting population genetic patterns and evolutionary histories among sympatric Sonoran Desert cactophilic Drosophila. Mol Ecol 13: 1365–1375. 60. Russo CAM, Takezaki N, Nei M (1995) Molecular Phylogeny and Divergence Times of Drosophilid Species. Mol Biol Evol 12: 391–404. p p 61. Cutter AD (2008) Divergence times in Caenorhabditis and Drosophila inferred from direct estimates of the neutral mutation rate. Mol Biol Evol 25: 778–786. p p 70. Markow TA, Castrezana S, Pfeiler E (2002) Flies across the water: genetic differentiation and reproductive isolation in allopatric desert Drosophila. Evolution 56: 546–552. 62. Obbard DJ, Maclennan J, Kim KW, Rambaut A, OGrady PM, et al. (2012) Estimating Divergence Dates and Substitution Rates in the Drosophila Phylogeny. Mol Biol Evol Adance Access doi:10.1093/molbev/mss150. 71. Markow TA, Fogleman JC, Heed WB (1983) Reproductive Isolation in Sonoran Desert Drosophila. Evolution 37: 649–652. 63. References Wittkopp PJ, Carroll SB, Kopp A (2003) Evolution in black and white: genetic control of pigment patterns in Drosophila. Trends Genet 19: 495–504. ( ) g 53. Drummond CS, Eastwood RJ, Miotto ST, Hughes CE (2012) Multiple continental radiations and correlates of diversification in Lupinus (Leguminosae): testing for key innovation with incomplete taxon sampling. Syst Biol 61: 443– 460. 25. Morales-Hojas R, Vieira CP, Reis M, Vieira J (2009) Comparative analysis of five immunity-related genes reveals different levels of adaptive evolution in the virilis and melanogaster groups of Drosophila. Heredity 102: 573–578. 54. Wang BC, Park J, Watabe HA, Gao JJ, Xiangyu JG, et al. (2006) Molecular phylogeny of the Drosophila virilis section (Diptera : Drosophilidae) based on mitochondrial and nuclear sequences. Mol Phylogen Evol 40: 484–500. 26. Hollocher H, Hatcher JL, Dyreson EG (2000) Evolution of abdominal pigmentation differences across species in the Drosophila dunni subgroup. Evolution 54: 2046–2056. 55. Hatadani LM, McInerney JO, de Medeiros HF, Junqueira ACM, de Azeredo- Espin AM, et al. (2009) Molecular phylogeny of the Drosophila tripunctata and closely related species groups (Diptera: Drosophilidae). Mol Phylogen Evol 51: 595–600. 27. Jaenike J (1995) Interactions between Mycophagous Drosophila and Their Nematode Parasites - from Physiological to Community Ecology. Oikos 72: 235–244. November 2012 \| Volume 7 \| Issue 11 \| e49552 PLOS ONE \| www.plosone.org 14 Drosophila Diversification Patterns Grimaldi DA (1987) Amber fossil Drosophilidae (Diptera), with particular reference to the Hispaniolan taxa. Am Mus Novit 2880: 1–23. 63. Grimaldi DA (1987) Amber fossil Drosophilidae (Diptera), p 72. Wagner CE, Harmon LJ, Seehausen O (2012) Ecological opportunity and sexual selection together predict adaptive radiation. Nature 487: 366–369. 72. Wagner CE, Harmon LJ, Seehausen O (2012) Ecological oppo reference to the Hispaniolan taxa. Am Mus Novit 2880: 1–23 selection together predict adaptive radiation. Nature 487: 366 64. Oliveira DCSG, Almeida FC, O’Grady PM, Armella MA, DeSalle R, et al. (2012) Monophyly, divergence times, and evolution of host plant use inferred 73. Venditti C, Meade A, Pagel M (2010) Phylogenies reveal new interpretation of speciation and the Red Queen. Nature 463: 349–352. 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W4388108943.txt	http://elibrary.vdi-verlag.de/10.37544/1436-4980-2023-10-60.pdf	de		Zyklische Scherprüfung metallischer Folien/Cyclic shear testing for metallic foils	Werkstattstechnik	2,023	cc-by	3,394	T I TE L T H E MA – FACHAUFSATZ Bei diesem Beitrag handelt es sich um einen wissenschaftlich begutachteten und freigegebenen Fachaufsatz („reviewed paper“). doi.org/10.37544/1436–4980–2023–10–60 Nutzen von Daten in der Umformtechnik – Potenziale der Digitalisierung Zyklische Scherprüfung metallischer Folien C. Karadogan, M. Beck, P. Cyron, K. R. Riedmüller, M. Liewald Die Bemühungen zur Emissionsreduktion in den Bereichen Energie und Mobilität führen zu einer steigenden Nachfrage nach geformten Bauteilen aus dünnen Metallfolien. Die Herstellung und Auslegung solcher Komponenten gestalten sich als äußerst anspruchsvoll. Herkömmliche Charakterisierungsversuche für die digitale Auslegung dieser Bauteile stoßen an ihre Grenzen. Daher wird in diesem Kontext ein innovativer Versuchsaufbau für den zyklischen Scherversuch vorgestellt, der die strukturelle Stabilität der Probe erhöht und eine Modellierung des Verfestigungsverhaltens ermöglicht. Cyclic shear testing for metallic foils Efforts to reduce emissions in the fields of energy and mobility lead to an increasing demand for shaped components made from thin metal foils. Manufacturing and designing such components is highly challenging. Conventional characterization tests for designing these components digitally are reaching their limits. In this context, an innovative setup for cyclic shear testing is introduced, enhancing the structural stability of the specimen and enabling modeling of the material‘s strengthening behavior. STICHWÖRTER Brennstoffzelle, Finite-Elemente-Methode (FEM), Simulation 1 Einleitung Die steigende Nachfrage nach Elektromobilität, Mikroelektronik und erneuerbaren Energien hat zu einer erheblichen Zunahme der Produktionsvolumina für umgeformte Komponenten aus dünnen metallischen Folien (Dicke ≤ 100 µm) geführt. Solche geringen Materialdicken sind eine enorme Herausforderung für die Machbarkeit und die Robustheit von Umformprozessen, da dünne metallische Folien insbesondere zu Defekten aufgrund von Falten- oder Reißerbildung neigen [1–3]. Zudem ist bis heute die Rückfederung eine wesentliche Herausforderung bei der Erreichung dimensionaler Genauigkeit von Bauteilen aus dünnen Metallfolien [4–6]. In modernen Brennstoffzellen beispielsweise kommen Bipolarplatten mit äußerst komplexen Flussfeldgeometrien zum Einsatz, die aus dünnen Edelstahlfolien mittels Hohlprägen hergestellt werden. Die Flussfeldgeometrien beziehungsweise die zugehörigen Strömungskanäle auf diesen geprägten Platten müssen sehr enge Toleranzbereiche innerhalb weniger Mikrometer in vertikaler und lateraler Richtung erfüllen [4, 7]. Andernfalls können aufgrund dimensionaler Ungenauigkeiten Schwierigkeiten beim Fügen oder Stapeln der Bipolarplatten, dem sogenannten Stacking, auftreten, die zu einer verringerten Leistung und Effizienz der gesamten Brennstoffzelle führen. Finite-Elemente-Simulationen (FE) können zur Auslegung solch komplexer Umformverfahren herangezogen werden, um Verfahrensschritte in einem frühen Entwicklungsstadium zu optimieren. Die Genauigkeit dieser Simulationsergebnisse hängt aber stark von der Modellierung des mechanischen Verhaltens des Materials ab. Dazu werden in der Regel eine Reihe von Charakte- 438 risierungsversuchen durchgeführt, welche anschließend in mathematische Formulierungen überführt werden [1, 8]. Um die Vorhersagequalität von FEM-Simulationen, insbesondere bei der zu erwartenden dimensionalen Genauigkeit und Rückfederung des Bauteils, zu verbessern, empfiehlt es sich, neben Spannungs-Dehnungs-Kurven auch Belastungssequenzen mit Lastumkehr zu berücksichtigen [9]. Das Verhalten bei Lastumkehr kann dann in komplexen phänomenologischen Materialmodellen wie dem Yoshida-Uemori-Modell zur Modellierung der kinematischer Verfestigung [10] oder dem Chaboche-Modell [11–12] berücksichtigt werden. Auf diese Weise ist es möglich, die Materialverfestigung sowie den Bauschingereffekt in der Umformsimulation sehr genau zu berücksichtigen und eine verbesserte Prognose des Rückfederungsverhaltens zu erzielen. Neben dem zyklischen Zug-Druck-Versuch, der für dünne Folien nur wenig praktikabel ist, kann das Materialverhalten bei zyklischer Last beziehungsweise bei Lastumkehr in einem zyklischen In-Plane-Scherversuch [13–14] oder in einem zyklischen In-Plane-Torsionsversuch [15] abgebildet werden. In diesem Beitrag wird der zyklische In-Plane-Scherversuch an dünnen metallischen Folien im Detail untersucht. Dieser Versuch kann, bei Nutzung einer entsprechenden Vorrichtung, in einer konventionellen Zugprüfmaschine umgesetzt werden und ermöglicht das Abbilden von zyklischen Belastungssequenzen mit Lastumkehr. Typischerweise wird der zyklische In-Plane-Scherversuch (im Folgenden als zyklischer Scherversuch bezeichnet), wie in Bild 1 dargestellt, durchgeführt. Um den nachteiligen Einfluss von Spannungsinhomogenitäten in der Nähe der freien Kanten zu verringern, wird lediglich ein https://doi.org/10.37544/1436-4980-2023-10-60, am 13.07.2024, 18:04:58 Open Access – - https://elibrary.vdi-verlag.de/agb WT WERKSTATTSTECHNIK BD. 113 (2023) NR. 10 TITELTHEMA – FACHAU FS A T Z Bild 1. a) Schematischer rechteckiger Probenkörper für den zyklischen Scherversuch mit der Dicke t. L = Probenlänge und W = Probenbreite; b) Probe in Belastungsrichtung und unter Lastumkehr. w = Breite des Messbereichs; c) Schematische Spannungs-Dehnungs-Kurve, die durch den Scherversuch ermittelt wird. Grafik: IFU Universität Stuttgart schmaler mittiger Bereich der Probe mit einer zyklischen beziehungsweise einer Scherbelastung in der Ebene (in-plane) mit Lastumkehr beansprucht. Unter der Annahme, dass die geklemmten Bereiche der Probe starr verbleiben und die Inhomogenitäten an den freien Kanten vernachlässigt werden können, werden die auftretende wahre Dehnung und Spannung mittels der Gleichungen (1) und (2) ermittelt. (1) (2) Zur Erfassung der tatsächlich auftretenden Scherdehnung innerhalb des Probenbereichs wird empfohlen, die Dehnungsverteilung mit einer optischen Dehnungsmessung zu erfassen. Außerdem sollte der Spannungszustand im Messbereich der Probe idealerweise ein ebener sein. Dies ist im Falle von Faltenbildung nicht gewährleistet. Daher muss die Faltenbildung im Messbereich der Probe unbedingt unterbunden werden. Konventionelle Vorrichtungen für Scherversuche mit einem breiteren Messbereich (wenige Millimeter breit) eignen sich nicht zur Charakterisierung von dünnen Metallfolien, da diese aufgrund ihrer geringen strukturellen Stabilität zur Bildung von Falten und Reißern neigen [1–3]. Vor allem beim Auftreten von Falten im Messbereich liegt der geforderte ebene Spannungszustand, nämlich eine reine Scherbeanspruchung, nicht mehr vor und es kommt zu einer Überlagerung verschiedener Spannungszustände. Eine Ermittlung der Scherspannungen und -dehnungen nach Gleichung (1) und (2) ist dann nicht mehr zulässig. Um die strukturelle Stabilität der metallischen Folie und insbesondere des Messbereichs sicherzustellen, muss die Breite des Messbereichs w proportional zur Materialdicke reduziert werden. Eine solche proportionale Reduktion der Breite des Messbereichs würde im Fall von dünnen metallischen Folien dazu führen, dass sie für herkömmliche optische Messsysteme zu klein ist. Diese Herausforderungen haben zur Entwicklung neuer Ansätze für die Durchführung zyklischer Scherversuche an dünnen metallischen Folien geführt. Sie wurden in zahlreichen ForWT WERKSTATTSTECHNIK BD. 113 (2023) NR. 10 schungsarbeiten vorgestellt und befassen sich hauptsächlich mit dem Problem der Faltenbildung im Messbereich [9, 16]. Ein Lösungsansatz, um die Faltenbildung während des Scherversuchs zu unterbinden, besteht darin, einen Normaldruck im Messbereich der Probe mithilfe einer sogenannten Anti-FaltenVorrichtung aufzubringen. Hierzu verwenden Pham et al. [9] transparente Platten aus Plexiglas, die während des zyklischen Scherversuchs einen Normaldruck auf den Messbereich der metallischen Folie bewirken. Nach Aussage der Autoren kann auf diese Weise die Faltenbildung effektiv vermieden werden, während gleichzeitig optische Dehnungsmessungen aufgrund der transparenten Platten möglich sind. Dennoch sind die Anbringung solcher Vorrichtungen zur Vermeidung von Faltenbildung, das genaue Aufbringen einer definierten Flächenpressung innerhalb des Messbereichs sowie das Einsetzen der Probe und der Anti-Falten-Vorrichtung in den Prüfstand bedeutende Herausforderungen. Insbesondere wenn die geringe strukturelle Stabilität von dünnen metallischen Folien bedacht wird. Obwohl die Autoren gezeigt haben, dass die Reibung zwischen der Spannvorrichtung und der Folie keinen wesentlichen Einfluss auf die gemessene Kraft hat, werden keine konkreten Angaben zum Einfluss der Reibung auf das stochastische Muster auf der Probenoberfläche gemacht [9]. Dieses stochastische Muster ist nötig, um die Dehnungen mittels einer optischen DIC (Digital Image Correlation)-Dehnungsmessung erfassen zu können. Es wird angenommen, dass die auftretende Reibung zwischen der transparenten Plexiglasplatte und dem aufgedruckten beziehungsweise lackierten stochastischen Muster die Messung des Verzerrungsfeldes verzerren kann. Basierend auf dem aktuellen Stand der Technik stellt dieser Beitrag eine neuartige Prüfvorrichtung zur Durchführung von zyklischen Scherversuchen an dünnen metallischen Folien vor. Die vorgeschlagene Prüfmethode verbessert die strukturelle Stabilität der Scherprobe im Messbereich und verhindert auf diese Weise die Faltenbildung während des Versuchs, ohne dass eine zusätzliche Vorrichtung zur Unterdrückung von Falten erforderlich ist. Die strukturelle Stabilität wird durch eine leichte Krümmung des Messbereichs erzielt, wodurch die Neigung zur Faltenbildung drastisch reduziert wird. Auf diese Weise wird eine konventionelle optische Dehnungsmessungen ermöglicht und die https://doi.org/10.37544/1436-4980-2023-10-60, am 13.07.2024, 18:04:58 Open Access – - https://elibrary.vdi-verlag.de/agb 439 T I TE L T H E MA – FACHAUFSATZ Bild 2. a) Simulationsmodell für den konventionellen Scherversuch, b) Simulationsmodell mit dem neuem Prüfaufbau in isometrischer Ansicht und in Draufsicht. Grafik: IFU Universität Stuttgart Notwendigkeit einer Vorrichtung zur Vermeidung von Falten entfällt. Zudem können auf diese Weise potenzielle Reibungseffekte zwischen einem potenziellen stochastischen Muster auf dem Messbereich und einer zusätzlichen Vorrichtung unterbunden werden. Zur Prüfung dieses neuartigen Ansatzes wurden FiniteElemente-Simulationen mit „LS-Dyna“ durchgeführt. Zunächst wurden die Simulationen darauf ausgerichtet, das Auftreten von Falten in konventionellen Scherversuchen nachzubilden, um geeignete numerische Parameter zu erhalten. Für die Simulation kam ein Materialmodell für einen austenitischen Edelstahl (1.4404/316L) mit einer Dicke von 100 µm zum Einsatz [4]. Anschließend wurden Untersuchungen mit geeigneten Werkzeugwirkflächen und Stützflächen sowie einem gekrümmten Messbereich durchgeführt. Dabei zeigte sich, dass eine leichte Krümmung des Messbereichs der Folie die Faltenbildung im Messbereich effektiv unterdrücken konnte. Darüber hinaus ergab eine Analyse der resultierenden Kräfte und der Verschiebungen beziehungsweise Dehnungen, dass die geringen Biegespannungen, die in die Probe eingebracht werden, keine signifikanten Auswirkungen auf die gemessenen Kräfte haben und daher keine nachteiligen Auswirkungen auf die Messergebnisse zu erwarten sind. 2 FE-Modellierung des zyklischen Scherversuchs und numerische Studien Um die auftretende Faltenbildung während konventioneller Scherversuche an dünnen metallischen Folien zu eliminieren, wurde in den vorliegenden Untersuchungen ein neuer Prüfaufbau verwendet, der die Scherprobe leicht krümmt und dadurch ihre strukturelle Stabilität während der Belastungsphasen erhöht, ohne dass zusätzliche Vorrichtungen auf den gesamten Messbereich 440 einwirken. Die Hypothese, dass Faltenbildung bei dünnen metallischen Folien durch eine leichte Krümmung der Vorrichtung während des Scherversuchs verhindert werden kann, wurde durch eine Simulation für einen Messbereich von 2 × 30 mm (siehe Bild 1) an einer Probe aus Edelstahl (1.4404/316L) mit einer Foliendicke von 0,1 mm geprüft. Dafür wurden zwei Simulationen mit LS-Dyna durchgeführt: eine mit dem konventionellen Ansatz unter Verwendung einer flachen Scherprobe und eine zweite mit dem vorgeschlagenen Ansatz, bei dem die Probe mit einer leichten Krümmung von rc = 5 mm versehen wurde und von hinten durch eine starre zylindrische Stützfläche im Messbereich gestützt wurde(Bild 2 b) rote Fläche). Eine solche unterstützende Fläche von einer Seite ist sowohl für die Einbringung der geringfügigen Biegung als auch für die Beibehaltung der gekrümmten Form der Probe beim Durchführen der Scherprüfung notwendig. Die beiden verwendeten Simulationsmodelle sind in Bild 2 dargestellt, wobei das verwendete Materialmodell MAT-36 auf Daten aus [4] basiert. Die Simulationen wurden mit Schalenelementen des Typs 16 durchgeführt, die 11 Integrationspunkte in Richtung der Foliendicke umfassen. Bild 2 stellt die Prüfkörper beziehungsweise Folienproben in brauner Farbe dar. Die beiden virtuellen Scherversuche wurden einer identischen zyklischen Scherbeanspruchung ausgesetzt. In Bezug auf die gewählte Breite des Messbereichs verformte sich die Probe zunächst mit einer Scherdehnung von g = 0,3 nach oben und dann in umgekehrter Richtung bis zu einer Scherdehnung von g = −0,3 (wobei gxy = 2exy). In den FE-Modellen werden die Knoten außerhalb des Messbereichs, die in den realen Tests durch Spannbacken fixiert werden, durch knotenbasierte Randbedingungen gesperrt oder in die entsprechenden Richtungen bewegt, um die Scherdeformation herbeizuführen. https://doi.org/10.37544/1436-4980-2023-10-60, am 13.07.2024, 18:04:58 Open Access – - https://elibrary.vdi-verlag.de/agb WT WERKSTATTSTECHNIK BD. 113 (2023) NR. 10 TITELTHEMA – FACHAU FS A T Z Bild 3. Vergleich der Faltenbildung und des -wachstums während des Scherversuchs mit freiem und ebenem Messbereich entsprechend Aufbau Bild 2a) und finales Ergebnis mit Aufbau Bild 2b) mit zylindrischer Stützfläche. Grafik: IFU Universität Stuttgart Der konventionelle ebene Scherversuch (Bild 2 a) zeigte dabei eine starke Faltenbildung im Messbereich, die den beabsichtigten Spannungszustand stark verfälschte. Eine zuverlässige Messung der reinen Scherdehnung mit einem optischen Messsystem wäre in diesem Fall nicht gewährleistet. Der modifizierte Scherversuch gemäß Bild 2 b) weist eine zylindrische Stützfläche mit einem Radius von rc = 5 mm auf, welche sich auf der Rückseite des Messbereichs befindet. In der Simulation wurde der Messbereich der Probe (braun) mithilfe dieser zylindrischen Stützfläche in einem ersten Schritt leicht gekrümmt. Das Einbringen dieser Krümmung beziehungsweise dieses initiale Biegen wurden in der FEM-Simulation ebenfalls simuliert. So werden die Auswirkungen der geringfügig eingebrachten Spannungen, welche durch die Biegung entstehen, über die 11 Integrationspunkte in der Dickenrichtung berücksichtigt. Die Vorderseite der Probe blieb dabei frei von jeglichem Werkzeugkontakt. Es wurden lediglich zwei ebenfalls zylindrische Flächen (in Bild 2 b) in blau dargestellt) oberhalb und unterhalb des Messbereichs in einem geringen Abstand zur Probenoberfläche positioniert. Diese zylindrischen Flächen, auch Faltenschilde genannt, sind so konstruiert, dass das Wachstum von Falten, das normalerweise am oberen und unteren Rand des Messbereichs aufgrund der Belastung initiiert wird, auf eine unkritische maximale Höhe begrenzt wird. Umfangreiche Simulationen im Rahmen dieser Studie haben gezeigt, dass das Begrenzen des Faltenwachstums im oberen und unteren Bereich der Probe (die nicht zwingend zur Bewertung der Scherdehnung durch das optische System verwendet werden) eine weitere Ausbreitung von Falten in den zentralen Messbereich der Probe unterbinden. Der zentrale Messbereich der Probe kann dabei nach wie vor für eine optische Messung der sich einstellenden Dehnungen genutzt werden. Es kann angenommen werden, dass der visuell freie Messbereich der Probe repräsentativ für den kompletten Messbereich gültig ist. Ein faltenfreier mittiger Messbereich kann auch durch den alleinigen Einsatz der gekrümmten Stützfläche erreicht werden (siehe unten Bild 3). WT WERKSTATTSTECHNIK BD. 113 (2023) NR. 10 3 Diskussion der Ergebnisse Die Simulationsergebnisse zeigen, wie in Bild 2 a) dargestellt, dass es beim konventionellen Scherversuch zu einer übermäßigen Faltenbildung in der flachen Probe kommt. Im Gegensatz dazu ist im Messbereich der leicht gekrümmten Probe, wie Bild 2 b) zeigt, keine Faltenbildung zu beobachten. Dies resultiert aus der erhöhten strukturellen Stabilität des belasteten Querschnitts der Probe gegenüber Faltenbildung. Die Verwendung von sogenannten zusätzlichen Faltenschildern kann mit der nachfolgenden Argumentation gerechtfertigt werden: Die Faltenbildung wird beim zyklischen Scherversuch in den freien Kanten am oberen und unteren Ende des Messbereichs, wie in Bild 3 dargestellt, initiiert. Diese Falten propagieren anschließend mit zunehmender Scherdehnung in den Bereich der Probenmitte beziehungsweise in die Mitte des Messbereichs. Durch eine Begrenzung der Höhe der initialen Falten durch sogenannte Faltenschilder kann ein weiteres Anwachsen sowie Propagieren der Falten verhindert werden. Dabei wird initial kein Druck auf die Probenoberfläche aufgebracht und erst im weiteren Verlauf des Scherversuchs werden Falten im Messbereich durch eine lokale und leichte Berührung unterbunden. Der Einfluss der Faltenschilder auf das finale Ergebnis ist hierbei marginal, wie im weiteren Verlauf der Untersuchungen gezeigt wird. Um sicherzustellen, dass die leichte Krümmung des Messbereichs der Probe vernachlässigbare Auswirkungen auf die Messgenauigkeit und die Auswertung von Schubspannungen und -dehnungen hat, wurden die Kräfte und die Verschiebungen sowohl aus dem konventionellen Scherversuch (siehe Bild 2 a) und aus dem neuen Ansatz mit gekrümmten Messbereich (siehe Bild 2 b) miteinander verglichen. Für einen geeigneten Vergleich wurde zusätzlich eine ideelle Probe mit ebenem und freiem Messbereich ohne Stützfläche herangezogen. In dieser ideellen Version wird die Faltenbildung durch das Sperren des Freiheitsgrades senkrecht zur Probenoberfläche verhindert. Ein Vergleich der resultierenden Kräfte im ideellen, im konventionellen und im https://doi.org/10.37544/1436-4980-2023-10-60, am 13.07.2024, 18:04:58 Open Access – - https://elibrary.vdi-verlag.de/agb 441 T I TE L T H E MA – FACHAUFSATZ Bild 4. Vergleich der berechneten Kraftwerte und der Scherdehnungen für eine ideelle ebene Probe mit einer gekrümmten Probe mit Stützfläche und mit einer ebenen Probe ohne Stützfläche. Grafik: IFU Universität Stuttgart neuen Versuchsaufbau mit gekrümmtem Messbereich ist in Bild 4 dargestellt. Es zeigt sich, dass sich zwischen den Kraftverläufen der ideellen Version und dem Versuchsaufbau mit gekrümmtem Probenbereich nur eine vernachlässigbare Abweichung einstellt. Im Gegensatz zur konventionellen ebenen Probe (rote Kurve in Bild 4) zeigt die gekrümmte Probe einen nahezu gleichen Kraftverlauf, wie auch die Probe im ideellen Szenario. Außerdem zeigen die Scherdehnungen g, die auf Grundlage der Knotenverschiebungen der Probe in der Simulation berechnet werden, keine signifikanten Unterschiede zwischen der gekrümmten und der idealisierten Probe. Die Ergebnisse zeigen, dass der vorgeschlagene neue Prüfaufbau mit einem gekrümmten Probenbereich eine effektive Methode zur Reduktion der Faltenbildung beim Scherversuch an dünnen metallischen Folien ist. 4 Zusammenfassung und Ausblick Dieser Beitrag präsentiert einen neuartigen Ansatz zur Durchführung von zyklischen Scherversuchen an dünnen metallischen Folien. Durch die Einführung einer leichten Krümmung der Probe im Messbereich kann die Bildung unerwünschter Falten vermieden werden, wodurch eine zuverlässige Messung und Berechnung von Schubspannungen und -dehnungen im Messbereich möglich wird. Zur Überprüfung dieses Ansatzes wurden FiniteElemente-Simulationen unter Verwendung vom LS-Dyna durchgeführt, um den Machbarkeitsnachweis zu erbringen. Die Simulationen konventioneller Scherversuche zeigten, dass die Faltenbildung in der Regel an den freien Kanten des Messbereichs der Probe oben und unten beginnt und sich anschließend nach innen ausbreitet. Durch die Krümmung des Messbereichs der Probe in eine leicht zylindrische Form unter Verwendung einer Stützfläche und die zusätzliche Nutzung sogenannter Faltenschilder wird ein faltenfreier Messbereich sichergestellt, der optisch zuverlässig vermessen werden kann. Ein Vergleich der Kraft- und Scherdehnungsverläufe der unterschiedlichen Versuchsaufbauten hat gezeigt, dass die beim Krümmen der Probe auftretende Biegespannung keinen Einfluss hat und daher vernachlässigt werden kann (siehe Bild 4). Zukünftige Arbeiten 442 werden sich auf weitere Untersuchungen mit unterschiedlichen Biegeradien sowie mit verschiedenen Folienmaterialien unterschiedlicher Dicke konzentrieren. Zur Validierung des vorgeschlagenen Konzepts wird eine geeignete Testeinrichtung für experimentelle Untersuchungen aufgebaut werden. DANKSAGUNG Diese Arbeit wurde im Rahmen des Arbeitskreises „Folienumformung“ am Institut für Umformtechnik sowie im Rahmen des trilateralen Transferprojekts AKS-Bipolar der DFG (Deutsche Forschungsgemeinschaft) und der Fraunhofer-Gesellschaft (DFG-Projekt 460294948) durchgeführt. Die Autoren danken der DFG und dem Arbeitskreis Folienumformung für Ihre Unterstützung. L i tera tu r [1] Vollertsen, F.; Biermann, D.; Hansen, H.N. et al.: Size effects in manufacturing of metallic components. CIRP Ann Manuf Technol 58 (2009) 2, pp. 566–587 [2] Neto, D. M.; Oliveira, M. C.; Alves, J. L. et al.: Numerical study on the formability of metallic bipolar plates for proton exchange membrane (PEM) fuel cells. Metals 9 (2019) 7, # 810 [3] Hu, Q.; Zhang, D.; Fu, H. et al.: Investigation of stamping process of metallic bipolar plates in PEM fuel cell – numerical simulation and experiments. International Journal of Hydrogen Energy 39 (2014) 25, pp. 13770–13776 [4] Beck, M.; Riedmüller, K. R.; Liewald, M. et al.: Investigation on the influence of geometric parameters on the dimensional accuracy of highprecision embossed metallic bipolar plates. In: Liewald, M. (Hrsg.): Production at the Leading Edge of Technology. Proceedings of the 12th Congress of the German Academic Association for Production Technology (WGP), Stuttgart, 2022 pp. 427–438 [5] Bauer, A.: Experimentelle und numerische Untersuchungen zur Analyse der umform-technischen Herstellung metallischer Bipolarplatten. Dissertation, TU Chemnitz, 2020 [6] Adzima, F.; Balan, T.; Manach, P. Y.: Springback prediction for a mechanical micro connector using CPFEM based numerical simulations. International Journal of Material Forming 13 (2020) pp. 649–659 [7] Porstmann, S.; Wannemacher, T.; Drossel, W. G.: A comprehensive comparison of state-of-the-art manufacturing methods for fuel cell bi- https://doi.org/10.37544/1436-4980-2023-10-60, am 13.07.2024, 18:04:58 Open Access – - https://elibrary.vdi-verlag.de/agb WT WERKSTATTSTECHNIK BD. 113 (2023) NR. 10 TITELTHEMA polar plates including anticipated future industry trends. Journal of Manufacturing Processes 60 (2020) pp. 366–383 [8] Vollertsen, F.; Schulze Niehoff, H.; Hu, Z.: State of the art in micro forming. I International Journal of Machine Tools and Manufacture 46 (2006) 11, pp. 1172–1179 [9] Pham, C.; Adzima, F.; Coër, J. et al.: Anti-buckling device for ultra-thin metallic sheets under large and reversed shear strain paths. Experimental Mechanics 57 (2017) pp. 593–602 [10] Yoshida, F.; Uemori, T.: A model of large-strain cyclic plasticity and its application to springback simulation. International Journal of Mechanical Sciences 45 (2003) 10, pp. 1687–1702 [11] Chaboche, J.L.: Time-independent constitutive theories for cyclic plasticity. International Journal of Plasticity 2 (1986) 2, pp. 149–188 [12] Chaboche, J.L.: Constitutive equations for cyclic plasticity and cyclic viscoplasticity. International Journal of Plasticity 5 (1989) 3, pp. 247–302 [13] Bouvier, S.; Haddadi, H.; Levée, P. et al.: Simple shear test: experimental techniques and characterization of the plastic anisotropy of rolled sheets at large strains. Journal of Materials Technology 172 (2006) pp. 96–103 [14] Yin, Q.; Zillmann, B.; Suttner, S. et al.: An experimental and numerical investigation of different shear test configurations for sheet metal characterization. International Journal of Solids and Structures 51 (2014) 5, pp. 1066–1074 [15] Brosius, A.; Yin, Q.; Güner, A. et al.: A new shear test for sheet metal characterization. Steel Research International 82 (2011) 4, pp. 323–328 [16] Zhang, P.; Pereira, M. P.; Abeyrathna, B. et al.: Improving the shear test to determine shear fracture limits for thin stainless steel sheet by shape optimisation. International Journal of Mechanical Sciences 164 (2019), #105116 – FACHAU FS A T Z Dr. sc. techn. C e l a l e t t i n K a ra do ga n Foto: IFU Stuttgart M ax i m B e c k , M. Sc. Pa t ri ck C y r o n , M. Sc. Dr.-Ing. K i m R o u ve n R ie d m ül l e r Univ.-Prof. Dr.-Ing. M a t h i a s L i e w a l d MBA Institut für Umformtechnik (IFU), Universität Stuttgart Holzgartenstr. 17, 70174 Stuttgart Tel. +49 711 / 985-838 40 sekretariat@ifu.uni-stuttgart.de www.ifu.uni-stuttgart.de LIZENZ Dieser Fachaufsatz steht unter der Lizenz Creative Commons Namensnennung 4.0 International (CC BY 4.0) WT WERKSTATTSTECHNIK BD. 113 (2023) NR. 10 https://doi.org/10.37544/1436-4980-2023-10-60, am 13.07.2024, 18:04:58 Open Access – - https://elibrary.vdi-verlag.de/agb 443
https://openalex.org/W2906727739	https://www.ped-perinatology.ru/jour/article/download/778/727	Russian	null	Dental health of adolescents with arterial hypertension: rationale for approaches to prevention using calcium glycerophosphate and magnesium chloride	Rossijskij vestnik perinatologii i pediatrii	2,018	cc-by	4,164	L.R. Kolesnikova1,2, A.V. Pogodina1, M.V. Fedotova3, L.V. Rychkova1, O.V. Valyavskaya1 1Scientific Center for Family Health and Human Reproduction, Irkutsk, Russia; 2Irkutsk State Medical University, Irkutsk, Russia; 3Lyutikov’s Clinics, Irkutsk, Russia Резюме. Цель исследования: охарактеризовать состояние стоматологического здоровья подростков с артериальной ги- пертензией и разработать на основе полученных результатов комплекс лечебно-профилактических мероприятий, позво- ляющий повысить его уровень. В исследование включено 130 подростков (65 – с артериальной гипертензией, 65 – кон- трольной группы) в возрасте 14,3 ± 2 года. Уровень систолического артериального давления при офисном измерении у подростков в основной группе был выше такового в группе контроля (126,6 ± 12,6 мм рт. ст. против 113,2 ± 7,4 мм рт. ст., p=0,00009), при том что уровень диастолического давления был сопоставимым. Полученные данные свидетельствуют о том, что подростков с артериальной гипертензией характеризуют неудовлетворительные показатели гигиены полости рта и значительно большая частота основных стоматологических заболеваний: кариеса зубов и пародонтита. Это обосно- вывает выделение подростков с артериальной гипертензией в группу, уязвимую как по развитию новых, так и по прогрес- сированию имеющихся заболеваний зубочелюстной системы, что может оказывать существенное ухудшающее влияние на кардиоваскулярное здоровье. Все это диктует необходимость разработки системы профилактических действий, кото- рые должны строиться на основании междисциплинарного подхода с активным участием специалистов соматического и стоматологического профиля. Ключевые слова: подростки, артериальная гипертензия, кариес зубов, заболевания пародонта, глицерофосфат кальция, хло- рид магния. Для цитирования: Колесникова Л.Р., Погодина А.В., Федотова М.В., Рычкова Л.В., Валявская О.В. Стоматологическое здоровье под- ростков с артериальной гипертензией: обоснование рациональных подходов к профилактике с использованием глицерофосфата каль- ция и хлорида магния. Рос вестн перинатол и педиатр 2018; 63:(6): 98–102. DOI: 10.21508/1027–4065–2018–63–5–98–102 Summary. Objective: to characterize the dental health of adolescents with arterial hypertension and to increase its level with a com- plex of therapeutic and prophylactic measures based on the results obtained. The study included 130 adolescents (65 – with arterial hypertension and 65 – control group) of 14.3 ± 2 years old. The level of systolic blood pressure in the main group was higher than that of the adolescents in the control group (126.6 ± 12.6 mm Hg vs 113.2 ± 7.4 mm Hg, p=0.00009), while the level of diastolic blood pressure was comparable. The data obtained suggest that adolescents with arterial hypertension are characterized by unsatisfactory oral hygiene indicators and a significantly higher incidence of major dental diseases: dental caries and periodontitis. Стоматологическое здоровье подростков с артериальной гипертензией: обоснование рациональных подходов к профилактике с использованием глицерофосфата кальция и хлорида магния Л.Р. Колесникова1,2, А.В. Погодина1, М.В. Федотова3, Л.В. Рычкова1, О.В. Валявская1 1ФГБНУ «Научный центр проблем здоровья семьи и репродукции человека», г. Иркутск, Россия; 2ФГБОУ ВО «Иркутский государственный медицинский университет» Минздрава России, г. Иркутск 3Клиника доктора Лютикова, г. Иркутск, Россия ОБМЕН ОПЫТОМ ОБМЕН ОПЫТОМ Стоматологическое здоровье подростков с артериальной гипертензией: обоснование рациональных подходов к профилактике с использованием глицерофосфата кальция и хлорида магния L.R. Kolesnikova1,2, A.V. Pogodina1, M.V. Fedotova3, L.V. Rychkova1, O.V. Valyavskaya1 It justifies the selection of adolescents with arterial hypertension in a group that is vulnerable both to the development of new and to the progression of existing diseases of the dental-maxillary system, which can significantly worsen cardiovascular health. Therefore, there is a need to develop a system of preventive actions built on the basis of an interdisciplinary approach with the active participation of somatic and dental specialists. For citation: Kolesnikova L.R., Pogodina A.V., Fedotova M.V., Rychkova L.V., Valyavskaya O.V. Dental health of adolescents with arterial hyper- tension: rationale for approaches to prevention using calcium glycerophosphate and magnesium chloride. Ros Vestn Perinatol i Pediatr 2018; 63:(6): 98–102 (in Russ). DOI: 10.21508/1027–4065–2018–63–5–98–102 Рычкова Любовь Владимировна – д.м.н., проф., директор Научного цен- тра проблем здоровья семьи и репродукции человека 664003 Иркутск, ул. Тимирязева, д. 16 Валявская Ольга Владимировна – к.м.н., врач отделения функциональ- ной диагностики Клиники Научного центра проблем здоровья семьи и репродукции человека, ORCID: 0000-0001-9727-2229 664043 Иркутск, ул. Дальневосточная, д. 67А Федотова Марина Викторовна – к.м.н., стоматолог-ортодонт стоматоло- гической клиники доктора Лютикова, ORCID: 0000-0002-4667-1497. 664011 Иркутск, ул. Российская, д. 13 adolescents, arterial hypertension, dental caries, periodontal disease, calcium glycerophosphate, magnesium chloride. Key words: adolescents, arterial hypertension, dental caries, periodontal disease, calcium glycerophosphate, magnesiu Характеристика детей и методы исследования Основная группа (n=65) была сформирована из числа подростков, направленных в клинику Науч- ного центра проблем здоровья семьи и репродукции человека по поводу повышения уровня артериально- го давления. Критериями включения были: возраст 10–17 лет; уровень систолического и/или диасто- лического артериального давления за любой период суток выше 95-го процентиля для данного возраста, роста и пола или выше уровня, принятого за отрез- ную точку диагностики артериальной гипертензии для взрослых (суточное 130/80 мм рт. ст., дневное 135/85 мм рт. ст., ночное 125/75 мм рт. ст.) [22]. Под- ростки, у которых в ходе обследования был установ- лен вторичный характер повышения артериального давления, из исследования исключались. Группа контроля (n=65) была сформирована из чис- ла подростков, направленных в клинику по поводу со- стояний, не ассоциированных с повышением уровня артериального давления, а также из школьников, про- ходивших плановый осмотр у стоматолога. Критерием включения в группу контроля было наличие нормаль- ного уровня артериального давления при трехкрат- ном измерении. Формирование групп осуществлялось по принципу «случай–контроль», так что каждому подростку основной группы (случай) соответствовал подросток в группе контроля того же пола и возраста (контроль). Родители / законные представители под- ростков, а также подростки старше 15 лет подписали информированное добровольное согласие на участие в исследовании. Общими критериями исключения были: наличие тяжелых соматических заболеваний; проводимое в настоящее время ортодонтическое лече- ние; наличие сахарного диабета; аномалии прикуса. До недавних пор в медицине доминировало пред- ставление о стоматологических заболеваниях как о локальной патологии. В настоящее время проведен- ные исследования меняют парадигму взглядов и про- слеживается иная концепция, согласно которой су- ществует очевидное взаимовлияние соматического здоровья и стоматологических заболеваний. Высокая распространенность и постоянный прирост основных стоматологических заболеваний, их роль в формиро- вании хронического одонтогенного очага инфекции, а также разработка планов лечения и профилактики патологии вызывают постоянный интерес к указанной проблеме [11–13]. В ряде исследований показаны зна- чимые ассоциации высокого артериального давления и стоматологических заболеваний [14–16]. Многочис- ленные зарубежные и отечественные исследования, в том числе и экспериментальные, демонстрируют влияние преморбидного фона на морфофункциональ- ной состояние челюстно-лицевой области [17–19]. Несмотря на то что этиологические факторы кариеса зубов и заболеваний пародонта изучались длительный период, парадигма стоматологии лишь в последнее время стала склоняться не только к учету коморбид- ных и полиморбидных состояний у пациентов сто- матологического профиля, но и к их интерпретации как постоянного и важного звена в непрерывной цепи стоматологической и соматической патологии. © Коллектив авторов, 2018 Адрес для корреспонденции: Колесникова Лариса Романовна – к.м.н., н.с. лаборатории педиатрии и кардиоваскулярной патологии Научного центра проблем здоровья семьи и репродукции человека, асс. кафедры стоматологии детского возраста Иркутского государственного медицин- ского университета, ORCID: 0000-0003-2161-6034 664003 Иркутск, ул. Красного Восстания, д. 1 Погодина Анна Валерьевна – д.м.н., гл.н.с. лаборатории педиатрии и кар- диоваскулярной патологии Научного центра проблем здоровья семьи и репродукции человека РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2018; 63:(6) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2018; 63:(6) РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2018; 63:(6) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2018; 63:(6) Колесникова Л.Р. и соавт. Стоматологическое здоровье подростков с артериальной гипертензией... А р я А ртериальная гипертензия у детей и подростков является актуальной проблемой современной клинической медицины. Многочисленными иссле- дованиями показано, что истоки возникновения сер- дечно-сосудистых заболеваний у взрослых следует искать именно в детском и подростковом возрасте, однако многие аспекты этой проблемы в данный воз- растной период остаются недостаточно изученными [1–5]. По материалам популяционных исследований, проведенных в нашей стране, распространенность повышенного артериального давления в детском и подростковом возрасте варьирует от 2,4 до 18% [6–9]. Артериальная гипертензия сопровождается многочисленными полисистемными нарушениями, снижением иммунитета, ранним возникновением атерогенных сдвигов, значительным дисбалансом нейровегетативных и эндокринных влияний, суще- ственными изменениями центральной и региональ- ной гемодинамики [10]. Результаты и обсуждение В исследование включены 130 подростков в возра- сте 14,3 ± 2 года. Подростки основной группы значи- тельно чаще, чем подростки контрольной группы, стра- дали хроническим тонзиллитом – 8 (12,3%) и 1 (1,5%), p=0,033 и ожирением – 28 (43,1%) и 4 (6,4%), p=0,00001 соответственно. Уровень систолического артериально- го давления при офисном измерении ожидаемо был выше у подростков в группе контроля (126,6 ± 12,6 мм рт.ст. против 113,2 ± 7,4 мм рт. ст., p=0,00009), при том что уровень диастолического артериального давления был сопоставимым. Сравнивая индексные показатели, получен- ные при обследовании тканей пародонта, выявлено, что подростки в контрольной группе в 3 раза чаще имеют здоровый пародонт, чем подростки с артери- альной гипертензией (41,5 и 13,8% соответственно; p=0,0004). И если число подростков, которых можно отнести к группе риска по развитию заболеваний па- родонта, было сопоставимым в обеих группах, то кли- нически выраженный пародонтит у подростков с арте- риальной гипертензией наблюдался значительно чаще и включал среднетяжелые формы заболевания, кото- рые в группе контроля не диагностировались (табл. 2). При стоматологическом обследовании в груп- пе контроля интактный зубной ряд был выявлен в 50,8% случаев, что в 2,2 раза превышало аналогич- ный показатель у подростков с артериальной гипер- тензией – 23,1% (р=0,001). Соответственно частота встречаемости кариеса зубов в группе подростков с артериальной гипертензией была значительно выше таковой в контроле (табл. 1). Следует отметить, что указанные различия реализовались за счет боль- Таблица 1. Частота встречаемости кариеса зубов в группах обследованных подростков, абс (%) Table 1. Frequency of occurrence of dental caries in the groups of the examined adolescents, аbs (%) Таблица 1. Частота встречаемости кариеса зубов в группах обследованных подростков, абс (%) Table 1. Frequency of occurrence of dental caries in the groups of the examined adolescents, аbs (%) Показатель Контрольная группа (n=65) Основная группа (n=65) р Частота встречаемости в группе В том числе: 1-я степень активности кариеса 2-я степень активности кариеса 3-я степень активности кариеса 32 (49,2) 9 (13,8) 14 (21,5) 9 (13,8) 50 (76,9) 21 (32,3) 19 (29,2) 10 (15,4) 0,001 0,012 0,314 0,804 П * С Показатель Контрольная группа (n=65) Основная группа 100 РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2018; 63:(6) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2018; 63:(6) Таблица 2. Частота встречаемости заболеваемости пародонта в группах обследованных подростков, абс (%) Table 2. ОБМЕН ОПЫТОМ Для анализа полученных данных использовали статистический пакет STATISTICA 6.1 StatSoftInc, США. Для определения близости к нормальному закону распределения количественных признаков применяли визуально графический метод и крите- рии согласия Колмогорова–Смирнова с поправкой Лиллиефорса и Шапиро–Уилка. Проверка равенства генеральных дисперсий осуществлялась с помощью критерия Фишера (F-test). При анализе межгруппо- вых различий по количественным переменным ис- пользовали непараметрический критерий Манна– Уитни, по качественным переменным – критерий χ2 и двусторонний критерий Фишера при малой чис- ленности групп. Критический уровень значимости принимался равным 5 % (0,05). шей частоты у подростков с артериальной гипертен- зией кариозного процесса 1-й степени активности. Тогда как частота кариеса 2-й и 3-й степени активно- сти была сопоставимой в обеих группах. При оценке гигиенического состояния полости рта получены следующие результаты: хорошее со- стояние полости рта выявлено в контрольной группе у 35,4%, что в 2,8 раза превышало аналогичный по- казатель в основной группе – 12,3% (р=0,002). Удо- влетворительное состояние полости рта отмечено в контрольной и основной группах в 43,1 и 44,6% слу- чаев соответственно. Неудовлетворительная гигиена полости рта в группе с артериальной гипертензией была определена у 32,3 %, что достоверно отличалось от контрольной группы – у 15,4% (р=0,024). Индекс гигиены с критерием «плохо» зарегистрирован в ос- новной группе у 10,8% подростков, в контрольной группе – у 6,1%. Исходя из того, что зубной налет является местным фактором, способствующим раз- витию и кариеса зубов, и заболеваний пародонта, по- лученные результаты обосновывают необходимость профилактической работы, направленной на пред- упреждение возникновения и прогрессирования имеющихся стоматологических заболеваний в когор- те подростков с артериальной гипертензией. Характеристика детей и методы исследования В кон- тексте современного представления «стомато-сомати- ческого здоровья» прослеживается междисциплинар- ный подход взаимосвязанных состояний, что еще раз подтверждает отрицательную коморбидность патоло- гии стоматологического и терапевтического профиля [20, 21]. Клиническое стоматологическое обследование каждого подростка проводилось по общеприня- той методике и включало: опрос, внешний осмотр, осмотр полости рта с записью зубной формулы и определением индекса интенсивности кариеса. Для оценки гигиенического состояния полости рта использовался индекс гигиены по Федорову–Волод- киной в модификации Пахомова, учитывающий пло- щадь зубного налета на вестибулярной поверхности зубов 16, 11, 21, 26, 36, 33, 32, 31, 41, 42, 43, 46. Окра- шивание проводилось раствором Шиллера–Писа- рева. Оценка гигиенического состояния полости рта характеризовалась с помощью полученных результа- тов: 1,1–1,5 – хорошо; 1,6–2,0 – удовлетворитель- но; 2,1–2,5 – неудовлетворительно; 2,6–3,4 – плохо; 3,5–5,0 – очень плохо. Комплексный пародонталь- ный индекс (Леус П.А., 1988) определяли при обсле- довании зубов 16, 11, 26, 31, 36, 46. Полученные зна- чения ранжировали следующим образом: 0 – норма; 0,1 – 1 – риск заболевания; 1,1 – 2,1 – легкая степень поражения; 2,1 – 3,5 – средняя степень поражения; 3,6 – 5,0 – тяжелая степень поражения. Цель работы: охарактеризовать состояние стома- тологического здоровья подростков с артериальной гипертензией и разработать на основе полученных результатов комплекс лечебно-профилактических мероприятий, позволяющих повысить его уровень. РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2018; 63:(6) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2018; 63:(6) РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2018; 63:(6) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2018; 63:(6) ОБМЕН ОПЫТОМ Заключение 2. Плановая санация полости рта. Полученные данные свидетельствуют о том, что подростков с артериальной гипертензией харак- теризуют плохие показатели гигиены полости рта и значительно большая частота основных стоматоло- гических заболеваний: кариеса зубов и пародонтита. Это обосновывает выделение подростков с артери- альной гипертензией в группу, уязвимую как по раз- витию новых, так и по прогрессированию имеющих- ся заболеваний зубочелюстной системы, что в свете современных представлений может оказывать суще- ственное ухудшающее влияние на кардиоваскуляр- ное здоровье. Все это диктует необходимость разра- ботки системы профилактических действий, которые должны строиться на основании междисциплинар- ного подхода с активным участием специалистов со- матического и стоматологического профиля. 3. Профессиональная гигиена полости рта с обучени- ем и коррекцией индивидуальной гигиены полости рта. 3. Профессиональная гигиена полости рта с обучени- ем и коррекцией индивидуальной гигиены полости рта. 4. Подбор средств гигиены с учетом состояния эмали зуба и тканей пародонта. 4. Подбор средств гигиены с учетом состояния эмали зуба и тканей пародонта. В настоящее время имеется широкий ассортимент средств по гигиеническому уходу за полостью рта. Рекомендуемые нами средства подобраны с учетом процессов созревания и формирования тканей в под- ростковом возрасте: • зубная паста R.O.C.S. PRO Young&White Enamel – двухразовая чистка зубов. Использование данной па- сты оказывает положительное влияние на незрелую эмаль зуба, увеличивая минерализующую функцию ротовой жидкости, а также способствует снижению скорости образования зубного налета, благоприятно воздействует на ткани пародонта; Результаты и обсуждение Осмотр стоматологом 3 раза в год. 1. Осмотр стоматологом 3 раза в год. Результаты и обсуждение Frequency of occurrence of periodontal morbidity in groups of examined adolescents, abs (%) Комплексный пародонтальный индекс Контрольная группа (n=65) Основная группа (n=65) р Частота встречаемости в группе В том числе: риск заболевания легкая степень средняя степень 38 32 6 0 58,5* 49,2 9,2* 56 30 21 5 86,1* 46,1 32,3* 7,7 0,0004 0,725 0,001 0,058** Примечание. Статистически значимые различия между группами. *Двусторонний Фишера. лица 2. Частота встречаемости заболеваемости пародонта в группах обследованных подростков, абс (%) le 2. Frequency of occurrence of periodontal morbidity in groups of examined adolescents, abs (%) 100 РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2018; 63:(6) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2018; 63:(6) РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2018; 63:(6) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2018; 63:(6) Колесникова Л.Р. и соавт. Стоматологическое здоровье подростков с артериальной гипертензией... • гель R.O.C.S. Medical Minerals – аппликации в те- чение 15 мин утром и вечером после чистки зубов. Является источником легкоусвояемых соединений кальция, фосфора и магния, обладает адгезивными свойствами, позволяющими продлить время экс- позиции активных компонентов. Введенный в его состав ксилит повышает реминерализирующий по- тенциал и подавляет активность кариесогенных ми- кроорганизмов; • гель R.O.C.S. Medical Minerals – аппликации в те- чение 15 мин утром и вечером после чистки зубов. Является источником легкоусвояемых соединений кальция, фосфора и магния, обладает адгезивными свойствами, позволяющими продлить время экс- позиции активных компонентов. Введенный в его состав ксилит повышает реминерализирующий по- тенциал и подавляет активность кариесогенных ми- кроорганизмов; В результате проведенного исследования в груп- пах детей, идентичных по полу, возрасту, стадии по- лового созревания, но различающихся по основному соматическому заболеванию, установлено, что у под- ростков с артериальной гипертензией частота встре- чаемости основных стоматологических заболеваний достоверно превышает таковую у подростков с нор- мальным уровнем артериального давления. Получен- ные результаты свидетельствуют о том, что подрост- ки с артериальной гипертензией представляют собой группу повышенного риска формирования как ка- риеса зубов, так и заболеваний пародонта. Следова- тельно, для этих пациентов необходима разработка дополнительных профилактических мероприятий, направленных на предупреждение появления и про- грессирования имеющихся стоматологических забо- леваний. • жевательные таблетки витаминно-минерального комплекса R.O.C.S. Medical – по 1 таблетке 3 раза в день во время еды для насыщения зубов минерала- ми. Потребность в витаминах в детском и подростко- вом возрасте значительно выше, чем у взрослых. Это объясняется особенностями растущего организма – напряжением обменных процессов, быстрым ростом и развитием [23]. Предлагаемая нами схема включает ледующие этапы: Все средства рекомендовано использовать в тече- ние 30 дней 3 раза в год с перерывом 3 мес. 1. Uric acid and cardiometabolic risk factors for hypertension in adolescents. 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Клинико- эпидемиологическая характеристика развития основных стоматологических заболеваний среди детского и под- росткового населения Таджикистана. Мат. II Междунар. науч. конф. «Новые задачи современной медицины». Санкт-Петербург, май 2013 г. СПб: Реноме 2013:41–43. [Djuraeva Sh.F., Turdyev B.Z, Sharipov H.S. Clinical and epidemiological characteristics of the development of major dental diseases among children and adolescents in Tajikistan. Materials II International. Sci. Conf. «New problems of mod- ern medicine». ЛИТЕРАТУРА (REFERENCES): Uric acid and cardiometabolic risk factors for hypertension in adolescents. Kardiologiya (Cardiology) 2014; 54(7): 36–42 (in Russ)]. 1. Долгих В.В., Леонтьева И.В., Рычкова Л.В., Погодина А.В., Мандзяк Т.В., Бугун О.В. и др. Алгоритмы диагностики и лечения, принципы профилактики артериальной ги- пертензии у подростков. Иркутск: ООО «Аспринт» 2008: 26. 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Зыкеева С.К., Ургенишбаева Ж.Р. Профилактика и ле- чение заболеваний пародонта у детей и подростков. Вестник КазНМУ 2016; 3: 97–100. [Zykeeva S.K., Urgen- ishbaeva Zh.R. Prevention and treatment of periodontal dis- eases in children and adolescents. Vestnik KazNMU 2016; 3: 97–100. (in Russ)] 13. Колесникова Л.Р., Власов Б.Я., Натяганова Л.В., Долгих Л.Г. Ассоциация эссенциальной артериальной гипертензии 13. Колесникова Л.Р., Власов Б.Я., Натяганова Л.В., Долгих Л.Г. Ассоциация эссенциальной артериальной гипертензии 13. Колесникова Л.Р., Власов Б.Я., Натяганова Л.В., Долгих Л.Г. Ассоциация эссенциальной артериальной гипертензии Received on 2018.10.03 Конфликт интересов: Conflict of interest: The authors of this article confirmed the lack of conflict of interest and financial support, which should be reported. Авторы данной статьи подтвердили отсутствие кон- фликта интересов и финансовой поддержки, о которых необходимо сообщить. РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2018; 63:(6) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2018; 63:(6)
https://openalex.org/W2081013412	https://hal.archives-ouvertes.fr/hal-00957607/file/Ratier2011.pdf	English	null	Analog Computing of Control Laws by Periodic Network of Resistances	null	2,010	cc-by	5,844	Analog computing of control laws by periodic network of resistances Nicolas Ratier Nicolas Ratier To cite this version: Nicolas Ratier. Analog computing of control laws by periodic network of resistances. Hardware and Software Implementation and Control of Distributed MEMS (DMEMS), Jan 2010, Besançon, France. pp.64-71, 10.1109/dMEMS.2010.17. hal-00957607 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-00957607 https://hal.science/hal-00957607v1 Submitted on 11 Apr 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License I. INTRODUCTION Actuation, sensoring and control of very large sized arrays of MEMS, says 1000×1000 MEMS or cells on the same chip, are out of reach of digital computing (microprocessor). The revival of analog computing might be the recourse for ﬁlling the gap of our computing power. Control laws are formulated in terms of ordinary or partial differential equations. The electronic analog computers used in the seventies were limited to solve ordinary differential equations. They were composed of operational ampliﬁers and capacitances. Unfortunatly, the use of numerous capacitances renders them unsuitable for high density integration. Today, advent of nanotechnology could make conceivable molecular or quantum computing [1]. Analog Computing of Control Laws by Periodic Network of Resistances Nicolas Ratier FEMTO–ST Institut Time–Frequency department 32 avenue de l’Observatoire, 25044 Besanc¸on Cedex, France nicolas.ratier@femto-st.fr We now present how to construct an electronic circuit of a linear PDE up to the fourth order. The discrete solution at each voltage node of the center of each cell composing this circuit converges towards the solution of the PDE, with its boundary conditions, for a given input signal (the right hand side of the PDE) when the number of periodic cells increases. The choice of the ﬁnite difference scheme used to represent the PDE is a mathematical problem and is not relevant here. It is clear that if this ﬁnite difference scheme is stable and accurate, its electronic implementation will have a good behaviour too. Abstract—We show in this paper that a class of Periodic Networks of Resistances (called PNR) can be used to solve partial differential equations. More precisely, each cell of this periodic network computes locally a ﬁnite difference approximation of the partial differential equation. The PNR circuits are composed of current source, voltage–controlled voltage source and positive and negative resistances. An example of application of PNR could be the real time control of distributed MEMS. Keywords-analog computing; partial differential equations; PDE; ﬁnite difference; periodic networks of resistances; PNR; The 14+1 partial differential operators up to order 4 are summarized in table I. It is sufﬁcient to study 9 of them (at the left side of the table), all the other ones can be deduced by symmetry. TABLE I PARTIAL DIFFERENTIAL OPERATORS UP TO ORDER 4. ∂0 ∂x0∂y0 ∂ ∂x ∂ ∂y ∂2 ∂x ∂2 ∂x∂y ∂2 ∂y ∂3 ∂x3 ∂3 ∂x2∂y ∂3 ∂x∂y2 ∂3 ∂y3 ∂4 ∂x4 ∂4 ∂x3∂y ∂4 ∂x2∂y2 ∂4 ∂x∂y3 ∂4 ∂y4 ∂0 ∂x0∂y0 ∂ ∂x ∂ ∂y ∂2 ∂x ∂2 ∂x∂y ∂2 ∂y ∂3 ∂x3 ∂3 ∂x2∂y ∂3 ∂x∂y2 ∂3 ∂y3 ∂4 ∂x4 ∂4 ∂x3∂y ∂4 ∂x2∂y2 ∂4 ∂x∂y3 ∂4 ∂y4 We propose in this paper a much simpler solution to bring back to life analog computing. Our method is based on the design of electronic circuits which “computes” locally the ﬁnite difference approximation of a partial differential equation. These unit cell circuits must have the properties of being possibly assembled then periodically recopied. The whole periodic network is then composed of the numerous repeated identical cells, except at the boundaries where the cell takes into account the boundary conditions. So we can say that this Periodic Network or Resistances (PNR) “solves” or “is described” by a PDE. The following sections propose a study of eight unit circuits which “code” a ﬁnite difference (FD) approximation of these 8 partial differential operators. The section names “Molecule αβ” refers to the centered FD approximation of the derivative ∂α+β ∂xα∂yβ with an error proportional to the step size of order 2. The calculation of the harmonic derivative (Molecule 20) is well known, all the other proposed circuits are originals. The study of each molecule is presented through an increasing difﬁculty and by gathering the similar solutions. The unit cell involves only a current source, VCVSs (Voltage–Controlled Voltage Sources) and positive and neg- ative resistances. The pratical realization of a PNR is not an issue of this paper, but we can just say that the designs of negative resistances can be based on variety of techniques, for example negative input impedance [2][3][4], negative impedance converters and inverters [5] or translinear circuits [6]. The last reference is itself electronically tunable. Next, we present how to mix these elementary cells to construct the circuit of a linear partial differential equation, and ﬁnally we show how to implement the boundary conditions in a circuit form. We introduce now some notations that we will use through- out the paper. In order to use the ﬁnite difference schemes, we divide the domain Ω= (0, 1)2 into (N −1)2 interior points with spatial step size h = 1/N in both x– and y– directions. TABLE I PARTIAL DIFFERENTIAL OPERATORS UP TO ORDER 4. ∂2v ∂x2 = ρ0i0 (3) ∂2v ∂x2 = ρ0i0 (3) (3) The values of the resistances inside a cell depend only on the circuit topology and are easily expressed as a function of ρ1, here r1 = r2 = ρ1/2. The values of the resistances inside a cell depend only on the circuit topology and are easily expressed as a function of ρ1, here r1 = r2 = ρ1/2. III. COMPUTATION OF ∂x4 (MOLECULE 40) The computational molecule of the fourth derivative is given by (4). The molecule is symmetrical and expands over two steps at each side. ∂4v ∂x4 ≈ 1 h4 ?>=< 89:; 1 ?>=< 89:; −4 ?>=< 89:; 6 ?>=< 89:; −4 ?>=< 89:; 1 (4) 1 2 2 1 Vi+2 Vi+1 r1 i0 r4 Vi r2 r5 r3 r6 Vi−2 Vi−1 Fig. 3. Network of molecule 40. ∂4v ∂x4 ≈ 1 h4 ?>=< 89:; 1 ?>=< 89:; −4 ?>=< 89:; 6 ?>=< 89:; −4 ?>=< 89:; 1 (4) (4) Fig. 1. Two–dimensional grid used in the ﬁnite difference approximation. TABLE I PARTIAL DIFFERENTIAL OPERATORS UP TO ORDER 4. The grid points (x, y) are given by xi = ih, i = −1, 0, 1, 2, . . . , N +1 and yj = jh, j = −1, 0, 1, 2, . . . , N +1. The uppercase letter Vi,j is used to denote the ﬁnite difference approximation of the function v (ih, jh). The two–dimensional grid used in the ﬁnite difference approximation is represented on Fig. 1 in the case N = 4. The points of the domain are labeled by a ﬁlled circle, those on the boundary are labeled by a ﬁlled square, and ﬁnally the point labeled by a cross are used to compute Neumann boundary conditions. along the paths located by the index 1 in the scheme and link- ing two adjacent potentials (i.e. ρ1 is the resistance between Vi and Vi−1 or Vi and Vi+1). By applying the Kirchhoff’s Current Law (KCL) at node Vi, rearranging some terms, the equation of the cell i can be written under the form (2). 1 ρ1 (Vi−1 −2Vi + Vi+1) = −i0 (2) (2) If we choose resistances which scaled down in h2 with the cell size, i.e. ρ1 = −h2ρ0, the potential of a cell i estimates the (second) derivative of the function v(x) at a single point. The whole electronic circuit composed of N cells computes a ﬁnite difference approximation of the differential equation (3). The particular choice to take ρ1 = −h2ρ0 instead of ρ1 = h2ρ0 is very important, it allows to superpose the electrical network of this molecule to other one (all the circuit equations must have the same left hand side to be superposed). If we choose resistances which scaled down in h2 with the cell size, i.e. ρ1 = −h2ρ0, the potential of a cell i estimates the (second) derivative of the function v(x) at a single point. The whole electronic circuit composed of N cells computes a ﬁnite difference approximation of the differential equation (3). The particular choice to take ρ1 = −h2ρ0 instead of ρ1 = h2ρ0 is very important, it allows to superpose the electrical network of this molecule to other one (all the circuit equations must have the same left hand side to be superposed). D Di Ne h x y h (N, 0) (N, N) (0, N) (0, 0) ∈∂Ω ∈Ω Fig. 1. Two–dimensional grid used in the ﬁnite difference approximation. IV. COMPUTATION OF ∂x (MOLECULE 10) (11) Vi 1 r0 + 2 ρ1 + 1 ρ2 −Vi−1 1 ρ1 + Vi+1 1 ρ2 −1 ρ1 i If we choose ρ1 = 2h3ρ0, ρ2 = 2h3ρ0/2, ρ3 = 2h3ρ0/2, ρ4 = 2h3ρ0/4 and r0 = −2h3ρ0/12, then the potential at node Vi estimates the third derivative of the function v(x) at a given point (Eq. (12)). If we choose ρ1 = 2h3ρ0, ρ2 = 2h3ρ0/2, ρ3 = 2h3ρ0/2, ρ4 = 2h3ρ0/4 and r0 = −2h3ρ0/12, then the potential at node Vi estimates the third derivative of the function v(x) at a given point (Eq. (12)). (8) ∂3v ∂x3 = ρ0i0 (12) (12) If we choose ρ1 = 2hρ0, ρ2 = 2hρ0/2, and r0 = −2hρ0/4, then the potential at node Vi estimates the ﬁrst derivative of the function v(x) at a given point (Eq. (9)). The resistances of the cells can be taken as r{1,3,7,9} = ρ1/4, r{4,6,10,12} = ρ2/4, r{2,8} = ρ3/2 and r{5,11} = ρ4/2. The resistances of the cells can be taken as r{1,3,7,9} = ρ1/4, r{4,6,10,12} = ρ2/4, r{2,8} = ρ3/2 and r{5,11} = ρ4/2. ∂v ∂x = ρ0i0 (9) (9) VI. COMPUTATION OF ∂xy (MOLECULE 11) The resistances of the cells can be taken as r1 = r3 = ρ1/2 and r2 = r4 = ρ2/2. The resistances of the cells can be taken as r1 = r3 = ρ1/2 and r2 = r4 = ρ2/2. The molecule of the 11 derivative is represented by (13). This molecule is the tensor product of two asymmetrical molecules (the molecules 10 and 01) and is therefore sym- metrical. IV. COMPUTATION OF ∂x (MOLECULE 10) IV. COMPUTATION OF ∂x (MOLECULE 10) inverted between these outer and inner values, this is translated into a direction change in the network (compare paths 2 and 4). The molecule of the ﬁrst derivative is represented by (7). This molecule is not symmetrical and thus cannot be repre- sented by a symmetrical network. 1 1 2 3 3 4 Vi−2 −Vi−2 Vi−1 −Vi−1 r3 r2 r8 r9 r6 r5 r11 r12 i0 Vi e0 r1 r4 r7 r10 −Vi Vi+1 −Vi+1 Vi+2 −Vi+2 r0 Fig. 5. Network of molecule 30. ∂v ∂x ≈ 1 2h n ?>=< 89:; −1 ?>=< 89:; 0 ?>=< 89:; 1 o (7) (7) To implement the molecule, we must construct an asymmet- rical circuit. The proposed one uses the potential −Vi,j. It is obtained by a linear VCVS e0 of value −1. (VCVS stands for Voltage–Controlled Voltage Source). The circuit is drawn on Fig. 4. Fig. 5. Network of molecule 30. 1 1 2 r1 i0 r0 r4 e0 Vi −Vi Vi+1 Vi−1 −Vi−1 −Vi+1 r2 r3 Fig. 4. Network of molecule 10. 1 1 2 r1 i0 r0 r4 e0 Vi −Vi Vi+1 Vi−1 −Vi−1 −Vi+1 r2 r3 Fig. 4. Network of molecule 10. The resistances ρ1 connect the potentials Vi and Vi±2 and the resistance ρ2 connects the potentials Vi and −Vi+2. In a similar way, the resistances ρ3 connect the potentials Vi and Vi±1 and the resistance ρ4 connects the potentials Vi and −Vi−1. Applying KCL at node Vi and after some algebraic manipulations, one can obtain (11). Fig. 4. Network of molecule 10. Vi 1 r0 + 2 ρ1 + 1 ρ2 + 2 ρ3 + 1 ρ4 −Vi−2 1 ρ1 + Vi+2 1 ρ2 −1 ρ1 (11) +Vi−1 1 ρ4 −1 ρ3 −Vi+1 1 ρ3 = i0 The resistances ρ1 connect the potentials Vi and Vi±1 and the resistance ρ2 connects the potentials Vi and −Vi+1. Apply- ing KCL at node Vi and after some algebraic manipulations, one can obtain (8). II. COMPUTATION OF ∂x2 (MOLECULE 20) The easiest computational molecule to implement under the form of an electronic circuit is the one associated with the second derivative. The molecule is symmetrical. Fig. 3. Network of molecule 40. The elementary cell which “calculates” (4) is represented on Fig. 3 with its four adjacent cells. The resistances ρ1 connect the potentials Vi and Vi±2 and the resistances ρ2 connect the potentials Vi and Vi±1. Applying KCL at node Vi and doing some algebraic manipulations leads to (5). ∂2v ∂x2 ≈ 1 h2 n ?>=< 89:; 1 ?>=< 89:; −2 ?>=< 89:; 1 o (1) (1) We recall that a computational molecule is a graphical depic- tion of an approximate partial derivative formula. The previous one stands for: 2Vi 1 ρ1 + 1 ρ2 −1 ρ1 Vi−2 −1 ρ2 Vi−1 −1 ρ2 Vi+1 −1 ρ1 Vi+2 = i0 (5) ∂2v ∂x2 ≈ 1 h2 (vi−1,j −2vi,j + vi+1,j) 1 1 r1 i0 r2 Vi Vi+1 Vi−1 Fig. 2. Network of molecule 20. (5) If we choose ρ1 = −h4ρ0 and ρ2 = h4ρ0/4, then the potential at node Vi estimate the fourth derivative of the function v(x) at a given point (Eq. (6)). ∂4v ∂x4 = ρ0i0 (6) Fig. 2. Network of molecule 20. (6) The elementary cell which “calculates” (1) is represented on Fig. 2 with its too adjacent cells. We call ρ1 the resistances The resistances of the cells can be taken as r1 = r3 = r4 = r6 = ρ1/4 and r2 = r5 = ρ2/2. The resistances of the cells can be taken as r1 = r3 = r4 = r6 = ρ1/4 and r2 = r5 = ρ2/2. V. COMPUTATION OF ∂x3 (MOLECULE 30) The molecule of the third derivative is represented by (10). Like every odd order derivative, the third molecule is not symmetrical and thus, it cannot be represented by a symmetrical network. Moreover, it expands over two steps at each side. ∂2v ∂x∂y ≈ 1 4h2                ?>=< 89:; −1 ?>=< 89:; 0 ?>=< 89:; 1 ?>=< 89:; 0 ?>=< 89:; 0 ?>=< 89:; 0 ?>=< 89:; 1 ?>=< 89:; 0 ?>=< 89:; −1                (13) (13) ∂3v ∂x3 ≈ 1 2h3 ?>=< 89:; −1 ?>=< 89:; 2 ?>=< 89:; 0 ?>=< 89:; −2 ?>=< 89:; 1 (10) (10) The proposed circuit is given on Fig 6. Following the same convention and notation as before, ρ1 corresponds to the resistance values between the potentials Vi,j and Vi±1,j±1, and ρ2 are the resistance values between the potentials Vi,j and Vi∓1,j±1. The electronic network which evaluates the molecule 11 is drawn on the center cell of Fig. 6, its eight The proposed circuit is given on Fig 6. Following the same convention and notation as before, ρ1 corresponds to the resistance values between the potentials Vi,j and Vi±1,j±1, and ρ2 are the resistance values between the potentials Vi,j and Vi∓1,j±1. The electronic network which evaluates the molecule 11 is drawn on the center cell of Fig. 6, its eight The proposed circuit is represented on Fig. 5. The circuit is a combination of the method used in molecule 40 to obtain the value of the potential, and those used in molecule 10 to get an asymmetrical value in the molecule. The paths 1 and 2 code the outer values of the molecule {−1, 1}, while the paths 3 and 4 code its inner values {2, −2}. Note that the minus sign is 1 ρ1/2 ρ1/2 ρ1/2 ρ1/2 ρ1/2 ρ1/2 ρ1/2 Vi+1,j+1 Vi,j ρ1/2 Fig. 7. Path linking Vi,j and Vi+1,j+1. adjacent cells are also represented. To facilitate the under- standing and the analysis of the network, only the center cell is completely drawn. On all the other cells, only the resistances along the paths which link there cell center (Vi−1,j+1, Vi,j+1, etc) to Vi,j have been represented. Fig. 7. Path linking Vi,j and Vi+1,j+1. VII. COMPUTATION OF ∂x2y2 (MOLECULE 22) The molecule 22 derivative is represented by (16). This molecule is the tensor product of two symmetrical molecules (the molecules 20 and 02) and is therefore symmetrical. ∂4v ∂x2∂y2 ≈ 1 h4                ?>=< 89:; 1 ?>=< 89:; −2 ?>=< 89:; 1 ?>=< 89:; −2 ?>=< 89:; 4 ?>=< 89:; −2 ?>=< 89:; 1 ?>=< 89:; −2 ?>=< 89:; 1                (16) (16) The proposed circuit is given on Fig 8. As shown in the network, ρ1 correspond to the resistance values between the potentials Vi,j and Vi±1,j and between Vi,j and Vi,j±1, and ρ2 are the resistance values between the potentials Vi,j and Vi±1,j±1 and between Vi,j and Vi∓1,j±1. The electronic net- work which evaluates the molecule 22 is drawn on the center cell of Fig. 8, its eight adjacent cells are also represented. To facilitate the understanding and the analysis of the network, only the center cell is completely drawn. Fig. 6. Network of molecule 11. Applying KCL at node Vi and after some algebraic manip- ulations, one can obtain (14). Vi,j 2 ρ1 + 2 ρ2 −1 ρ1 Vi+1,j+1 −1 ρ2 Vi−1,j+1 −1 ρ1 Vi−1,j−1 −1 ρ2 Vi+1,j−1 = i 2 2 1 1 2 1 2 1 i0 Vi,j Vi−1,j+1 Vi,j+1 Vi+1,j+1 Vi−1,j Vi+1,j Vi−1,j−1 Vi−1,j Vi+1,j−1 Fig. 8. Network of molecule 22. (14) = i0 (14) If we choose ρ1 = −4h2ρ0 and ρ2 = 4h2ρ0, the potential at node Vi estimates the 1–1 partial derivative of the function v(x) at a given point (Eq. (15)). If we choose ρ1 = −4h2ρ0 and ρ2 = 4h2ρ0, the potential at node Vi estimates the 1–1 partial derivative of the function v(x) at a given point (Eq. (15)). ∂2v ∂x∂y = ρ0i0 (15) (15) The actual resistances are represented by thick vertical and horizontal segments. The resistances represented by long segments can be taken as r{long} = ρ1/2, while the ones represented by short segments can be taken as r{short} = ρ2/2. For example, along the path from Vi,j to Vi+1,j+1, there are 4 resistances (of the same value) in series which are in parallel with 4 others, so the equivalent resistance is (4 × ρ1/2) //(4 × ρ1/2) = ρ1. V. COMPUTATION OF ∂x3 (MOLECULE 30) 2 2 1 1 Vi−1,j−1 Vi−1,j+1 Vi,j+1 Vi+1,j+1 Vi−1,j Vi,j Vi+1,j Vi−1,j Vi+1,j−1 i0 Fig. 6. Network of molecule 11. in practical circuits. It is not difﬁcult to derive an equivalent network, from the proposed one, with a much lower number of resistances. VII. COMPUTATION OF ∂x2y2 (MOLECULE 22) The network has been designed in such a way that one gets a maximum number of resistances having the same value, up to the sign. It seems that such resistances are easier to integrate Fig. 8. Network of molecule 22. nip- 17) tial ion 18) and 3 4 1 5 6 3 2 1 5 Vi−1,j+1 Vi,j+1 Vi+1,j+1 Vi+1,j−1 Vi−1,j Vi−1,j−1 Vi+1,j Vi−1,j Vi,j −Vi,j i0 e0 r0 −Vi−1,j+1 −Vi,j+1 −Vi+1,j+1 −Vi−1,j−1 −Vi−1,j −Vi−1,j −Vi+1,j −Vi+1,j−1 Fig. 9. Network of molecule 21. Applying KCL at node Vi and after some algebraic manip- ulations, one can obtain (17). Vi,j 4 ρ1 + 4 ρ2 −1 ρ1 Vi+1,j −1 ρ1 Vi,j+1 −1 ρ1 Vi−1,j −1 ρ1 Vi,j−1 −1 ρ2 Vi+1,j+1 −1 ρ2 Vi−1,j+1 −1 ρ2 Vi−1,j−1 −1 ρ2 Vi+1,j−1 = i (17) (17) If we choose ρ1 = h4ρ0/2 and ρ2 = −h4ρ0, the potential at node Vi estimates the 2–2 partial derivative of the function v(x) at a given point (Eq. (18)). ∂4v ∂x2∂y2 = ρ0i0 (18) (18) Fig. 9. Network of molecule 21. Fig. 9. Network of molecule 21. The actual resistances are represented by thick vertical and horizontal segments. The eight resistances represented by very long segments can be taken as r{very long} = ρ1/2, while all the other ones (i.e. resistances represented by long and short segments) can be taken as r{long,short} = ρ2/2. The explanation of this choice is identical to that of the molecule 11. ulations, one can obtain (20). ulations, one can obtain (20). Vi,j 1 r0 + 2 ρ1 + 1 ρ2 + 2 ρ3 + 1 ρ4 + 2 ρ5 + 1 ρ6 +Vi,j−1 1 ρ2 −1 ρ1 −Vi,j+1 1 ρ1 +Vi−1,j+1 1 ρ4 −1 ρ3 −Vi+1,j−1 1 ρ3 +Vi+1,j+1 1 ρ6 −1 ρ5 −Vi−1,j−1 1 ρ5 = i0 ( VIII. COMPUTATION OF ∂x2y (MOLECULE 21) The molecule 21 derivative is represented by (19). This molecule is the tensor product of one symmetrical molecule and one asymmetrical molecule (the molecules 20 and 01) and is therefore asymmetrical. (20) ∂3v ∂x2∂y ≈ 1 2h3                ?>=< 89:; 1 ?>=< 89:; −2 ?>=< 89:; 1 ?>=< 89:; 0 ?>=< 89:; 0 ?>=< 89:; 0 ?>=< 89:; −1 ?>=< 89:; 2 ?>=< 89:; −1                (19) If we choose r0 = −2h3/12 and the sets of ρα so that, If we choose r0 = −2h3/12 and the sets of ρα so that, If we choose r0 = −2h3/12 and the sets of ρα so that, ρ1 = 2h3ρ0/2 ρ2 = 2h3ρ0/4 ρ3 = 2h3ρ0 ρ4 = 2h3ρ0/2 ρ5 = 2h3ρ0 ρ6 = 2h3ρ0/2 (19) the potential at node Vi estimates the 2–1 partial derivative of the function v(x) at a given point (Eq. (21)). the potential at node Vi estimates the 2–1 partial derivative of the function v(x) at a given point (Eq. (21)). ∂3v ∂x2∂y = ρ0i0 (21) To deﬁne its electronic circuit, the molecule is splitted into 3 smaller molecules crossing the point (i, j), the vertical one (−2, 0, 2), and the molecules along the two diagonals (−1, 0, 1). Each submolecule can then be implemented by the same method as the one used for the molecule 10. (21) The actual resistances are represented by segments between two dots. The 6 resistances along the paths 1 and 2 can be taken as r⟨1⟩,⟨2⟩= ρ1/2, while the resistances along the paths 3, 4, 5 and 6 can be taken as r⟨1⟩,⟨2⟩,⟨3⟩,⟨4⟩,⟨5⟩,⟨6⟩= ρ2/3. The value of the actual resistances along the paths deﬁne, by periodicity, the resistances in the elementary cell. A careful drawing of the location of resistances to obtain a periodicity of the cells leads to the network of Fig. 9. Only the center cell is completely drawn to facilitate the reading of the network. The resistance between one of the 9 paths linking the potentials of adjacent cell to Vi,j are called ρα. The bold index α on the scheme indicates their corresponding path. X. COMPUTATION OF A PDE A linear PDE up to the order four can be written under the general form (25). The coefﬁcient ρij in front of each derivative is the factor ρ0 deﬁned in the study of the circuit of the corresponding molecule. The coefﬁcient aij is simply a multiplicative term. For the PDEs coming from physics (Poisson’s equation, deﬂection of plates, etc... ) the ratio aij/ρij is usually an integer. 1 2 1 2 Vi−2,j+1 Vi−1,j+1 Vi,j+1 Vi+1,j+1 Vi+2,j+1 Vi−1,j−1 Vi−2,j−1 Vi,j−1 Vi+1,j−1 Vi+2,j−1 Vi+2,j Vi+1,j Vi−1,j Vi−2,j Vi,j i0 Fig. 10. Network of outer submolecule of molecule 31. a00 ρ00 V + a10 ρ10 ∂V ∂x + a01 ρ01 ∂V ∂y + · · · + a04 ρ04 ∂4V ∂y4 = i0 (x, y) (25) (25) The elementary cell of a linear PDE is obtained by superposing the corresponding cell of each of its terms. This superposition is carried out in the following way: • The nodes on the boundaries of each collected cell must remain disjoint. • The nodes on the boundaries of each collected cell must remain disjoint. • The voltage nodes Vi,j, as the current sources i0, of all the collected cell must be merged. • The resistances of each assembled cell must be scaled with the corresponding factor aij. Fig. 10. Network of outer submolecule of molecule 31. In the case of nonlinear PDE, the parts under the form of a product of derivative, could be implemented by multiplying the voltage nodes Vi,j of each term and then by connecting the result to the current source i0. The circuit of the molecule 31 is the “schematic sum” of the network of Fig. 10 and the network of molecule 11 (with resistances renamed ρ′ 1 and ρ′ 2). A “schematic sum” means that the two circuits must be superposed with separated dots in the cell border, and that these two circuits have the same current source i0. Applying KCL at node Vi and after some algebraic manipulations, one can obtain (23). IX. COMPUTATION OF ∂x3y (MOLECULE 31) The molecule 21 derivative is represented by (22). This molecule is the tensor product of two asymmetrical molecules Applying KCL at node Vi and after some algebraic manip- (the molecules 30 and 01) and is therefore symmetrical. (the molecules 30 and 01) and is therefore symmetrical. given point (Eq. (24)). given point (Eq. (24)). ∂4v ∂x3∂y = ρ0i0 (24) ∂4v ∂x3∂y ≈ 1 4h4          ?>=< 89:; −1 ?>=< 89:; 2 ?>=< 89:; 0 ?>=< 89:; −2 ?>=< 89:; 1 ?>=< 89:; 0 ?>=< 89:; 0 ?>=< 89:; 0 ?>=< 89:; 0 ?>=< 89:; 0 ?>=< 89:; 1 ?>=< 89:; −2 ?>=< 89:; 0 ?>=< 89:; 2 ?>=< 89:; −1          ( (24) (22) The actual resistances of Fig. 10 are represented by segments between two dots. The 4 resistances along the paths 1 and 2 can be taken as r⟨1⟩,⟨2⟩= ρ1/4. The value of the actual resis- tances along the paths deﬁne, by periodicity, the resistances in the elementary cell. The molecule is separated into two submolecules arround the center point (i, j). The ﬁrst submolecule is the inner one (−2, 2, −2, 2), it can be deﬁned under a circuit form from the circuit of the molecule 11 by inverting and scaling the resistance ρα. The circuit of the second molecule, the outer one (1, −1, 1, −1) is drawn on Fig 10. As indicated in the network, ρ1 correspond to the resistances between the potentials Vi,j and Vi±2,j±1 and ρ2 are the resistances between the potentials Vi∓2,j∓1. XI. BOUNDARY CONDITIONS Consider for example the deﬂection of a square clamped plate (26–28) to illustrate the implementation of the boundary conditions. The Dirichlet boundary conditions (27) impose the value of the function along the boundary. The Neumann boundary conditions (28) impose the normal derivative along the boundary. Vi,j 2 ρ1 + 2 ρ2 + 2 ρ′ 1 + 2 ρ′ 2 −1 ρ1 Vi+2,j+1 −1 ρ2 Vi−2,j+1 −1 ρ1 Vi−2,j−1 −1 ρ2 Vi+2,j−1 (23) −1 ρ′ 1 Vi+1,j+1 −1 ρ′ 2 Vi−1,j+1 −1 ρ′ 1 Vi−1,j−1 −1 ρ′ 2 Vi+1,j−1 = i0 ∂4V ∂x4 + 2 ∂4V ∂x2∂y2 + ∂4V ∂y4 = i0 (x, y) , (26) V (x, y) = VB on ∂Ω, (27) ∂V ∂n (x, y) = 0 on ∂Ω. (28) (23) (28) The cell of the boundary conditions is constructed by doing a “schematic sum” of the boundary cells of each term of the PDE. The cell which implements the Dirichlet boundary conditions is built by simply replacing the current source i0 of each elementary cell by a constant voltage source v0 of value VB. (v0 = Vi,j with i and j null or equal to N in Fig. 11) This imposes the voltage VB along the boundaries. The cell The cell of the boundary conditions is constructed by doing a “schematic sum” of the boundary cells of each term of the PDE. The cell which implements the Dirichlet boundary conditions is built by simply replacing the current source i0 of each elementary cell by a constant voltage source v0 of value VB. (v0 = Vi,j with i and j null or equal to N in Fig. 11) This imposes the voltage VB along the boundaries. The cell If we choose ρ1 = −4h4ρ0, ρ2 = 4h4ρ0 and the corre- sponding resistances ρ′ 1 and ρ′ 2 of the molecule 11 so that ρ′ 1 = 4h2ρ0/2 and ρ′ 2 = −4h2ρ0/2, the potential at node Vi estimates the 3–1 partial derivative of the function v(x) at a of the Dirichlet boundary conditions for the fourth derivative term is given in Fig. 11. These cells are located around a ﬁlled square on Fig. 1. The case α = β = 0 corresponds to the Molecule 00, which is the trivial molecule ?>=< 89:; 1 . The unit circuit which “codes” the equation Vi = ρ0i0 is given in Fig 13. XI. BOUNDARY CONDITIONS This case is important, it allows to manage the terms of PDE which involves the function itself (or derivative of order 0 of a function to justify the terminology Molecule 00). 1 2 2 1 r1 r4 r2 r5 r3 r6 v0 = VB V0 = VB Fig. 11. Cell of the Dirichlet boundary conditions for the fourth derivative term. i0 Vi ρ0 Fig. 13. Network of molecule 00. Fig. 11. Cell of the Dirichlet boundary conditions for the fourth derivative term. Fig. 13. Network of molecule 00. The cell which implements the Neumann boundary condi- tions is built by simply replacing the current source i0 of each elementary cell by a voltage–controlled voltage source v−1, in the case of condition (28), the VCVS has the value of the voltage node V1 at the inner side of the boundary. This is the PNR-circuit counterpart of how the ﬁnite difference method imposes a null derivative of a function along the boundaries. Other Neumann conditions are built in the same way. The cell of this Neumann boundary conditions for the fourth derivative term is given in Fig. 12. These cells are located arround a cross on Fig. 1. Notice that for the cell at the left part of the boundary the resistances r1, r2 and r3 are not connected and could be removed. XIII. CONCLUSION We have proposed a concept of periodic network of re- sistances (PNR) to deﬁne electronic circuits which are able to solve PDE. We adopt an electronician point of view, i.e. designing the electronic circuits and simulating them. The matrix equations of our periodic circuit are the same as the corresponding set of ﬁnite difference equations to resolve a given PDE. Hovewer, the accuracy and the stability of the numerical schemes have to be carrefully studied. By doing a “schematic sum” of some elementary networks deﬁned in the previous sections, it is possible to build the 2D electronics circuit which is a solution of a given linear partial differential equation, for example the plate equation: 1 2 2 1 r1 r4 r2 r5 r3 r6 v−1 = V1 V−1 = V1 Fig. 12. Cell of the Neumann boundary conditions for the fourth derivative term. ∂4u ∂x4 + 2 ∂4u ∂x2∂y2 + ∂4u ∂y4 = P D. Moreover, generalization to non constant coefﬁcient PDE, non–elliptic PDE or PDE with an arbitrary function in the right hand side is faisable by the same method. By using multiplior, even nonlinear PDE could be manageable. All these generalizations suppose implicitly that the ﬁnite difference equations relative to the periodic network are mathematicaly valid. Fig. 12. Cell of the Neumann boundary conditions for the fourth derivative term. XII. SIMULATION AND COMMENTS An example of application of PNR could be the real time control of a system, with a PDE control law implemented under the form of an analog integrated circuit instead of a numerical one. It is difﬁcult to quatify the gain in speed, that may be of a factor 1000. We have developed a program which generates the Spice network corresponding to the chosen ﬁnite difference discreti- sation scheme from a (linear) partial differential equation. The generated ﬁle contained the N or N 2 cells of the domain, and the cells at the boundaries to take account of the boundary conditions. All the circuits deﬁned in the previous sections have been independently checked by simulating the PDE: Of course, a practical implementation of the method de- scribed in this paper under the form of integrated circuits is challenging, some of the numerous problems are the integra- tion of the many NIC (Negative Impedance Convertor) to sim- ulate the negative resistances: How to choose the magnitude of the elements ? Should the scaling operate on the resistances or on the current sources ? What is the effect of the scattering of the component values (some ﬁnite difference schemes are very sensitive to it, but ﬁnite difference schemes deriving from physical equations have “generally” a good behavior). ∂α+βv ∂xα∂yβ = α!β!, with α+β ⩽4 on the domain Ω= [0, 1]×[0, 1], with the right boundary conditions. The Spice solution for the potential Vi,j has been compared with the PDE solutions v (x, y) = xαyβ for different mesh sizes. On these academic examples, there is no signiﬁcant difference between the solutions. [4] L. Chua, “Bipolar–jfet–mosfet negative resistance devices,” IEEE Trans. Circuits Syst., vol. CAS–32, no. 1, pp. 46–61, Jan. 1985. REFERENCES [4] L. Chua, “Bipolar–jfet–mosfet negative resistance devices,” IEEE Trans. Circuits Syst., vol. CAS–32, no. 1, pp. 46–61, Jan. 1985. [1] S. Shukla and R. I. Bahar, Nano, Quantum and Molecular Computing. Kluwer Academic Publishers, 2004. [5] C. Toumazou and F. Lidgey, “Current–conveyor basic and applications,” in Proc. IEEE International Symposium on Circuits and Systems (ISCAS 1994), London, England, May 30–June 2 1994, pp. 569–585. [2] K. Lehovec and R. Zuleeg, “Negative resistance of a modiﬁed insulated– gate ﬁeld–effect transistor,” Proc. IEEE, vol. 62, pp. 1163–1165, Aug. 1974. [6] W. Kiranon, C. Loescharataramdee, N. Kiatwarin, and P. Wardkein, “Elec- tronically controlled negative resistance based on translinear circuits,” in Proc. of ISPACS’99, Phuket, Thailand, Dec. 1999, pp. 645–648. [3] H. Takagi and G. Kano, “Complementary jfet negative resistance devices,” IEEE J. Solid–State Circuit, vol. SC–10, pp. 509–515, Dec. 1975.
https://openalex.org/W4206586095	https://sciforum.net/paper/download/10686/manuscript	English	null	Seed biopriming of durum wheat with purified and combined halotolerant bacteria improved seed germination under salinity conditions	null	2,021	cc-by	4,767	Seed biopriming of durum wheat with purified and combined bacterial culture improved seed germination under salt stress Adel Hadj Brahima, Lobna Daouda , Manel Ben Alia,b , Asmahen Akremia, Mouna Jlidia , Houda Hmania ,Naser Aliye Fetoc, Mamdouh Ben Alia,b E-mail addresses: adelhadjibrahim@gmail.com a Laboratory of Microbial Biotechnology, Enzymatic, and Biomolecules (LMBEB), Centre of Biotechnology of Sfax (CBS), University of Sfax, P.O. Box 1177, Sfax 3018, Tunisia b A Bi h B i i b C f Bi h l f Sf (CBS) U i i f Sf R d f Sidi b Astrum Biotech, Business incubator, Center of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour km 6, PO Box 1177 Sfax 3018, Tunisia. c OMICS Research Group, Department of Biotechnology, Vaal University of Technology, Johannesburg- Vanderbijlpark, South Africa MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 1 MDPI MOL2NET, International Conference Series on Multidisciplinary Sciences MODEC-05: Nat. Prod. & Agro-Indust. Proc. in Amazon, UEA, Puyo, Ecuador, 2020 1. Introduction To date, soil salinity becomes a huge obstacle for food production worldwide since salt stress is one of the major factors limiting agricultural productivity. It is estimated that a significant loss of crops (20– 50%) would be due to drought and salinity. Salinity not only hampers crop productivity, but also threatens the sustainability of agro-ecosystems worldwide. The osmotic stress caused by high salinity (100–200 mM) is originated from the reduction in solute potential of soil solution [1]. Further, a salt- affected area in Tunisia is fast escalating due to intrusion of saline water on arable land and the use of chemical fertilizers and pesticides [2]. Moreover, climate change scenarios showed the increased risk of salinization at different latitudes. Therefore, a great effort is required for maintaining crop production under limiting factors. Due to the rising severity of salinity on global food production, numerous strategies have been offered to cope with the increasing challenging soil conditions. Along with plant breeding [3], plant genetic engineering [4], and genetic transformation [5], agricultural practices have dramatically contributed to the improvement of plant tolerance to salinity stress. Among these agricultural approaches, the bacterization of plant crops with PGPB and the implementation of these useful rhizobacteria in seed biopriming have demonstrated their beneficial properties in enhancing plant growth and development, and in augmenting plant salt stress tolerance through different mechanisms [6-9]. Therefore, the present study was conducted to isolate and identify PGPB associated with the halophyte salicornia brachiata from the coastal saline sites and evaluation their bacterization effect on germination rate and explants growth of durum wheat seeds with “Biopriming” approach. Abstract: Background and aim: Improved crop productivity under stressful conditions is a major asset of global agriculture. Salinity is one of the most severe abiotic stresses limiting crop yield. Further, a salt- affected area in Tunisia is fast escalating due to intrusion of saline water on arable land and the use of chemical fertilizers and pesticides. Moreover, climate change scenarios showed the increased risk of salinization at different latitudes. Therefore, a great effort is required for maintaining crop production under limiting factors. The present study was conducted to isolate and identify PGPB associated with the halophyte Salicornia brachiata from the coastal saline sites and evaluation their bacterization effect of durum wheat seeds with “Biopriming” technology. Methods: The selection parameters of PGPB strains were based on the ability to promote the growth of plants under stressful conditions. On the other hand, the effect of selected isolates on germination of durum wheat (Triticum durum) was assessed in vivo conditions. The treatment with bacteria was applied on purified and combined strain under 25 and 125 mM NaCl. Results: A total of 22 isolates were selected in-vitro for studying their plant growth-promoting (PGPB) ability including, tolerance with salt concentration, ACC deaminase activity, N2 fixation, phosphorus solubilization, and indole-3-acetic acid (IAA), out of which, three strains (MA9, MA32, and SA62) were selected. Our data revealed that experiments using treated with NaCl and bioprimed seed permitted us to identify the most efficient isolates in a combined culture which offered the best rate of germination and the highly vegetable growth of explants (roots and shoots). In fact, the benefic effect of seed biopriming was more pronounced in samples added with NaCl than that of untreated samples. Conclusion: Seed biopriming with efficient PGPB strains induced salinity tolerance of wheat and therefore enhanced their rate of germination and growth of explants under salinity. ords: Co-inoculation; PGPB; halotolerant bacteria; seed biopriming; durum wheat; MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 2 2. 1. Materials 2. 1. Materials Roots of salicornia brachiata and soil samples were collected from sabkha site in Tunisia (34°46’16’’N10°48’24’’E). Many laboratories materials were used such as rotary spectrophotometer, shecker, Marie bath, Bacterial temperature Incubator, Erlenmeyer flasks,… MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 3 Then, a 10-fold serial dilution was prepared, and 0.1 mL aliquots were spread in Petri plates in duplicate over the Burk’s N-free medium. The plates were kept at 30◦ C for 7 days. The macerate of washed roots were inoculated in a culture flask containing Burk’s N-free medium and incubated for 7days at 30°C. Then, a 6-fold serial dilution was prepared, and 0.1 mL aliquots were spread in Petri plates in duplicate over the Burk’s N-free medium. The plates were incubated for 7 days at 30°C and morphologically different colonies appearing on the medium were isolated and subcultured for further analysis. 2.2.2. Diazotrophic potential of the isolates Bacterial isolates were examined for their nitrogen-fixation (diazotrophic) potential first by testing their growth on the liquid and solid mineral nitrogen free medium [10] ( medium removing NaNO3) with oil as a sole source of carbon and energy. Isolates were then tested for nitrogenase, using the method of Quantification of N fixing capacity. 2.2.3. Effect of physiological conditions on the growth of potent N fixing Bacteria Effect of various growth conditions such as temperature, salt tolerance and pH on the growth of the most potent N fixers were checked in nutrient broth. For studying the effect of temperature, potent bacteria were incubated at temperatures viz., 25oC, 30oC, 35oC, and 40oC for 24 h at 150 rpm. Nutrient broth supplemented with different concentrations of NaCl (ranging from 50-400mM) was used for salt tolerance studies and the hydrogen ion concentration in the range of 5-8 was selected for pH studies. The flasks were incubated at corresponded temperature for 24 h in a rotary shaker at 150 rpm. The growth and activity of the potent N fixing bacteria in the given growth conditions were observed by taking the optical density of the medium. Screening of phosphate solubilizing Bacteria Modified Pikovskaya agar plates were prepared and test isolates were streaked on plates, then the plates were the incubated at 37°C and observed for 2-7 days [11]. The strains forming zone of clearance were maintained by streaking on nutrient agar slants and stored at 4°C. 2.2.1. Sampling and isolation of diazotrophs bacteria Ten grams of soil collected from a native site of sabkha in Tunisia (34°46’16’’ N10°48’24’’ E) were added into 90 mL of sterile saline (NaCl, 0.85%) in Erlenmeyer flasks, which were shaken for 30 min. Hydrogen cyanide (HCN) production HCN production was determined by color change of filter paper [14]. Loopful of bacterial suspension was inoculated on nutrient agar medium (Merck, Germany) containing 4.4 g L-1 glycine. Filter papers were soaked in a reagent solution (sodium carbonate 2% and picric acid 0.5%) and placed in the upper lid of Petri dishes. To prevent volatilization, the plates were sealed with parafilm and incubated at 37°C for 7 days. One plate without inoculation of bacterium was considered as control. If HCN was produced, yellow filter papers changed to cream, light brown, dark brown and eventually turn into reddish-brown. Siderophore production Production of siderophore was detected by standard method Schwyn and Neiland (1987) [13] using chrome azurol S (CAS) as indicator. The isolates were spot inoculated at the center of the plate and incubated for 7 days. The change in the color of the medium around the bacterial spot was an indication of siderophore production. Production of Ammonia Bacterial isolates were tested for the production of ammonia in peptone water. Freshly grown cultures were inoculated in 10 ml peptone water in each tube and incubated for 7 days at 37°C. Nessler’s reagent (0.5 ml) was added in each tube. Development of brown to yellow colour was a positive test for ammonia production [15]. MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 4 Indole acetic acid (IAA) production was detected as described by Patten et al. (2002) [12]. Bacterial cultures were grown for 7 days in halophilic medium containing supplement of 20g NaCl at 37°C. Fully grown cultures were centrifuged at 3,000 rpm for 30 min. The supernatant (2 mL) was mixed with 2 drops of orthophosphoric acid and 4 mL of Salkowski reagent (50 mL, 35% of perchloric acid and 1 mL 0.5 M FeCl3solution). Development of pink color indicated IAA production. Exopolysaccharide production The qualitative determination of exopolysaccharide production was performed according to Paulo et al. (2012) [16]. Each strain was inoculated onto 5-mm diameter paper discs disposed in a medium (2% yeast extract; 1.5% K2HPO4; 0.02% MgSO4; 0.0015% MnSO4; 0.0015% FeSO4; 0.003% CaCl2; 0.0015% NaCl; 1.5% agar) modified by the addition of 10% of saccharose, pH value of 7.5. The production was characterized by the size of the halo produced and its slime appearance. IAA production MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 Antifungal assay The agar well diffusion method as adopted earlier by Mehmood et al., (1999) [17] was used with minor modification. The bacterial isolates tested for their antifungal activity were fully grown in LB medium. Wells of 8mm diameter of test fungus were punched into in the Potato dextrose agar (PDA) slants and filled with 200 ml (2. 107 CFU/ml) of bacterial culture. Potato dextrose broth was taken as negative MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 5 control. The plates were incubated for 5–6 days at 28°C. The antifungal activity was evaluated by measuring the growth inhibition zone against test fungi. 2.2.4. Preparation of inoculum and seed coating: Seeds of wheat variety “aouija” were obtained from Agricultural Research Institute INGREF Tunis- Tunisia. Bacterial strains were grown overnight in LB broth at 28±2°C with constant shaking. Cells were harvested by centrifugation and re-suspended in normal saline to get an optimum growth (OD 108 cells per mL at λ600). Seeds were constantly shaken along-with the bacterial suspension with continuous addition of the sterile carrier material until the seeds become coated with a thin film of bacterial suspension and carrier material. Coated seeds were air-dried before sowing. After seed coating with potent PGPB strains, uniform and en seeds were sowed in pots and germination of seeds was observed after 3th day. The seed germination was observed 3 Results and Discussion 3. Results and Discussion Table 1: Bacterial growth of selected isolates under different temperature, pH, and salt concentrations. Table 1: Bacterial growth of selected isolates under different temperature, pH, and salt concentrations. Isolate code Salt tolerance (mM) Temperature tolerance(°C) pH tolerance 50 150 200 400 25 30 35 40 5 6 7 8 MA9 + ++ + + ++ +++ + + + ++ +++ + MA32 ++ ++++ +++ ++ ++ +++ ++ ++ ++ +++ ++++ ++ hese strains (MA9, and MA32) were found to be able to metabolize sucrose, mannitol Accordingly, these strains (MA9, and MA32) were found to be able to metabolize sucrose, mannitol, glucose, starch, citrate, and nitrate with API 20E (data not shown). The isolates were also evaluated for their tolerance to different concentration of salt and growth at different temperature and pH values. We found that almost all bacterial strains were able to grow under all of the NaCl concentrations tested (50 to 400 mM) (Table 1), with an optimal growth at 30oC temperature and a pH of 6.5-7 and 150 mM of NaCl for respectively, MA9, and MA32. glucose, starch, citrate, and nitrate with API 20E (data not shown). The isolates were also evaluated for their tolerance to different concentration of salt and growth at different temperature and pH values. We found that almost all bacterial strains were able to grow under all of the NaCl concentrations tested (50 to 400 mM) (Table 1), with an optimal growth at 30oC temperature and a pH of 6.5-7 and 150 mM of NaCl for respectively, MA9, and MA32. MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 6 Table 2: Plant growth-promoting properties of selected strains isolated from coastal saline soil site in Tunisia. Table 2: Plant growth-promoting properties of selected strains isolated from coastal saline soil site in Tunisia. Isolate code PGP properties Mycelial growth inhibition HCN Ammonium Siderophores Diazotrophic potentiel ACC deaminase EPS Rhizoctonia solani Fusarium solani Fusarium oxysporum Fusarium graminarum MA9 - +++ + + +++ ++++ +++ + - + MA32 - ++++ +++ +++ +++++ ++++ +++ ++++ ++++ ++++ Mycelial growth inhibition Mycelial growth inhibition In addition, the strains were positive for phosphate solubilisation, production of antimicrobial compounds, production of auxin and siderophores, as well as for important plant growth promoting (PGP) properties including dinitrogen fixation ability (Table 2), and hydrolytic potential of 1- aminocyclopropane-1-carboxylate (ACC deaminase) (Table 2; Fig. 1). 3. Results and Discussion In an effort to decrease the widespread use of chemicals as a means of preventing phytopathogen damage to plants, scientists have been developing the use of certain environmentally friendly PGPB as biocontrol agents The antifungal activity of all strains was checked against fusarium solani, fusarium oxysporum, fusarium graminearum and rhizoctonia solani using PDA medium (Table 2). The antifungal activity of the tested strains varied according to PGPB and phytopathogenic fungal strain whose MA32 as the most effective against all fungal strains. It should be noted that only with MA32 this activity was efficient against fusarium oxysporum. Moreover, no antifungal activity was noticed with MA9 for fusarium oxysporum, whereas the strain MA9 had the same activity of MA32 under rhizoctonia solani. PGPB as biocontrol agents with differents pathways: antibiotics production, cell wall degrading enzymes, competition, hydrogen cyanide, induced systemic resistance (ISR), quorum quenching, and bacteriophages. MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 7 The biodegradation of ACC, precursor of ethylene which had a negative impact on bacterial growth under stress and therefore, limits the plant growth and productivity, was assessed with another laboratory assay for better understand the bacterial biological pathways of ACC. The results shown in Fig. 2 affirmed that the strain MA32 was a more activated biological pathway of ACC assimilation than MA9 Figure 1: Bacterial growth in N free medium (A) and LB (B) under optimum of osmotic presure (mM NaCl) à 30°C et pH 7. 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 MA9 MA32 Bacterial growth PGPB strain MM MM+ACC A 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 0% 1 2 4% Bacterial growth NaCl % MA9 MA32 B 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 0% 1 2 4% Bacterial growth NaCl % MA9 MA32 B 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 MA9 MA32 Bacterial growth PGPB strain MM MM+ACC A Figure 1: Bacterial growth in N free medium (A) and LB (B) under optimum of osmotic presure (mM NaCl) à 30°C et pH 7. The bacterial growth of the isolates was put under their optimum salt at 30°C and pH 7 under two media: one rich LB and the other poor lacking a nitrogen source to enhance the growth capacity of the strains to support stressful and non-stressful conditions. Growth on LB (Fig 1B) as well on N-free medium ( Fig. 1A) shows that MA32 exhibits the best growth ability. Therefore, MA32 had higher growth in a ACC medium than MA9. Ethylene synthesis in a particular plant is affected by the presence and concentration of other plant hormones, temperature, gravity, light, nutrition, and the presence of various degrees of biotic/abiotic stress which the plant may be subjected [18]. Its production more than its threshold level by the action of ACC oxidase enzyme in plant tissues causes “stress ethylene” which affects the root and shoot development in plants. Colonization of “stress ethylene” plant rhizosphere by ACC deaminase producing PGPB help to alleviate this situation and restores normal plant development. In order to test MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 8 the ability of strain to assimilate 1-aminocyclopropane-1-carboxylate (ACC), precursor of ethylene responsible of senescence and stress hormone in plants, bacterial growth is measured in the presence of ACC and another source of inorganic nitrogen ((NH4)2SO4) in the minimum medium. Our results show that the growth of MA9, using both sources, is minimal for ACC and maximal for (NH4)2SO4 and contradictory in MA32 whose activity is maximal for ACC and minimal for (NH4)2SO4 (Fig.2). Whereas, the strain, which able to assimilate ACC, have active pathway of secretion of hydrolytic enzyme of ACC named ACC deaminase. This biological pathway is active in other tested strain MA9, but not in the same rate of secretion. the ability of strain to assimilate 1-aminocyclopropane-1-carboxylate (ACC), precursor of ethylene responsible of senescence and stress hormone in plants, bacterial growth is measured in the presence of ACC and another source of inorganic nitrogen ((NH4)2SO4) in the minimum medium. Our results show that the growth of MA9, using both sources, is minimal for ACC and maximal for (NH4)2SO4 and contradictory in MA32 whose activity is maximal for ACC and minimal for (NH4)2SO4 (Fig.2). Whereas, the strain, which able to assimilate ACC, have active pathway of secretion of hydrolytic enzyme of ACC named ACC deaminase. This biological pathway is active in other tested strain MA9, but not in the same rate of secretion. In this regard, bacteria that express ACC deaminase, by lowering plant ACC levels (and subsequently plant ethylene levels) can decrease the detrimental effect on plants from different stresses [19]. The ACC is being converted by ACC deaminase in the PGPB to α-Ketobutyrate and ammonia. Figure 2: ACC deaminase activity expressed in bacterial growth of selected bacteria on N-free medium (NFMM) supplemented with ACC (1-aminocyclopropane-1- carboxylate) and (NH4)2SO4 as nitrogen source. 0 0,2 0,4 0,6 0,8 1 1,2 1,4 ACC (NH4)2SO4 Bacterial growth Nitrogen source MA9 MA32 0 0,2 0,4 0,6 0,8 1 1,2 1,4 ACC (NH4)2SO4 Bacterial growth Nitrogen source MA9 MA32 Figure 2: ACC deaminase activity expressed in bacterial growth of selected bacteria on N-free medium (NFMM) supplemented with ACC (1-aminocyclopropane-1- carboxylate) and (NH4)2SO4 as nitrogen source. The tested strains were found to produce variable amounts of auxins (Fig. 3A) ranging from 82 to 209 µg/ml in a free-tryptophan medium. The two strains MA9 and MA32 produced relatively more auxin with tryptophan at 202 and 376 µg/ml, respectively. MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 9 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,45 MA9 MA32 IAA (mg/ml) PGPB strain IAA-Trypt IAA+Trypt A 0 10 20 30 40 50 60 MA9 MA32 Phosphate soluble (µg/ml) PGPB strain phosphat… B 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,45 MA9 MA32 IAA (mg/ml) PGPB strain IAA-Trypt IAA+Trypt A 0 10 20 30 40 50 60 MA9 MA32 Phosphate soluble (µg/ml) PGPB strain phosphat… B Figure 3: (A) IAA production, (B) P-solubilization of selected strains in the presence of 2% NaCl. Error bars show the standard deviation of the mean values of three replicates (P<0.05). Figure 3: (A) IAA production, (B) P-solubilization of selected strains in the presence of 2% NaCl. Error bars show the standard deviation of the mean values of three replicates (P<0.05). Figure 3: (A) IAA production, (B) P-solubilization of selected strains in the presence of 2% NaCl. Error bars show the standard deviation of the mean values of three replicates (P<0.05). The idea of eliminating the use of all chemicals inputs in agriculture which are sometimes environmentally unsafe is slowly becoming a reality because of the emergence of microorganisms that can serve the same purpose or even do better [20]. Depletion of soil nutrients through leaching into the waterways and causing contamination are some of the negative effects of these chemical inputs that prompted the need for suitable alternatives. This brings us to the idea of using microbes that can be developed for use as biological inputs (biofertilizers; biostimulators; bioinductors of defense system;…) [20]. They are environmentally friendly as they are natural living organisms. They increase crop yield and production and, in addition, in developing countries, they are less expensive compared to chemical fertilizers. These biofertilizers are typically called plant growth-promoting bacteria (PGPB). MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 10 0 20 40 60 80 100 120 MA9 MA32 Control MA9/MA32 % Germination PGPB strain 0mM NaCl 125 mM NaCl Wheat (Triticum aestivum L. subseq. durum) represents a major renewable resource for food, feed, and industrial raw material and it is the most widely grown worldwide crop. For this, it is interesting to develop new techniques, such as seed biopriming, for improving and protecting crops against limiting factors. Seed biopriming proved the germination rate of wheat seeds after five days of obscurity incubation at 25°C. MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 Compared with unbioprimed seeds treated with salt, the germination rate is more pronounced in untreated and unbioprimed seeds. Therefore, germination is more favored after seed treatment with the strains. Under control conditions, after five days, the germination is improved in bioprimed seeds with MA9 and MA32 by 91 and 93% respectively. While, under treated seeds with 125 mM salt, the germination rate is 78 and 81% respectively seed biopriming with MA9 and MA32. Wheat (Triticum aestivum L. subseq. durum) represents a major renewable resource for food, feed, and industrial raw material and it is the most widely grown worldwide crop. For this, it is interesting to develop new techniques, such as seed biopriming, for improving and protecting crops against limiting factors. Seed biopriming proved the germination rate of wheat seeds after five days of obscurity incubation at 25°C. Compared with unbioprimed seeds treated with salt, the germination rate is more pronounced in untreated and unbioprimed seeds. Therefore, germination is more favored after seed treatment with the strains. Under control conditions, after five days, the germination is improved in bioprimed seeds with MA9 and MA32 by 91 and 93% respectively. While, under treated seeds with 125 mM salt, the germination rate is 78 and 81% respectively seed biopriming with MA9 and MA32. As it was mentioned that the two strains exhibit all the biochemical characteristics of plant growth promotion under 2% NaCl, it is interesting to exploit the combined effect of these two complementary strains to better have a detailed idea on the formulation of efficient bio-inoculants in agriculture. This combined effect is tested to germinate durum wheat seeds under stress to ascertain the effect of stress on growth and on the germination rate of seeds. As it was mentioned that the two strains exhibit all the biochemical characteristics of plant growth promotion under 2% NaCl, it is interesting to exploit the combined effect of these two complementary strains to better have a detailed idea on the formulation of efficient bio-inoculants in agriculture. This combined effect is tested to germinate durum wheat seeds under stress to ascertain the effect of stress on growth and on the germination rate of seeds. Our results reveal that the biopriming of the seeds with a combination of two relevant strains, markedly improves the germination rate that it was 93 and 88% respectively with MA9 and MA32 in untreated and treated seeds. MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 11 highest germination promotion and salinity tolerance. Hence, it would be worth to test this combination strains under field conditions as a step towards its commercial production. Moreover, these strains could be further assessed for its potential role in bioprotection and growth promotion of other crop plants. 16. Paulo, E.M., et al., An alternative method for screening lactic acid bacteria for the production of exopolysaccharides with rapid confirmation. Food Science and Technology, 2012. 32(4): p. 710-714. 5. References 1. Ha-Tran, D.M., et al., Roles of Plant Growth-Promoting Rhizobacteria (PGPR) in Stimulating Salinity Stress Defense in Plants: A Review. International Journal of Molecular Sciences, 2021. 22(6): p. 3154. 1. Ha-Tran, D.M., et al., Roles of Plant Growth-Promoting Rhizobacteria (PGPR) in Stimulating Salinity Stress Defense in Plants: A Review. International Journal of Molecular Sciences, 2021. 22(6): p. 3154. 2. Bai, Y.-C., et al., Soil chemical and microbiological properties are changed by long-term chemical fertilizers that limit ecosystem functioning. Microorganisms, 2020. 8(5): p. 694. 3. Fita, A., et al., Breeding and domesticating crops adapted to drought and salinity: a new paradigm for increasing food production. Frontiers in Plant Science, 2015. 6: p. 978. 4. Wang, W., B. Vinocur, and A. Altman, Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. Planta, 2003. 218(1): p. 1-14. 5. Jha, S., Transgenic approaches for enhancement of salinity stress tolerance in plants, in Molecular Approaches in Plant Biology and Environmental Challenges. 2019, Springer. p. 265-322. 6. Moeinzadeh, A., et al., Biopriming of Sunflower ('Helianthus annuus' L.) Seed with'Pseudomonas fluorescens' for Improvement of Seed Invigoration and Seedling Growth. Australian Journal of Crop Science, 2010. 4(7): p. 564. 7. Ansari, T., et al., Biopriming of wheat seeds with rhizobacteria containing ACC-deaminase and phosphorate solubilizing activities increases wheat growth and yield under phosphorus deficiency. Pakistan Journal of Agriculture, Agricultural Engineering and Veterinary Sciences, 2015. 31(1): p. 24-32. 8. Meena, S.K., et al., Effect of nitrogen levels and seed bio-priming on root infection, growth and yield attributes of wheat in varied soil type. Biocatalysis and Agricultural Biotechnology, 2017. 12: p. 172-178. 9. Brahim, A.H., et al., Seed-Biopriming of Durum Wheat with Diazotrophic Plant Growth Promoting Bacteria (PGPB) Enhanced Tolerance to Fusarium Head Blight (FHB) and Salinity Stress. 2019. 10. Sorkhoh, N., et al., Crude oil and hydrocarbon-degrading strains of Rhodococcus rhodochrous isolated from soil and marine environments in Kuwait. Environmental Pollution, 1990. 65(1): p. 1-17. 11. Mehta, S. and C.S. Nautiyal, An efficient method for qualitative screening of phosphate- solubilizing bacteria. Current microbiology, 2001. 43(1): p. 51-56. 12. Patten, C.L. and B.R. Glick, Role of Pseudomonas putida indoleacetic acid in development of the host plant root system. Appl. Environ. Microbiol., 2002. 68(8): p. 3795-3801. 12. Patten, C.L. and B.R. Glick, Role of Pseudomonas putida indoleacetic acid in development of the host plant root system. Appl. Environ. Microbiol., 2002. 68(8): p. 3795-3801. 13. Schwyn, B. 4. Conclusions The strains MA9 and MA32 were found to have PGPB characteristics as they produced indole-3-acetic acid, siderophores, and lytic enzymes, fixed free atmospheric nitrogen, and solubilized inorganic phosphate, in vitro. Yet, biopriming with combined bacterial formulation MA9/MA32 offered the MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 17. Mehmood, Z., et al., Indian medicinal plants: a potential source for anticandidal drugs. Pharmaceutical Biology, 1999. 37(3): p. 237-242. g , ( ) p 20. Olanrewaju, O.S., B.R. Glick, and O.O. Babalola, Mechanisms of action of plant growth promoting bacteria. World Journal of Microbiology and Biotechnology, 2017. 33(11): p. 1-16. 18. Gamalero, E. and B.R. Glick, Bacterial modulation of plant ethylene levels. Plant Physiology, 2015. 169(1): p. 13-22. 19. Glick, B.R., Bacteria with ACC deaminase can promote plant growth and help to feed the world. Microbiological research, 2014. 169(1): p. 30-39. Mehmood, Z., et al., Indian medicinal plants: a potential source for anticandida Pharmaceutical Biology, 1999. 37(3): p. 237-242. 5. References and J. Neilands, Universal chemical assay for the detection and determination of d h A l i l bi h i 198 160(1) 4 56 Schwyn, B. and J. Neilands, Universal chemical assay for the detection and determi siderophores. Analytical biochemistry, 1987. 160(1): p. 47-56. siderophores. Analytical biochemistry, 1987. 160(1): p. 47 56. 14. Alström, S. and R.G. Burns, Cyanide production by rhizobacteria as a possible mechanism of plant growth inhibition. Biology and Fertility of Soils, 1989. 7(3): p. 232-238. 15. Cappuccino, J. and N. Sherman, Biochemical activities of microorganisms. Microbiology, A Laboratory Manual. The Benjamin/Cummings Publishing Co. California, USA, 1992: p. 188- 247. 16. Paulo, E.M., et al., An alternative method for screening lactic acid bacteria for the production of exopolysaccharides with rapid confirmation. Food Science and Technology, 2012. 32(4): p. 710-714. MOL2NET, 2021, 7, ISSN: 2624-5078 http://sciforum.net/conference/mol2net-07 12 p 19. Glick, B.R., Bacteria with ACC deaminase can promote plant growth and help to feed the world. Microbiological research, 2014. 169(1): p. 30-39. g ( ) p 20. Olanrewaju, O.S., B.R. Glick, and O.O. Babalola, Mechanisms of action of plant growth promoting bacteria. World Journal of Microbiology and Biotechnology, 2017. 33(11): p. 1-16.
https://openalex.org/W2318281787	https://europepmc.org/articles/pmc4093304?pdf=render	English	null	Vaccination Recommendations for the Hematology and Oncology and Post–Stem Cell Transplant Populations	Journal of the advanced practitioner in oncology	2,012	cc-by	9,709	REVIEW REVIEW Vaccination Recommendations for the Hematology and Oncology and Post–Stem Cell Transplant Populations VIVIAN TSANG, PharmD, BCOP From Cedars-Sinai Medical Center, Los Angeles, California From Cedars-Sinai Medical Center, Los Angeles, California From Cedars-Sinai Medical Center, Los Angeles, California Author's disclosures of potential conflicts of interest are found at the end of this article. Correspondence to: Vivian Tsang, PharmD, BCOP, Cedars-Sinai Medi cal Center, 8700 Beverly Boulevard, Los Angeles, CA 90048. E-mail: tsangv@cshs.org © 2012 Harborside Press® Correspondence to: Vivian Tsang, PharmD, BCOP, Cedars-Sinai Medi cal Center, 8700 Beverly Boulevard, Los Angeles, CA 90048. E-mail: tsangv@cshs.org y Diseases While infection remains the leading cause of posttransplant complications, protection against vaccine-preventable infections remains a priority. Many patients have undergone childhood vaccina tion per the CDC guidelines. As an adult, the need for boosters is recommended based on a recent out break or the demonstration of a decrease or loss in immunity. In patients undergoing transplant, the loss of pretransplant immunity is inevitable. The de gree of immunity loss may be dependent on several factors such as the strength of the existing immunity, the type of transplant, the source of the stem cells, the conditioning regimen used, the presence and se verity of graft-vs.-host disease (GVHD), and the im munosuppression used (Ljungman et al., 2005). With a general 2-week lag time for an antibody response postvaccination, it is recommended that at-risk patients obtain the influenza vaccine prior to the peak of the season. Per the CDC, annual vac cination against influenza is recommended (CDC, 2012). Patients should be vaccinated at least 2 weeks before starting chemotherapy or in between cycles, with the former scenario likely more ben eficial (Sommer et al., 2006; Melcher, 2005). Following the suppression of the immune sys tem, the body’s natural course of recovery (other wise known as immune reconstitution) begins at the blood cell line level, followed by B-cell recov ery, and finally T-cell recovery. After high-dose cytotoxic therapy, once nadir is reached, blood cell line recovery begins at 2 to 4 weeks followed by B- and T-cell recovery at approximately 1 to 3 months posttransplant. As a result of the delayed recovery, a fully functional immune system is not obtained until approximately 6 to 12 months posttransplant (Singhal & Mehta, 1999). Despite eventual recovery of the immune system, some posttransplant patients are viewed as “never vac cinated,” thereby requiring specific reimmuniza tion for certain vaccines while avoiding others. i In the HSCT population, it is recommended that vaccination with the inactivated influen za vaccine should take place no sooner than 6 months posttransplant but within the first year after transplant. A second dose, 1 month later, should be considered, especially if it is the first time the patient has ever been exposed to the in fluenza vaccine. Due to a compromised immune system posttransplant, live attenuated influenza is contraindicated in this population. Abstract Vaccination is a simple yet important process used to prevent many infec tions in the general population. For patients with suppressed immune sys tems, especially those who are undergoing chemotherapy or who have un dergone stem cell transplant, repeat vaccination or boosters may be crucial in prolonging and/or extending immunity. The purpose of this review is to examine the need for each vaccine in two separate oncology populations: patients receiving concurrent chemotherapy and post–stem cell transplant patients. In addition, the importance of avoiding live vaccines and criteria for reconsideration at a future time will also be discussed. Author's disclosures of potential conflicts of interest are found at the end of this article. J Adv Pract Oncol 2012;3:71–83 J Adv Pract Oncol 2012;3:71–83 D D espite the implementa tion of appropriate vacci nations for a person with a healthy immune sys tem, immunity is not always achieved. For example, some individuals never establish immunity after receiving a hepatitis B vaccine (HBV) series (Egea et al., 1991; Wood et al., 1993). Similarly, oncology patients who have received chemotherapy or hematopoietic stem cell transplantation (HCST) may have poor uptake or may be unable to mount an effective immune response postvaccination. In order to accurately assess a patient’s response to a specific vaccination, it is suggested that a base line titer be drawn prior to treatment and compared to posttreatment levels (Ring et al., 2003). For a healthy immune system, it typically takes up to 2 weeks after vac cination for the adaptive immunity to respond to the exposed pathogen. In the oncology population, concurrent che motherapy and immune reconstitution posttransplant are two factors that may alter the effectiveness of vaccinations as well as the recovery process of the im mune system. As a result, the timing of vaccinations with respect to treatment may play a role in achieving extended immunity and better outcomes for oncol ogy patients (Pollyea et al., 2010). The Centers for Disease Control and Prevention (CDC) have established guidelines detailing recommended rou tine vaccination schedules for various populations. For healthy individuals, the recommended schedules for the dif 71 Vol 3  No 2  Mar/Apr 2012 Vol 3  No 2  Mar/Apr 2012 AdvancedPractitioner.com REVIEW TSANG ferent age groups are available through the CDC website (CDC, 2012). While these guidelines also include high-risk patients, the timing and specific recommendations for the oncology population are inadequate. y Diseases It is also recommended that family members and caretak ers also receive annual inactivated influenza vac cination if there is contact with those who are im munosuppressed (Ljungman et al., 2009).f The observed side effects of the inactivated influenza vaccine are similar in all populations in that they are generally mild and may include soreness or pain around the injection site, fever, fatigue, or myalgia. These symptoms may occur 6 to 12 hours postinjection and generally last no longer than 2 days. In a rare case scenario Guil lain-Barré Syndrome, a neurologic complication, may occur (Shoji & Kaji, 2003). Abstract This review will focus on the need for appropriate timing of specific vaccinations in two adult oncology populations: those who are receiving chemotherapy and those who have undergone stem cell transplantation. 2004). Influenza A and B are two subtypes responsi ble for this viral illness. Symptoms of influenza may include fever and myalgia, with or without lower re spiratory tract symptoms. Influenza A is further de fined based on surface antigens (hemagglutinin and neuraminidase), and influenza B by genetic lineages. Each year, the World Health Organization (WHO) and the CDC produce influenza vaccine targeting specific anticipated strains. In the general oncology population, the low vaccination rate and insufficient immunologic response to the influenza vaccine are two con tributing factors to the high influenza mortal ity rate: up to 90% (Loulergue et al., 2008) vs. < 10% in the nononcology population (CDC, 2012). In adults, the ability to mount a protective response to the influenza vaccine was observed in 17% to 76% of patients with hematologic malignancies and 41% to 83% of patients with solid tumors (Table 1). Author The two inactivated influenza vaccine types and dosing recommendations are presented in Table 2. Patients with a known allergic reaction or hypersen sitivity to eggs should not receive the influenza vac cine. While it is not contraindicated to vaccinate pa tients who present with neutropenia, timing of the influenza vaccination with respect to their acute or chronic conditions should be considered. at-risk patients, it would likely have a significant im pact on the improvement of mortality. The pneumo coccal vaccine is generally recommended for very young and very old individuals, and particularly for those who are immunosuppressed and asplenic. Currently, two types of pneumococcal vac cines are available. Pneumococcal conjugate vac cine (PCV7) and pneumococcal polysaccharide 23-valent vaccine (PPSV23) are recommended for pediatric and adult populations, respectively. In the general pediatric population, PCV7 serotype specif ic efficacy could reach upwards of 85% (Black et al., 2000). PPSV23 has an overall effectiveness of 50% to 70% in preventing pneumococcal bacteremia in the adult population (Mangtani, Cutts, & Hall, 2003; Fedson, 1999; Fine et al., 1994), but fails in the pre vention of nonbacteremia pneumonia or otitis me Influenza Vaccine According to the CDC, an estimated 5% to 20% of the general population is affected by influ enza each year. Despite the availability of vaccines, influenza still accounts for over 200,000 hospi talizations and roughly 35,000 deaths each year (> 90% in older adults) (Thompson et al., 2003 & 72 J Adv Pract Oncol AdvancedPractitioner.com VACCINATION RECOMMENDATIONS Table 1. Adult Response to Influenza Vaccine Author Population Measure of efficacy Response Hematology Stiver et al. (1978) Lymphoma, CLL 4× increase in HI titer 17% Schafer et al. (1979) NHL or lymphoproliferative disorders 4× increase in HI titer 36% Hodges et al. (1979) Hematologic malignancies 4× increase in HI titer 52% Lo et al. (1993) Lymphoma 4× increase in HI titer 42% after 1st shot 50% after 2nd shot Robertson et al. (2000) Multiple myeloma HI titer > 1/40 19% Rapezzi et al. (2003) Lymphoproliferative disorders HI titer > 1/40 65%–76% Solid tumors Ganz et al. (1978) Solid tumors HI titer > 1/40 41%–47% Stiver et al. (1978) Breast cancer 4× increase in HI titer 50% Anderson et al. (1999) Lung cancer HI titer > 1/40 83% Note. CLL = chronic lymphocytic leukemia; HI = hemagglutination inhibition; NHL = non-Hodgkin lymphoma. Pneumococcal Vaccine The National Foundation for Infectious Diseas es (NFID) reports that an estimated 175,000 hospi talizations each year are attributed to pneumococcal pneumonia. Streptococcus pneumonia and 90 other strains of pneumococcus are the primary culprits of such bacterial infections affecting many parts of the body. When this infection invades the lungs, blood, and brain, it results in pneumo nia, bacteremia, and meningitis, respectively. While the incidence of severe pneumococcal infec tion accounts for a small portion of all pneumococcal infections, the mortality rate remains at ap proximately 7% (NFID, 2011). If appropriate vaccination is rec ommended and instituted for Table 2. Inac Rec Vaccine type Trivalent influe Influenza A (H Note. IM = intr Table 2. Inactivated Influenza Vaccine Types and Dosing Recommendation Vaccine type Dose Route Population Trivalent influenza vaccine (TIV) 0.5 mL IM ≥ 36 mo Influenza A (H1N1) monovalent 0.5 mL IM ≥ 36 mo Note. IM = intramuscular. Table 2. Inactivated Influenza Vaccine Types and Dosing Recommendation Vaccine type Dose Route Population Trivalent influenza vaccine (TIV) 0.5 mL IM ≥ 36 mo Influenza A (H1N1) monovalent 0.5 mL IM ≥ 36 mo Note. IM = intramuscular. Table 2. Inactivated Influenza Vaccine Types and Dosing Recommendation 73 Vol 3  No 2  Mar/Apr 2012 AdvancedPractitioner.com REVIEW TSANG dia (Table 3). Due to the variation in immunogenic ity between PCV7 and PPSV23, there are different recommendations for these pneumococcal vaccines. cause PCV7 is more immunogenic. For this reason, PCV7 is the preferred agent, with a three-dose se ries followed by one dose of PPSV23 to broaden the immune response (Cordonnier et al., 2008; Kumar et al., 2007; Meisel et al., 2007; Molrine et al., 2003). Patients should begin receiving PCV7 at 6 months posttransplant with 1-month intervals between each of the first three doses, then a PPSV23 booster at 18 months after transplant to maximize the dura tion of the pneumococcal protection (Hilgendorf et al., 2011). If pneumococcal vaccination took place early posttransplant before the 6-month mark, the duration of antibody response could be suboptimal; therefore, antibody titer should be determined and revaccination implemented as deemed appropriate (Hilgendorf et al., 2011; Ljungman et al., 2009). Pneumococcal Vaccine In general, adult patients who are receiving chemotherapy treatment for both hematologic (with some exceptions) and oncologic condi tions should receive the pneumococcal vaccine (PPSV23) 4 to 6 weeks (minimum 2 weeks) prior to starting chemotherapy; revaccination should occur once 5 years after the initial dose, then as recommended by vaccination guidelines (CDC, 2012; Arrowood, 2002). Due to a lower immuno genic response, patients receiving treatment for multiple myeloma, Hodgkin and non-Hodgkin lymphomas, and chronic graft-vs.-host disease (cGVHD), as well as those who have undergone total-body irradiation, should be vaccinated with PCV7 instead of PPSV23 (Mangtani, Cutts, & Hall, 2003; Fedson, 1999; Fine et al., 1994). Common adverse reactions related to the pneu mococcal vaccines are soreness at the injection site and fever. Severe reactions are rare. Patients with an acute infection—particular oncology patients who are undergoing or have recently undergone chemo therapy—should have pneumococcal vaccination postponed until resolution of the infection. Approximately 1% to 10% of the HSCT popula tion will contract a serious pneumococcal infection, with a median onset of 1 year posttransplant (Engel hard et al., 2002; Kulkarni et al., 2000). Timing and vaccine formulation are critical for maximizing the efficacy of pneumococcal vaccines. In four prospec tive trials, HSCT recipients obtained better response from PCV7 as compared to PPSV23, perhaps be Diphtheria-Pertussis-Tetanus Vaccines Diphtheria-Pertussis-Tetanus Vaccines Diphtheria is a transmissible upper respiratory tract infection caused by Corynebacterium diphthe riae. It spreads through direct contact with aerosol ized and infected secretions. Since the implementa tion of vaccination programs, there have been fewer cases of tetanus and diphtheria cases each year in the United States. Pertussis, also known as whoop ing cough, is a highly contagious bacterial infection caused by Bordetella pertussis. Due to the potential for recurrence epidemiologically, an estimated 48 million cases and 295,000 deaths are attributed to pertussis worldwide (Bettiol et al., 2010). Tetanus, or lockjaw, is a preventable disease that affects the muscles and nerves caused by Clostridium tetani. The usual path of entry is a break in the skin. In all three instances, the primary method of prevention for tetanus, diphtheria, and pertussis is vaccination. Table 3. Comparison Between PCV7 and PPSV23 Type of vaccine Population PCV7 • Indicated in children < 2 years old • Efficacy upwards of 85% • More immunogenic • Preferred in HSCT population and all other oncology patients listed below PPSV23 • Indicated in adults • Efficacy 50%–70% (bacteremia pneumonia only) • Less effective than PCV7 at protecting immunosuppressed patients (particularly during treatment for multiple myeloma, Hodgkin disease, non-Hodgkin lymphoma, chronic GVHD, post-TBI) Note. GVHD = graft-vs.-host disease; HSCT = hematopoietic stem cell transplantation; TBI = total- body irradiation. Information from Black et al. (2000); Fedson (1999); Fine et al. (1994); Mangtani et al. (2003). Table 3. Comparison Between PCV7 and PPSV23 Type of vaccine Population PCV7 • Indicated in children < 2 years old • Efficacy upwards of 85% • More immunogenic • Preferred in HSCT population and all other oncology patients listed below PPSV23 • Indicated in adults • Efficacy 50%–70% (bacteremia pneumonia only) • Less effective than PCV7 at protecting immunosuppressed patients (particularly during treatment for multiple myeloma, Hodgkin disease, non-Hodgkin lymphoma, chronic GVHD, post-TBI) Note. GVHD = graft-vs.-host disease; HSCT = hematopoietic stem cell transplantation; TBI = total- body irradiation. Information from Black et al. (2000); Fedson (1999); Fine et al. (1994); Mangtani et al. (2003). Table 3. VACCINATION RECOMMENDATIONS REVIEW 1930s, the distinction was made between bacterial- induced H. influenzae and viral-induced influenzae. H. influenzae is often presented as a respiratory tract infection, but may rarely cause bacteremia, pneumo nia, and meningitis. Since the implementation of the Hib conjugate vaccine in the 1990s, the prevalence of invasive disease has dramatically decreased by 99% to less than 1 per 100,000 cases (CDC, 2008). the differences between these products. The lower case “d” in Tdap represents a reduced dose of diph theria toxoid as compared to the upper case “D” in DTap, which contains a full dose of diphtheria tox oid. The tetanus component is the same in Td, Tdap, and DTap, while acellular pertussis is included in the latter two products only. For patients with hematologic and oncologic malignancies, Td is recommended once every 10 years; this is the same as the recommendation for the general public (CDC, 2012). It is extremely important for patients who are receiving che motherapy to be updated with the tetanus-diph theria vaccination (Arrowood & Hayney, 2002). Due to the recent outbreak of pertussis, it is also recommended that a single dose of Tdap replace one of the routine Td boosters. DTap is approved for individuals less than 7 years old. Tdap is rec ommended for healthy adults who have received full-dose diphtheria as a child in order to avoid the potential for a local reaction from repeated exposure to full-dose diphtheria (CDC, 2012). In hematologic and oncologic adult patients, the need for a vaccine booster is based on antibody ti ter. It has been demonstrated that in patients with Hodgkin disease and leukemia, posttreatment anti body response to the Hib vaccine is suppressed as compared to pretreatment levels. In addition, an tibody titers used to confer protection are lower in cancer patients as compared to the general popula tion. In order to ensure optimal protection against H. influenzae, particularly for this population (Hodgkin disease and leukemic patients), patients should have titers tested both pre- and posttreatment; revaccina tion may be considered if the titers remain low post treatment (Pirofski & Casadevall, 1998). If the titer is low, revaccination should be administered at least 2 weeks prior to starting chemotherapy to ensure maximal response or at least 3 months after comple tion of chemotherapy (Arrowood & Hayney, 2002). In contrast to their oncology and hematology counterparts, posttransplant patients are poor re sponders to Tdap. VACCINATION RECOMMENDATIONS It is recommended that post transplant patients receive DTap due to its higher response rate as compared to Tdap (Ljungman et al., 2009; Hilgendorf et al., 2011). Post-HSCT patients are viewed as “never vaccinated” and are at a lower risk of local reaction from DTap than healthy indi viduals. As such, DTap may be more immunogenic and should be considered the initial vaccination posttransplant. Patients should receive the DTap three-dose series beginning 6 months posttrans plant in monthly intervals, followed by a booster 18 months after HSCT (Hilgendorf et al., 2011). In the HSCT population, a three-dose series beginning 6 months posttransplant, followed by a booster at 18 months posttransplant, is recommend ed (Hilgendorf et al., 2011; Ljungman et al., 2009). The response to postvaccination antibody level is augmented if HSCT recipients received donor-im munized hematopoietic stem cells as opposed to nonimmunized stem cells (Molrine et al., 1996). As such, it is extremely important that the donor re ceive up-to-date vaccination prior to stem cell col lection while the recipient receives a series of appro priate vaccinations posttransplant. As with all other vaccines, the common adverse events include local injection site reaction and fever. Additional adverse events may include drowsiness and anorexia, which may be self-limiting. Rare but severe adverse events may include Guillain-Barré syndrome, neurologic disorders, uncontrollable sei zures, prolonged convulsions, or acute encephalop athy. These serious and acute neurologic symptoms generally occur within the first 3 days after admin istration of DTP as compared to Td. Common adverse events associated with the Hib vaccine may include pain and swelling at the site of the injection. Other adverse events may in clude fever, drowsiness, anorexia, and vomiting. Hepatitis B Vaccine Hepatitis B virus (HBV)–related infection pri marily arises through the transmission of bodily fluids. While its incidence remains low, patients undergoing chemotherapy with or without HSCT may have the potential to contract HBV infection through blood transfusions. But in today’s practice, blood products routinely undergo a series of rigor Haemophilus influenzae Type B Conjugate Vaccine l Diphtheria-Pertussis-Tetanus Vaccines Comparison Between PCV7 and PPSV23 Type of vaccine Population PCV7 • Indicated in children < 2 years old • Efficacy upwards of 85% • More immunogenic • Preferred in HSCT population and all other oncology patients listed below PPSV23 • Indicated in adults • Efficacy 50%–70% (bacteremia pneumonia only) • Less effective than PCV7 at protecting immunosuppressed patients (particularly during treatment for multiple myeloma, Hodgkin disease, non-Hodgkin lymphoma, chronic GVHD, post-TBI) Note. GVHD = graft-vs.-host disease; HSCT = hematopoietic stem cell transplantation; TBI = total- body irradiation. Information from Black et al. (2000); Fedson (1999); Fine et al. (1994); Mangtani et al. (2003). In order to lessen the discomfort of receiving several vaccines at once, a series of combination vac cines have been made available. Currently, there are several combination products available as a single injection containing tetanus and diphtheria vaccines with or without pertussis vaccines: Td, Tdap, and DTap. In general, the abbreviation DTP is used to re fer to the triple combination products on the whole. The diphtheria toxoid component further defines Note. GVHD = graft-vs.-host disease; HSCT = hematopoietic stem cell transplantation; TBI = total- body irradiation. Information from Black et al. (2000); Fedson (1999); Fine et al. (1994); Mangtani et al. (2003). 74 J Adv Pract Oncol AdvancedPractitioner.com VACCINATION RECOMMENDATIONS Haemophilus influenzae Type B Conjugate Vaccine l Historically, Haemophilus influenzae was thought to be the primary cause of influenzae. In the 75 Vol 3  No 2  Mar/Apr 2012 AdvancedPractitioner.com REVIEW TSANG ous testing for the detection of potential infection that could be transferred to the recipient. At the in stance when the patient is most vulnerable—when the immune system is suppressed— the likelihood of contracting an acute HBV infection, as well as the potential for reactivating chronic liver infections, is increased. Table 4 reflects the different stages of in fection based on the various hepatitis parameters. receiving a donor-immunized graft helps augment the transfer of donor immunity in at least 50% of pa tients (Ljungman et al., 2005). i Specific patients without antibodies to hepati tis B or not at risk of reactivation prior to transplant may delay their hepatitis B vaccination until 6 to 12 months posttransplant as this population is not at immediate risk. In general, hepatitis B vaccine can reduce the risk of reverse seroconversion in patients with a history of hepatitis B infection. In patients who have resolved hepatitis B, or HBc Ab positive alone prior to transplant, prophylactic nucleoside analog should be initiated with the start of condition ing therapy at the latest. In addition, the response to early posttransplant vaccination with hepatitis B may be augmented if the donor is immunized prior to stem cell collection (Ljungman et al., 2005; Hil gendorf et al., 2011). Despite being seronegative re cipients at baseline, the population at greatest risk is those patients who will be receiving a HBs Ag (posi tive) graft. In this population, hepatitis B vaccination before transplant, as well as the initiation of prophy lactic hepatitis B immune globulin and nucleoside analog, is recommended (Hilgendorf et al., 2011). Hepatitis B virus vaccine is not required for previously vaccinated patients undergoing che motherapy alone, as immunogenicity is sustained throughout treatment, unless hepatitis B antigen is undetectable by titer (Arrowood & Hayney, 2002). In a study of patients who had not previ ously been exposed to hepatitis B, or who had an undetectable titer level, and received the HBV vaccine prior to chemotherapy, 70% achieved adequate antibody response during the initial 12-month observational period posttreatment. This immunogenicity to the HBV vaccine was as sociated with increased survival rate (Weitberg et al., 1985). Hepatitis A Vaccine produces inflammation along the spinal cord, caus ing muscle weakness and paralysis. Developed in the 1950s, inactivated poliovirus vaccine (IPV) was the first polio vaccine intended to protect individu als against polio. Through various manipulations, an enhanced-potency IPV was licensed in 1987 as the vaccine of choice against polio. Oral polio vaccine (OPV) is a live-attenuated vaccine that has excellent GI immunity compared to IPV (Pirofski, 1998). Ingestion of contaminated food or water is the usual route for hepatitis A infection. Direct contact with an infected individual could also lead to acute infection. Although it is self-limiting in most cases, rare but serious acute liver failure can occur. The incidence of hepatitis A infection is highest in underdeveloped countries and in areas of (or individuals with) poor hygiene prac tice. Vaccination plays a major role in preventing hepatitis A infection; its protective effects can last for more than 20 years. In healthy patients, two doses of hepatitis A vaccine will induce protec tion in up to 99% of recipients (CDC, 1999). In patients with hematologic and oncolog ic malignancies, revaccination with IPV is not necessary unless antibody titer is below detect able levels, in which case revaccination is rec ommended prior to chemotherapy (Arrowood & Hayney, 2002). Due to the loss of immunity to poliovirus, post-HSCT patients should receive a series of three IPV injections beginning 6 months posttransplant at monthly intervals plus a booster dose at 18 months post-HSCT. Only the IPV for mulation should be used in post-HSCT patients, as opposed to the live formulation (OPV). Oral polio vaccine should be avoided due to the risk of vaccine-associated paralytic episodes occur ring in immunocompromised patients (Pirofski & Casadevall, 1998; CDC, 2012). Unless one is traveling to endemic areas or requires postexposure prophylaxis, hepatitis A vaccine is optional. In general, two doses of monovalent hepatitis A vaccine given 6 months apart may be recommended. In posttransplant patients, hepatitis A vaccine recommendations are the same as for the general population. Com mon adverse events may include mild fever, head ache, and pain at the injection site. Patients with prior risk of anaphylactic reaction to this vaccine should not receive any further doses. Adverse events associated with IPV are generally mild. Oral polio vaccine is a live vac cine that has been associated with rare but seri ous vaccine-induced paralytic poliomyelitis in healthy recipients. VACCINATION RECOMMENDATIONS REVIEW Hepatitis A Vaccine Hypersensitivity is a side ef fect for both formulations as it contains traces of bacitracin (OPV only), streptomycin, and neomy cin (both OPV and IPV). The IPV formulation is the polio vaccine of choice for most patients. Haemophilus influenzae Type B Conjugate Vaccine l This demonstrated safe and effective ability of hepatitis B vaccine in inducing immu nity in patients undergoing chemotherapy who have not previously been vaccinated. In post- HSCT patients, there are different recommenda tions based on hepatitis B history (Table 5). Even with vaccination before transplant, post transplant vaccination will need to be instituted to ensure long-lasting immunity (Ilan, 2000). For this reason, posttransplant patients should re ceive the three-dose series of hepatitis B vaccine at 1-month intervals, followed by a booster at 18 months post-HSCT (Hilgendorf et al., 2011). Patients who were previously HBs Ag positive or HBc Ab positive alone or HBs Ag positive and HBc Ab positive have the potential to reactivate the hepatitis B virus, particularly while immunosup pressed or immediately posttransplant (Hilgendorf et al., 2011). With the different combinations of an tigen or antibody positivity, the recommendation and timing for hepatitis B vaccination differs. It is also important to note that early vaccination while Common adverse events related to hepatitis B vaccine include fever and pain at the injection site. Anaphylaxis and hypersensitivity are rare but serious adverse events that have been associ ated with the hepatitis B vaccine. Table 4. Hepatitis B Markers and Various Stages of Infection and Vaccination Acute infection Resolved infection Chronic infection History of vaccination HBs Ag Recent infection Detected Not detected Detected Not detected HBs Ab Produced to fight HBV Not detected Detected Not detected Detected HBc Ab History of infection Detected Detected Detected Not detected Note. HBc Ab = hepatitis B core antibody; HBs Ab = hepatitis B surface antibody; HBs Ag = hepatitis B surface antigen; HBV = hepatitis B virus. Information from Hepatitis B Foundation (2012). Table 4. Hepatitis B Markers and Various Stages of Infection and Vaccination Table 4. Hepatitis B Markers and Various Stages of Infection and Vaccination Note. HBc Ab = hepatitis B core antibody; HBs Ab = hepatitis B surface antibody; HBs Ag = hepatitis B surface antigen; HBV = hepatitis B virus. Information from Hepatitis B Foundation (2012). 76 J Adv Pract Oncol AdvancedPractitioner.com VACCINATION RECOMMENDATIONS Polio Vaccine Poliomyelitis (polio) is an acute viral infection that is spread by direct contact via the fecal-oral route of entry. Infantile paralysis is another term that has been used to refer to polio, as it caused det rimental paralysis during the infant years in up to 20,000 cases before the introduction of the polio vaccine. As the infection attacks the human body, it Table 5. Hepatitis B Vaccine Recommendations for Post-HSCT Patients HBc Ab positive/negative HBs Ag positive • Vaccination early posttransplant may be considered (particularly if receiving a seronegative donor immunized graft) • Consider prophylactic nucleoside analog with conditioning therapy, at the latest, to reduce risk of reverse seroconversion HBc Ab positive HBc Ab negative HBs Ag negative • Vaccination early posttransplant may be considered (particularly if receiving a seronegative donor immunized graft) • Consider prophylactic nucleoside analog • Vaccinate (6–12 mo post-HSCT) Recipient seronegative, but receiving HBs Ag positive graft: • Vaccinate before HSCT • Consider prophylactic hepatitis B immunoglobulin and nucleoside analog Note. HBc Ab = hepatitis B core antibody; HBs Ag = hepatitis B surface antigen; HSCT = hematopoietic stem cell transplant. Adapted from Hilgendorf et al. (2011). Table 5. Hepatitis B Vaccine Recommendations for Post-HSCT Patients 77 AdvancedPractitioner.com Vol 3  No 2  Mar/Apr 2012 7 Vol 3  No 2  Mar/Apr 2012 77 AdvancedPractitioner.com Vol AdvancedPractitioner.com REVIEW TSANG Human Papillomavirus Vaccine Human papillomavirus (HPV) is a sexually transmitted infection that has been associated with the development of genital warts, cervical cancer, and anal cancer. In 2009, a three-dose vaccine was developed to protect against the four most common strains of HPV leading to cervical cancer (types 6, 11, 16, and 18). Currently, the HPV vaccine is indicated for the prevention of cervical cancer and genital warts in women who are 9 to 26 years old who have not been exposed to the virus. In males, it is approved for the prevention of genital warts and anal cancer in individuals 9 to 26 years old (CDC, 2012). In the on cology population, it is optional, if age appropriate. There are currently two formulations of me ningococcal vaccine available: meningococcal con jugate quadrivalent vaccine (MCV4) and menin gococcal polysaccharide vaccine (MPSV4). MCV4 is administered intramuscularly while MPSV4 is given subcutaneously. Age is a major determinant for immunogenicity. A child will have an overall an tibody response that is only 10% of the adult vaccine (Lepow, 1994). Therefore, MCV4 is recommended for individuals aged 2 to 55 while MPSV4 is recom mended for those over 55. Several studies have dem onstrated that similar to the pneumococcal vaccine, the conjugated meningococcal vaccine is more im munogenic than the polysaccharide-based menin gococcal vaccine (CDC, 2012). The most common adverse events may in clude pain and swelling at the injection site, headache, fever, nausea, and fainting. While 92% of the reports were deemed noncritical, 8% of the reports were critical in nature (including death, life-threatening illness, and hospitalization). Guillain-Barré syndrome has also been reported after administration of the HPV vaccine. Meningococcal vaccines are recommended in many nononcologic conditions, such as for pa tients with anatomic or functional asplenia and for college freshmen living in dormitories. The meningococcal vaccines also play an important role for oncologic patients as well as for patients receiving eculizumab (Soliris), per the package insert recommendation (Alexion, 2011). Meningococcal Vaccine Common adverse events associated with the meningococcal vaccine are generally mild, in cluding fever and pain at the injection site. Other rare but serious adverse events may include burn ing, numbness, and breathing difficulty. Neisseria meningitides is a bacterial infection that affects the brain and spinal cord. The consequences of such an infection could lead to meningitis, an in flammation of the meninges surrounding the central nervous system, which can be life-threatening. Most often, patients will present with a series of symp toms that include but are not limited to headache, neck stiffness, fever, altered mental status, and vom iting. Due to the seriousness of the condition, the use of vaccine as prophylaxis may play a role in pre venting such a deleterious condition while ensuring long-term protective effects. VACCINATION RECOMMENDATIONS REVIEW The MMR vaccine is given as two separate sub cutaneous injections at least 1 month apart to pro duce long-lasting immunity. In general, the MMR vaccine is included in the childhood vaccination schedule before the age of 2. Repeat vaccination is not generally recommended in adults, unless there is evidence of a lack of immunity as determined by a low or absent titer. In patients undergoing chemo therapy, live vaccines should be avoided, especially in immunocompromised patients (Ljungman et al., 1989; Kroger et al., 2011). In order to ensure pro tection to individuals undergoing chemotherapy, household members and close contacts should be immunized with MMR. Although the evidence be hind the use of live MMR vaccine in cancer patients is not strong, the CDC’s Advisory Committee on Immunization Practices suggests the use of MMR vaccination for leukemic patients in remission no sooner than 3 to 6 months after completion of all chemotherapy treatment (CDC, 1998; Arrowood & Hayney, 2002). The varicella vaccine, introduced in the 1990s, is a live vaccine that provides protection against chickenpox and herpes zoster. It is generally recom mended for children under 13, with the first given dose at 12 to 15 months and a repeat dose given at 4 to 6 years. For individuals who are older than 13 but have never been introduced to chickenpox, the two doses should be administered 4 to 8 weeks apart. In patients undergoing chemotherapy, live varicella vaccine is not recommended, especially in immu nocompromised patients (CDC, 2012). In order to ensure protection to individuals undergoing che motherapy, household members and close contacts should be immunized with varicella vaccine (Marin, Güris, Chaves, Schmid, & Seward, 2007). Similar to the loss of MMR immunity, post-HSCT patients will undergo a decrease in and eventually a loss of immunity to varicella. Although vaccination with live attenuated varicella vaccine is generally not recommended immediately post-HSCT, it should be reconsidered at least 2 years posttransplant. Patients should be free from chronic GVHD and off immuno suppression at the time of revaccination (Ljungman et al., 1989; Kroger et al., 2011).f Unlike patients who receive chemotherapy without stem cell transplant, post-HSCT patients will experience in a decrease in and eventually a loss of MMR immunity from most previous vac cination within the first few months after trans plant. Live Vaccines In general, live vaccines (MMR, varicella, and intranasal influenza) should be avoided in patients undergoing concurrent chemotherapy and post transplant patients. The timing and method of pro tection differs between these two populations. The common adverse reactions to MMR vac cine may include fever, malaise, and rash up to 3 weeks postvaccination. Joint pain may be more evident in elderly women. In rare instances, ana phylaxis has been reported. Rare neurologic dis orders have also been reported. Live vaccines should be withheld in patients who are prior to or receiving concurrent chemo therapy. Instead, it is recommended that household members and close contacts be fully immunized to lessen the likelihood of the infection. Patients who are immunosuppressed, including posttransplant patients, should wait at least 24 months posttrans plant and until they are no longer receiving immu nosuppression, free from graft-vs.-host disease, and have immunologic response before receiving live vaccines. As previously discussed, due to the dramat ic decrease and eventually a complete loss of immu nity from previous vaccinations, live vaccines may be reconsidered when the above criteria are met in order to reinitiate lifelong protection after transplant (Ljungman et al., 2009; Hilgendorf et al., 2011). VACCINATION RECOMMENDATIONS Although vaccination with live attenuated MMR vaccine is generally not recommended im mediately post-HSCT, it should be reconsidered at least 2 years posttransplant. Patients should be free from chronic GVHD and off immunosup pression at the time of revaccination (Ljungman et al., 1989; Kroger et al., 2011). Common side effects may include soreness and redness at the injection site, fever, and rash. Seizures, pneumonia, and anaphylaxis are rare but serious side effects that have been reported. Measles-Mumps-Rubella Vaccinef Measles is a viral infection that affects the respi ratory system. Mumps is a viral condition inducing inflammation of the salivary gland and the testicles. Rubella, also known as German measles, is a viral infection that presents as a rash on the face, trunk, and limbs. In some cases, it can also cause joint pain, swollen glands, and conjunctivitis. Together, all three highly contagious conditions have led to wide spread outbreaks causing hundreds of thousands of deaths. In the 1960s, the introduction of a measles vaccine diminished the number of reported cases each year. In 2000, the United States declared the elimination of measles. Rubella and mumps cases also sharply declined following the introduction of their respective vaccines. In general, when live vac cines are given individually, they must be given on three separate occasions. Since the introduction of the combined measles, mumps, and rubella (MMR) vaccine, individuals can receive the three vaccines as a single injection, inducing quicker immunity while minimizing pain (CDC, 2012). In pediatric patients, meningococcal vaccine produced variable responses among acute leuke mics and suggested the likelihood of response based on proximity to chemotherapy (Yu et al., 2007). Pa tients with hematologic or oncologic malignancies (particularly Hodgkin disease) who are at high risk of infection should receive the meningococcal vac cine at least 1 week prior to starting therapy. Revac cination is recommended 5 years after the comple tion of therapy for Hodgkin disease, and then every 5 years after that (Ambrosino & Molrine, 1993). In post-HSCT patients, vaccination with the conju gated meningococcal vaccine starting 6 to 12 months posttransplant may be considered if the likelihood of contracting Neisseria meningitides is high. 78 J Adv Pract Oncol AdvancedPractitioner.com VACCINATION RECOMMENDATIONS Donor Vaccination the donor is immunized prior to stem cell collection (Ljungman et al., 2005; Hilgendorf et al., 2011). Although there are no specific recommenda tions, donor vaccination with Td, PCV7, and Hib has been associated with an improvement in post transplant immunity for the recipient of the stem cell transplant (Ljungman et al., 2009). In addition, early administration of the hepatitis B vaccine post transplant may assist with the transfer of immunity from the donor to the recipient of the transplant, if RECOMMENDED RECOMMENDED Negative marker: Hep B 3-dose series (begin 6 mo posttransplant) plus a booster (18 mo posttransplant) Resolved infection: with concurrent nucleoside analog; consider early posttransplant vaccination Seronegative recipient receiving HBsAg positive graft: vaccinate before HSCT; with concurrent nucleoside analog and HBIG; also should receive posttransplant vaccination for long- lasting immunity Seronegative recipient receiving HBsAg positive graft: vaccinate before HSCT; with concurrent nucleoside analog and HBIG; also should receive posttransplant vaccination for long- lasting immunity Notes. cGVHD = chronic graft-vs.-host disease; HBIG = hepatitis B immunoglobulin; HSCT = hematopoietic stem cell transplant; IM = intramuscular; IPV = inactivated polio vaccine; OPV = oral poliomyelitis vaccine; PCV7 = pneumococcal conjugate vaccine; PPSV23 = pneumococcal polysaccharides 23-valent vaccines; SC = subcutaneous; TBI = total-body irradiation. aConcurrent chemotherapy. bPosttransplant and on immunosuppression. Notes. cGVHD = chronic graft-vs.-host disease; HBIG = hepatitis B immunoglobulin; HSCT = hematopoietic stem cell transplant; IM = intramuscular; IPV = inactivated polio vaccine; OPV = oral poliomyelitis vaccine; PCV7 = pneumococcal conjugate vaccine; PPSV23 = pneumococcal polysaccharides 23-valent vaccines; SC = subcutaneous; TBI = total-body irradiation. aConcurrent chemotherapy Hematology/oncology patientsa Post-HSCT patientsb RECOMMENDED Annually (given at least 2 wk prior to chemotherapy) RECOMMENDED Annually (begin no sooner than 6 mo posttransplant); may require a second dose for first-time recipient RECOMMENDED PCV7 3-dose series 1 mo apart (begin 6 mo posttransplant); give booster dose of PPSV23 (18 mo post- transplant) RECOMMENDED DTap 3-dose series 1 mo apart (begin 6 mo posttransplant) plus a booster (18 mo posttransplant) RECOMMENDED DTap 3-dose series 1 mo apart (begin 6 mo posttransplant) plus a booster (18 mo posttransplant) RECOMMENDED Hib 3-dose series (begin 6 mo post- transplant) plus a booster (18 mo posttransplant) RECOMMENDED Hib 3-dose series (begin 6 mo post- transplant) plus a booster (18 mo posttransplant) Varicella Vaccine Chickenpox is a highly contagious condition induced by the varicella zoster virus (VZV). It pres ents as an itchy skin rash, particularly on the body and head. It is spread by direct exposure to the rash secretions. There is generally a lag time of a few days to weeks after the individual has been exposed be fore the rash presents. Chickenpox appearing late in life is a reactivation of the VZV, otherwise known as shingles. In addition to proper hygiene, vaccination plays a role in providing protection (CDC, 2012). 79 Vol 3  No 2  Mar/Apr 2012 AdvancedPractitioner.com REVIEW TSANG REVIEW TSANG Table 6. Recommendations and Timing for Vaccinations for Hematology/Oncology and Post-HSCT Patients Vaccine type Hematology/oncology patientsa Post-HSCT patientsb Inactivated and conjugated vaccines Influenza (IM) • Inactivated form only • Avoid live intranasal vaccine RECOMMENDED Annually (given at least 2 wk prior to chemotherapy) RECOMMENDED Annually (begin no sooner than 6 mo posttransplant); may require a second dose for first-time recipient Pneumococcal (IM or SC) • PCV7 is different from PPSV23 RECOMMENDED PPSV23 (general hem/onc population, unless in the specific category immediately below) PCV7 (patients receiving treatment for multiple myeloma, Hodgkin, and non-Hodgkin, lymphomas, cGVHD and post-TBI) given at least 2 wk prior to chemotherapy; repeat once with PPSV23 in 5 yr after initial dose RECOMMENDED PCV7 3-dose series 1 mo apart (begin 6 mo posttransplant); give booster dose of PPSV23 (18 mo post- transplant) Diphtheria-Tetanus- Pertussis (IM) • DTap: full dose of diphtheria • Tdap: reduced dose of diphtheria OPTIONAL No additional dose needed if up to date with vaccination Td every 10 yr; replace a single dose of Td with Tdap RECOMMENDED DTap 3-dose series 1 mo apart (begin 6 mo posttransplant) plus a booster (18 mo posttransplant) Haemophilus influenzae (IM) OPTIONAL None needed, unless posttreatment titers are undetectable (If low titer, given at least 2 wk prior to chemotherapy) RECOMMENDED Hib 3-dose series (begin 6 mo post- transplant) plus a booster (18 mo posttransplant) Hepatitis B (IM) OPTIONAL None needed, unless posttreatment titers are undetectable May be considered if at risk of HBs Ag positive contacts Hep B 3-dose series, not while on immunosuppressive therapy RECOMMENDED Negative marker: Hep B 3-dose series (begin 6 mo posttransplant) plus a booster (18 mo posttransplant) Resolved infection: with concurrent nucleoside analog; consider early posttransplant vaccination Seronegative recipient receiving HBsAg iti ft i t b f Notes. cGVHD = chronic graft-vs.-host disease; HBIG = hepatitis B immunoglobulin; HSCT = hematopoietic stem cell transplant; IM = intramuscular; IPV = inactivated polio vaccine; OPV = oral poliomyelitis vaccine; PCV7 = pneumococcal conjugate vaccine; PPSV23 = pneumococcal polysaccharides 23-valent vaccines; SC = subcutaneous; TBI = total-body irradiation. aConcurrent chemotherapy. bP tt l t d i i Conclusion In most instances, patients undergoing chemo therapy should receive both influenza and pneu mococcal vaccine at least 2 weeks before starting chemotherapy with potential additional vaccines based on the clinical picture (see Table 6). If possi 80 J Adv Pract Oncol AdvancedPractitioner.com VACCINATION RECOMMENDATIONS REVIEW Table 6. Recommendations and Timing for Vaccinations for Hematology/Oncology and Post-HSCT Patients (Cont.) Vaccine type Hematology/oncology patienta Post-HSCT patientb Inactivated and conjugated vaccines Polio vaccine (IM or SC) • Only give IPV in post- HSCT patients. • IPV and OPV are not interchangeable OPTIONAL Not routinely needed unless post- treatment titers are undetectable RECOMMENDED IPV 3-dose series (begin 6 mo post- transplant) plus a booster (18 mo posttransplant) Hepatitis A (IM) OPTIONAL Only recommended if traveling to endemic areas or as postexposure prophylaxis OPTIONAL Only recommended if traveling to endemic areas or as postexposure prophylaxis Meningococcal vaccine (IM for MCV4, SC for MPSV4) • MCV4: for age 2–55 yr • MPSV4: 56 yr and older RECOMMENDED Hodgkin disease: or who are at high risk of infection: MCV4 (1 wk prior to starting therapy); repeat 5 yr after completion of therapy OPTIONAL Based on clinical consideratons MCV4 1–3 monthly doses Human papillovirus (HPV) quadrivalent vaccine (IM) • Age specific (9–26 yr old) for the prevention OPTIONAL If age appropriate HPV 3-dose series OPTIONAL If age appropriate HPV 3-dose series Table 6. Recommendations and Timing for Vaccinations for Hematology/Oncology and Post-HSCT Patients (Cont.) Vaccine type Hematology/oncology patienta Post-HSCT patientb Inactivated and conjugated vaccines RECOMMENDED IPV 3-dose series (begin 6 mo post- transplant) plus a booster (18 mo posttransplant) OPTIONAL Not routinely needed unless post- treatment titers are undetectable OPTIONAL If age appropriate HPV 3-dose series Live vaccines MMR (SC) • Live vaccine should be avoided in immunocompromised patients OPTIONAL None needed if childhood vaccination up to date If previously vaccinated, draw post- treatment titer If low titer, administer at least 3 mo after completion of chemotherapy (particular in leukemic in remission) Varicella (SC) • Live vaccine should be avoided in immunocompromised patients OPTIONAL If not previously exposed or vaccinated (should be considered before starting chemotherapy; withhold during concurrent chemotherapy) Varicella 2-dose series (at least 4–8 wk apart) Notes. cGVHD = chronic graft-vs.-host disease; HBIG = hepatitis B immunoglobuli IM = intramuscular; IPV = inactivated polio vaccine; OPV = oral poliomyelitis vacci Live vaccines MMR (SC) • Live vaccine should be avoided in immunocompromised patients OPTIONAL None needed if childhood vaccination up to date If previously vaccinated, draw post- treatment titer If low titer, administer at least 3 mo after completion of chemotherapy (particular in leukemic in remission) CONTRAINDICATED immediately post-HSCT May be considered at 24 mo posttransplant (no longer on immunosuppression and attains full immune system) Varicella (SC) • Live vaccine should be avoided in immunocompromised patients OPTIONAL If not previously exposed or vaccinated (should be considered before starting chemotherapy; withhold during concurrent chemotherapy) Varicella 2-dose series (at least 4–8 wk apart) CONTRAINDICATED immediately post-HSCT May be considered at 24 mo posttransplant (no longer on immunosuppression and attains full immune system) OPTIONAL None needed if childhood vaccination up to date If previously vaccinated, draw post- treatment titer If low titer, administer at least 3 mo after completion of chemotherapy (particular in leukemic in remission) OPTIONAL If not previously exposed or vaccinated (should be considered before starting chemotherapy; withhold during concurrent chemotherapy) Varicella 2-dose series (at least 4–8 wk apart) CONTRAINDICATED immediately post-HSCT May be considered at 24 mo posttransplant (no longer on immunosuppression and attains full immune system) CONTRAINDICATED immediately post-HSCT May be considered at 24 mo posttransplant (no longer on immunosuppression and attains full immune system) CONTRAINDICATED immediately post-HSCT May be considered at 24 mo posttransplant (no longer on immunosuppression and attains full immune system) REFERENCES g/ /j Engelhard, D., Cordonnier, C., Shaw, P. J., Parkalli, T., Guenther, C., Martino, R.,…Ljungman, P. (2002). Infectious Disease Working Party of the European Bone Marrow Transplanta tion (IDWP-EMBT). Early and late invasive pneumococcal infection following stem cell transplantation: A European Bone Marrow Transplantation survey. British Journal of Haematology, 117, 444–450. http://dx.doi.org/10.1046/ j.1365-2141.2002.03457.xf Alexion Pharmaceuticals. (2011). Eculizumab package insert. Retrieved from http://www.soliris.net/indications/soliris_ pi.pdf Ambrosino, D. M., & Molrine, D. C. (1993). Critical appraisal of immunization strategies for prevention of infection in the compromised host. Hematology Oncology Clinics of North America, 7, 1027–1050. Fedson, D. S. (1999). The clinical effectiveness of pneumococcal vaccination: A brief review. Vaccine, 17 (suppl 1), S85–S90. http://dx.doi.org/10.1016/S0264-410X(99)00113-9f Anderson, H., Petrie, K., Berrisford, C., Charlett, A., Thatcher, N., & Zambon, M. (1999). 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Respons es of patients with neoplastic diseases to influenza virus vaccine. Cancer, 42, 2233–2247. http://dx.doi. org/10.1002/1097-0142(197811)42:5<2244::AID- CNCR2820420523>3.0.CO;2-7 Black, S., Shinefield, H., Fireman, B., Lewis, E., Ray, P., Hansen, J. R.,…Edwards, K. (2000). Efficacy, safety and immu nogenicity of heptavalent pneumococcal conjugate vac cine in children. Northern California Kaiser Permanente Vaccine Study Center Group. Pediatric Infectious Disease Journal, 19, 187–195. http://dx.doi.org/10.1097/00006454- 200003000-00003 Hepatitis B Foundation. (2012). Retrieved from http://www. hepb.org Hilgendorf, I., Freund, M., Jilig, W., Einsele, H., Gea-Banacloche, J., Greinix, H.,…Meisel, R. (2011). Vaccination of alloge neic haematopoietic stem cell transplant recipients: Re port from the Internal Consensus Conference on Clinical Practice in chronic GVHD. Vaccine, 29, 2825–2833. http:// dx.doi.org/10.1016/j.vaccine.2011.02.018 Centers for Disease Control and Prevention. (1998). Measles, mumps and rubella—Vaccine use and strategies for elimi nation of measles, rubella, and congenital rubella syndrome and control of mumps: Recommendation of the Advisory Committee on Immunization Practice (ACIP). OPTIONAL Varicella (SC) • Live vaccine should be avoided in immunocompromised patients CONTRAINDICATED immediately post-HSCT May be considered at 24 mo posttransplant (no longer on immunosuppression and attains full immune system) CONTRAINDICATED immediately post-HSCT May be considered at 24 mo posttransplant (no longer on immunosuppression and attains full immune system) nated with certain vaccines, while some are optional and others should be avoided (see Table 6). ble, inactivated vaccines should be administered at a minimum of 2 weeks prior to starting therapy. If the previous scenario is not possible, then one should consider receiving vaccination in between cycles. Vaccinating too close to chemotherapy could lead to the loss of durable immunity (Hilgendorf et al., 2011; Ljungman et al., 2009). In most cases, many post- HSCT patients are viewed as “never vaccinated.” These individuals are recommended to be revacci For the general population, childhood vaccina tion has led to the elimination of many preventable diseases. Likewise, the same approach in the hema tology and oncology population has been beneficial. In the immunosuppressed population, appropriate timing and avoidance of live vaccines will not only provide protection against these diseases but also 81 Vol 3  No 2  Mar/Apr 2012 AdvancedPractitioner.com REVIEW TSANG lic Health Foundation. confer longer-lasting immunity. Advanced practi tioners can benefit from recognizing the importance and implications of each vaccine for the hematology, oncology, and posttransplant populations. Cordonnier, C., Labopin, M., Chesnel, V., Ribaud, P., Da La Ca mara, R., & Martino, R. (2008). Influence of immunisation timing on the response to conjugate-pneumococcal vac cine after allogeneic stem cell transplant: Final results of the EBMT IDWP01 Trial. Bone Marrow Transplantation, 45(suppl 1), S2. DISCLOSURE Egea, E., Iglesias, A., Salazar, M., Morimoto, C., Kruskall, M. S., Awdeh, Z.,…Yunis, E. J. (1991). The cellular basis for lack of antibody response to hepatitis B vaccine in humans. Jour nal of Experimental Medicine, 173, 531–538. http://dx.doi. org/10.1084/jem.173.3.531 The author has no conflicts of interest to disclose. VACCINATION RECOMMENDATIONS VACCINATION RECOMMENDATIONS REVIEW for active immunization of the immunocompromised host. Clinical Microbiology, 11, 1–26. for active immunization of the immunocompromised host. Clinical Microbiology, 11, 1–26. es, 45, 1576–1582. http://dx.doi.org/10.1086/523583 Lepow, M. L. (1994). Meningococcal vaccines. In S. A. Plotkin & E. A. Moritmer, Jr., (Eds.), Vaccines (2nd ed., pp. 503–515). Philadelphia, PA: W. B. Saunders. Pollyea, D. A., Brown, J. M. Y., & Horning, S. J. (2010). Utility of influenza vaccination for oncology patients. Journal of Clin ical Oncology, 28(14), 2481–2490. http://dx.doi.org/10.1200/ JCO.2009.26.6908 Ljungman, P., Fridell, E., Lonqvist, B., Bolme, P., Bottiger, M., Gahrton, G.,…Wahren, B. (1989). Efficacy and safety of vac cination of marrow transplant recipients with a live at tenuated measles, mumps, and rubella vaccine. Journal of Infectious Diseases, 159, 610–615. http://dx.doi.org/10/1093/ infdis/159.4.610 Rapezzi, D., Sticchi, L., Racchi, O., Mangerini, R., Ferraris, A. M., & Gaetani, G. F. (2003). Influenza vaccine in chronic lymphoproliferative disorders and multiple myeloma. Eu ropean Journal of Haematology, 70, 225–230. http://dx.doi. org/10.1034/j.1600-0609.2003.00028.x Ljungman, P., Cordonnier, C., Einsele, H., Englund, J., Mach ado, C. M., Storek, J., & Small, T. (2009). Vaccination of hematopoietic cell transplant recipients. Bone Marrow Transplantation, 44, 521–526. http://dx.doi.org/10.1038/ bmt.2009.263 Ring, A., Marx, G., Steer, C., Prendiville, J., & Ellis, P. (2003). Poor uptake of influenza vaccinations in patients receiving cyto toxic chemotherapy. International Journal of Clinical Prac tice, 57, 542–543. Ljungman, P., Engelhard, D., de la Camara, R., Einsele, H., Lo casciulli, A., Martino,…Cordonnier, C. (2005). Vaccina tion of stem cell transplant recipients: Recommendations of the Infectious Diseases Working Party of the EBMT. Bone Marrow Transplantation, 35, 737–746. http://dx.doi. org/10.1038/sj.bmt.1704870 Robertson, J. D., Nagesh, K., Jowitt, S. N., Anderson, H., Mutton, K., Zambon, M., & Scarffe, J. H. (2000). Immunogenicity of vaccination against influenza, Steptococcus pneumonia and Haemophilus influenzae type B in patients with multiple myeloma. British Journal of Cancer, 82, 1261–1265. http:// dx.doi.org/10.1054/bjoc.1999.1088 Lo, W., Whimbey, E., Elting, L., Couch, R., Cabanillas, F., & Bodey, G. (1993). Antibody response to a two-dose influ enza vaccine regimen in adult lymphoma patients on che motherapy. European Journal of Clinical Microbiology & Infectious Diseases, 12, 778–782. http://dx.doi.org/10.1007/ BF02098469 Schafer, A. I., Churchill, W. H., Ames, P., & Weinstein, L. (1979). The influence of chemotherapy on response of patients with hematologic malignancies to influenza vaccine. Can cer, 43, 25–30. http://dx.doi.org/10.1002/1097-0142l Shoji, H., & Kaji, M. (2003). The influenza vaccination and neu rological complications. Internal Medicine, 42, 139. VACCINATION RECOMMENDATIONS http:// dx.doi.org/10.2169/internalmedicine.42.139 Loulergue, P., Mir, O., Alexandre, J., Ropert, S., Goldwasser, F., & Launay, O. (2008) Low influenza vaccination rate among patients receiving chemotherapy for cancer. 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Stiver, H. G., & Weinerman, B. H. (1978). Impaired serum anti body response to inactivated influenza A and B vaccine in cancer patients. Canadian Medical Association Journal, 119, 733–738. Meisel, R., Kuypers, L., Dirksen, U., Schubert, R., Gruhn, B., & Strauss, G. (2007). Pneumococcal conjugate vaccine pro vides early protective antibody responses in children af ter related and unrelated allogeneic hematopoietic stem cell transplantation. Blood, 109, 2322–2326. http://dx.doi. org/10.1182/blood-2006-06-032284l Thompson, W. W., Shay, D. K., Weintraub, E., Brammer, L., Bridges, C. B., Cox, N. J., & Fukuda, K. (2004). Influenza- associated hospitalizations in the United States. Journal of the American Medical Association, 292, 1333–1340. http:// dx.doi.org/10.1001/jama.292.11.1333 g/ / Melcher, L. (2005). Recommendations for influenza and pneu mococcal vaccinations in people receiving chemotherapy. Clinical Oncology, 17, 12–15. http://dx.doi.org/10.1016/j. clon.2004.07.010 Thompson, W. W., Shay, D. K., Weintraub, E., Brammer, L., Cox, N., Anderson, L. J., & Fukuda, K. (2003). Mortality associ ated with influenza and respiratory syncytial virus in the United States. Journal of the American Medical Association, 289, 170–186. http://dx.doi.org/10.1001/jama.289.2.179 Molrine D. C., Guinan, E. C., Antin, J. H., Parsons, S. K., Wein stein, H. J., Wheeler, C.,…Ambrosino, D. M. (1996). Donor immunization with Haemophilus influenzae type b (HIB)- conjugate vaccine in allogeneic bone marrow transplanta tion. Blood, 87, 3012–3018.f Weitberg, A. B., Weitzman, S. A., Watkins, E., Hinkle, C., O’Rourke, S., & Dienstag, J. L. (1985). Immunogenicity of hepatitis B vaccine in oncology patients receiving chemo therapy. REFERENCES Morbidity and Mortality Weekly Report, 47(RR-8), 1–57. Hodges, G. R., Davis, J. W., Lewis, H. D., Whitter, F. C., Siegel, C., Chin, T. D. Y.,…Noble, G. R. (1979). Response to influ enza A vaccine among high-risk patients. 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https://openalex.org/W2148126506	https://dipot.ulb.ac.be/dspace/bitstream/2013/218207/3/doi_201834.pdf	English	null	Breakthrough in cardiac arrest: reports from the 4th Paris International Conference	Annals of intensive care	2,015	cc-by	22,501	© 2015 Kudenchuk et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Epidemiology of CA Epidemiology of CA Out‑of‑hospital cardiac arrest in 2014: incidence, outcome and disparities Abstract Jean-Luc Diehl The French Intensive Care Society organized on 5th and 6th June 2014 its 4th “Paris International Con‑ ference in Intensive Care”, whose principle is to bring together the best international experts on a hot topic in critical care medicine. The 2014 theme was “Breakthrough in cardiac arrest”, with many high-quality updates on epidemiol‑ ogy, public health data, pre-hospital and in-ICU cares. The present review includes short summaries of the major presentations, classified into six main chapters: • • Epidemiology of CA • • Pre-hospital management • • Post-resuscitation management: targeted temperature management • • Post-resuscitation management: optimizing organ perfusion and metabolic parameters • • Neurological assessment of brain damages • • Public healthcare • • Epidemiology of CA • • Pre-hospital management • • Post-resuscitation management: targeted temperature management • • Post-resuscitation management: optimizing organ perfusion and metabolic parameters • • Neurological assessment of brain damages • • Public healthcare Keywords: Cardiac arrest, Cardio-pulmonary resuscitation, Targeted temperature management, Therapeutic hypothermia, Persistent vegetative state, Minimally conscious state, Organ donation Keywords: Cardiac arrest, Cardio-pulmonary resuscitation, Targeted temperature management, Therapeutic hypothermia, Persistent vegetative state, Minimally conscious state, Organ donation said to be the only uniform aspect of cardiac arrest, which is otherwise riddled with disparities. The first of these disparities lies in how few patients who are successfully resuscitated from cardiac arrest ultimately survive to hos- pital discharge. In Seattle, approximately 60 % of patients in whom cardiac arrest presents as ventricular fibrillation (VF) are successfully resuscitated and admitted to hospital; yet only about half of these (30 %) typically survive to hos- pital discharge. When cardiac arrest presents as asystole or pulseless electrical activity (PEA), outcomes are strikingly worse: only 20–30 % of such patients are successfully resuscitated, and an even smaller proportion of these, as few as 2 in 10—ranging from 2 to 5 % of patients, survive to hospital discharge [1]. Breakthrough in cardiac arrest: reports from the 4th Paris International Conference Peter J. Kudenchuk1, Claudio Sandroni2, Hendrik R. Drinhaus3, Bernd W. Böttiger3, Alain Cariou4,5, Kjetil Sunde6, Martin Dworschak7, Fabio Silvio Taccone8, Nicolas Deye9, Hans Friberg10, Steven Laureys11, Didier Ledoux12, Mauro Oddo13, Stéphane Legriel14, Philippe Hantson15, Jean‑Luc Diehl16* and Pierre‑Francois Laterre17 Kudenchuk et al. Ann. Intensive Care (2015) 5:22 DOI 10.1186/s13613-015-0064-x REVIEW Breakthrough in cardiac arrest: reports from the 4th Paris International Conference Peter J. Kudenchuk1, Claudio Sandroni2, Hendrik R. Drinhaus3, Bernd W. Böttiger3, Alain Cariou4,5, Kjetil Sunde6, Martin Dworschak7, Fabio Silvio Taccone8, Nicolas Deye9, Hans Friberg10, Steven Laureys11, Didier Ledoux12, Mauro Oddo13, Stéphane Legriel14, Philippe Hantson15, Jean‑Luc Diehl16* and Pierre‑Francois Laterre17 Abstract Jean-Luc Diehl The French Intensive Care Society organized on 5th and 6th June 2014 its 4th “Paris International Con‑ ference in Intensive Care”, whose principle is to bring together the best international experts on a hot topic in critical care medicine. The 2014 theme was “Breakthrough in cardiac arrest”, with many high-quality updates on epidemiol‑ ogy, public health data, pre-hospital and in-ICU cares. The present review includes short summaries of the major presentations, classified into six main chapters: • • Epidemiology of CA • • Pre-hospital management • • Post-resuscitation management: targeted temperature management • • Post-resuscitation management: optimizing organ perfusion and metabolic parameters • • Neurological assessment of brain damages • • Public healthcare Keywords: Cardiac arrest, Cardio-pulmonary resuscitation, Targeted temperature management, Therapeutic hypothermia, Persistent vegetative state, Minimally conscious state, Organ donation Open Access Kudenchuk et al. Ann. Intensive Care (2015) 5:22 DOI 10.1186/s13613-015-0064-x REVIEW Breakthrough in cardiac arrest: reports from the 4th Paris International Conference Peter J. Kudenchuk1, Claudio Sandroni2, Hendrik R. Drinhaus3, Bernd W. Böttiger3, Alain Cariou4,5, Kjetil Sunde6, Martin Dworschak7, Fabio Silvio Taccone8, Nicolas Deye9, Hans Friberg10, Steven Laureys11, Didier Ledoux12, Mauro Oddo13, Stéphane Legriel14, Philippe Hantson15, Jean‑Luc Diehl16* and Pierre‑Francois Laterre17 Abstract Jean-Luc Diehl The French Intensive Care Society organized on 5th and 6th June 2014 its 4th “Paris International Con‑ ference in Intensive Care”, whose principle is to bring together the best international experts on a hot topic in critical care medicine. The 2014 theme was “Breakthrough in cardiac arrest”, with many high-quality updates on epidemiol‑ ogy, public health data, pre-hospital and in-ICU cares. The present review includes short summaries of the major presentations, classified into six main chapters: • • Epidemiology of CA • • Pre-hospital management • • Post-resuscitation management: targeted temperature management • • Post-resuscitation management: optimizing organ perfusion and metabolic parameters • • Neurological assessment of brain damages • • Public healthcare Keywords: Cardiac arrest, Cardio-pulmonary resuscitation, Targeted temperature management, Therapeutic hypothermia, Persistent vegetative state, Minimally conscious state, Organ donation Open Access Kudenchuk et al. Ann. Intensive Care (2015) 5:22 DOI 10.1186/s13613-015-0064-x Open Access Correspondence: jldiehl@invivo.edu 16 Medical Intensive Care Unit, AP‑HP, European Georges Pompidou Hospital, Paris Descartes University and Sorbonne Paris Cité-Medical School, Paris, France Full list of author information is available at the end of the article p gy Out‑of‑hospital cardiac arrest in 2014: incidence, outcome and disparities Peter Kudenchuk Out-hospital-cardiac arrest (OHCA) remains a common public health problem. Each year, car- diac arrest claims more than 424,000 lives in the United States, 300,000 lives in Europe, and upwards of 3.7 million lives worldwide. Resuscitation is typically conducted in accordance with a uniform algorithmic approach embod- ied in the “chain of survival” which emphasizes the impor- tance of early activation of emergency medical services, prompt CPR, rapid defibrillation, advanced cardiac life support and post-cardiac arrest care. This, in fact might be A second disparity lies in the changing incidence of rhythms that precipitate cardiac arrest. In Seattle dur- ing the decade of the 1970s, VF accounted for approxi- mately 60 % of all out-of-hospital cardiac arrests treated by emergency medical services (EMS), whereas the Correspondence: jldiehl@invivo.edu 16 Medical Intensive Care Unit, AP‑HP, European Georges Pompidou Hospital, Paris Descartes University and Sorbonne Paris Cité-Medical School, Paris, France Full list of author information is available at the end of the article Page 2 of 24 Kudenchuk et al. Ann. Intensive Care (2015) 5:22 in survival outcome from cardiac arrest include patient characteristics—such as age, gender, and co-morbidities; and by the circumstances of the arrest—such as whether witnessed by bystanders, the location of its occurrence, and by the presenting arrest rhythm. Most would regard these as “factors of fate” and as such not alterable. Con- versely, the components of prehospital emergency medi- cal care, including rapid dispatch, dispatcher-assisted CPR, EMS training and time-to-treatment, are all poten- tially correctable factors. Targeting these aspects of pre- hospital care affords an opportunity to change outcome for the better after cardiac arrest. Among these, perhaps the one that can be most readily and immediately imple- mented is high-performance CPR. remainder of acute rhythm presentations were equally divided between asystole and PEA. In the ensuing years, the proportion of cardiac arrests caused by VF has declined to 25–30 % of cases, such that now asystole and PEA represent the most common presenting rhythms. Extrapolating these incidence data from Seattle to the United States census, the annual rate of cardiac arrest due to ventricular fibrillation declined from about 85 persons per 100,000 in 1980 to 38/100,000 in 2000 [1]. Data from the Resuscitation Outcomes Consortium estimated a fur- ther decline in the incidence of VF cardiac arrest to 17.4 adults/100,000 in 2011 [2]. These statistics, coupled with the known worse survival prognosis of patients in whom cardiac presents as a non-shockable rhythm, pose a new and major challenge for the present and future treatment of this emerging “new wave” of cardiac arrest victims. g p High-performance CPR consists of training with meticulous attention to the details of performing CPR to the best known prescribed parameters of chest compres- sion rate, depth, full chest recoil, and minimized inter- ruptions, and applying strict compliance standards (e.g. a compression fraction of no less than 85 %) for their performance during resuscitation. In addition to striv- ing for “letter perfect” CPR performance, it also involves a system of accountability, whereby feed-back of CPR performance derived from a review of recordings of field resuscitations is consistently conveyed back to providers for further possible improvement. Deploying such a high- performance CPR protocol in King County, Washington, starting in 2005 resulted in a significant improvement in survival from both cardiac arrest due to ventricular fibril- lation (Fig. Effectiveness of rapid response systems for prevention of cardiac arrest Claudio Sandroni Despite the immediate availability of qualified life support, the outcome of in-hospital cardiac arrest (IHCA) remains poor. Survival to discharge after IHCA rarely exceeds 20 % [9] and it has remained stable in the last 25 years (Fig. 3). Rapid response systems (RRS) have been established to prevent IHCA in non-critical care areas of the hospital [10]. Those systems are based on timely detection of deteriorating patients by the ward personnel (the afferent limb of the system), who will there- fore summon a medical emergency team (MET; the effer- ent limb of the system), whose roles are to stabilize the patient in the ward or escalate the level of care. Although the theory underlying RRS is compelling, there is no defi- nite evidence that their implementation improves patient outcome. The major problem in evaluating the effective- ness of RRS is the choice of the outcome measure. The third, and most comprehensive endpoint for RRS effectiveness is hospital mortality. Unfortunately, meta- analyses of available evidence on this endpoint showed conflicting results, with some studies showing benefit and others showing no or only non-significant reduc- tion of hospital mortality after RRS implementation [13]. Moreover, the quality of evidence is relatively low, with almost all studies having a before-and-after design, which make them prone to bias due to secular trends unrelated to the study intervention or to changes in hospital case mix, a variable which is difficult to adjust for.hf The first endpoint for a study addressing the effective- ness of RRS could be the rates of unexpected cardiac arrests occurring outside intensive care units (ICUs), that is, the rates of cardiac arrest occurring in ward patients for whom there is no do-not-attempt-resusci- tation (DNAR) order. This endpoint, however, is poten- tially biased by the fact that one of the tasks of METs is to identify ward patients for whom a resuscitation would be fi The ultimate strategy for assessing RSS effectiveness would be a randomized trial with concurrent cohorts, which would allow the investigators to control for most possible confounders. However, this solution is ham- pered by both ethical and implementation issues. Ran- domization at individual patient level for interventions which are commonly believed to be beneficial would in fact be ethically questionable. 2) [7], as well as asystole/PEA [8] that has been sustained in the ensuing years. The attractiveness of high-performance CPR is that it is relatively inexpensive to deploy (can be easily added to existing EMS training programs), does not require special equipment (hands A third disparity lies in the marked differences in out- come from cardiac arrest between communities across Europe and the United States. For cardiac arrest due to VF, survival can vary significantly between major cities by many-fold (Fig. 1) [3–5]. To some extent, such differences in outcome are explained by inaccuracies in record keep- ing. For example, few communities actually report their incidence of cardiac arrest and outcome [6]. And even among those that do, complete capture of all cases of car- diac arrest and the reliability of their survival data can be questionable, accounting for some of the variability in outcomes that are reported. This said, record keeping alone does not entirely explain these discrepancies. Iden- tifying and targeting remediable causes of differences in survival between communities is imperative if we are to assure citizens that they are comparably “safe” from death by cardiac arrest in whatever locale they call home. Clinical factors that are known to account for differences Fig. 1 Survival to hospital discharge from out-of-hospital ventricular fibrillation in various communities outside the United States Fig. 2 The impact of instituting high-performance CPR in King County, Washington, in 2005 on survival from witnessed out-of- hospital cardiac arrest due to ventricular fibrillation Fig. 1 Survival to hospital discharge from out-of-hospital ventricular fibrillation in various communities outside the United States Fig. 2 The impact of instituting high-performance CPR in King County, Washington, in 2005 on survival from witnessed out-of- hospital cardiac arrest due to ventricular fibrillation Fig. 2 The impact of instituting high-performance CPR in King County, Washington, in 2005 on survival from witnessed out-of- hospital cardiac arrest due to ventricular fibrillation Fig. 2 The impact of instituting high-performance CPR in King County, Washington, in 2005 on survival from witnessed out-of- hospital cardiac arrest due to ventricular fibrillation Page 3 of 24 Kudenchuk et al. Ann. Intensive Care (2015) 5:22 only), and has demonstrated that it can improve outcome for virtually all presentations of cardiac arrest. inappropriate. Therefore, part of the observed reduction in the rate of unexpected cardiac arrest after the imple- mentation of a RRS is because the fraction of expected cardiac arrests is increased by placement of a DNAR order. While many of the identified disparities in cardiac arrest will continue to pose challenges for its manage- ment, a focus on improving systems-of-care, particularly deployment of high-performance letter-perfect CPR, offers the promise of a practical intervention that can be implemented immediately and an effort that promises to improve survival outcomes in any community. Another endpoint for measuring RRS effectiveness is the reduction of unplanned ICU admissions. The ration- ale is that the introduction of RRS should increase the number of ICU admissions that are planned early, before further deterioration occurs, and decrease those occur- ring as emergency admissions after resuscitation from cardiac arrest. This model has been indirectly demon- strated for ICU admissions from Emergency Depart- ment [11] where an earlier transfer to ICU has been demonstrated to decrease both ICU and hospital mor- tality. However, this is not always the case with RRS. In one large American before-and-after study [12] in which almost half of the MET interventions resulted in an ICU admission, the implementation of the RRS was followed by a reduction of non-ICU codes but it did not translate in a reduction of hospital mortality. In that study, mor- tality in patient transferred from ward to ICU was very high, and problems of patient selection, appropriateness and timeliness of ICU transfer have been advocated to explain these results.h Effectiveness of rapid response systems for prevention of cardiac arrest Cluster randomization is ethically acceptable, but difficult to implement because RRS intervention cannot be blinded, and contamination between the two study arms would be unavoidable, as did actually occur in the MERIT trial, the only randomized study conducted since now on RRS [14]. Another major implementation issue in that trial, as in general for RRS, was an afferent limb failure [15], due to an incomplete compliance of the ward personnel with the MET calling criteria. Fig. 3 The 25-year trend of rates of survival to discharge after resus‑ citation from in-hospital cardiac arrest in 100 observational studies, 1985–2010 Fig. 3 The 25-year trend of rates of survival to discharge after resus‑ citation from in-hospital cardiac arrest in 100 observational studies, 1985–2010 Page 4 of 24 Kudenchuk et al. Ann. Intensive Care (2015) 5:22 raised from 3 to 10 % during the intervention period. It is worth noting that the percentage of AED-use increased only from 1 to 2 %, which implies that the impressive improve- ment of outcomes of OHCA patients is rather due to prompt basic life support (as well as improved post-resuscitation care) than to AEDs. Similar initiatives are now being under- taken in Germany. Once CPR is started, its high quality is decisive for outcome. Compression depth must be sufficient (5–6 cm), frequency appropriate (100–120/min.), the chest must be released between compressions and interruptions of chest compressions must be kept as short as possible. Several studies have shown an association between these factors and survival [18–20]. During ALS by EMS-person- nel, further variables are part of CPR quality: in the OHCA- setting, automated chest compression devices have failed to prove superiority to manual CPR. There is still much debate on airway management and staffing of ambulance cars. Retrospective studies have shown higher survival rates in patients who received endotracheal intubation (ETI) as compared with extraglottic airways [21, 22]. In retrospec- tive studies, though, successful ETI might also be indica- tive of a generally higher skill-level of the EMS-personnel performing CPR. When using ETI during CPR, one needs to keep in mind that a high level of training is indispensa- ble and that prolonged ETI-attempts, which go along with long interruptions of chest compressions, must be avoided. Effectiveness of rapid response systems for prevention of cardiac arrest In a recent prospective, non-randomized trial, presence of a physician during CPR of OHCA-patients was associated with an impressively higher rate of survival than in patients being resuscitated by paramedics only [23], which confirms results of previous trials that observed higher survival rates in physician-staffed EMS [24]. Taken together, we can save thousands of lives each year if we manage to increase the likelihood of bystander-CPR and to optimize quality of ALS. A final issue is reproducibility. The vast majority of studies have been made in UK or Australian–New Zea- land systems, a minority of studies have been conducted in US and only very few studies have been conducted in other World areas as Continental Europe or Asia. The effectiveness of an RRS depends on the nature and the quantity of the urgent, unmet patients’ needs in general wards. This model may not work in places where the severity of patients in general wards, the education of the ward personnel or the resource availability is different from that of places where this model was developed. In summary, there are different ways of measuring the effectiveness of RRSs. The major include the rate of unexpected CA outside ICU, the rate of unplanned ICU admissions, and hospital mortality. All these outcome measures have limitations and are prone to bias. The level of evidence supporting the effectiveness of RRSs is rela- tively low and almost all studies have a before-and-after design. Despite the ethical and implementation difficul- ties, high-quality randomized trials are warranted to reli- ably assess the effectiveness of RRS. CPR quality has a deep impact y Hendrik Drinhaus and Bernd Böttiger Despite enormous efforts in recent years to improve quality of cardiopulmo- nary resuscitation (CPR) by the development of new CPR- guidelines and to enhance post-resuscitation care particu- larly by the introduction of mild therapeutic hypothermia and early coronary intervention, survival rates after OHCA remain unsatisfyingly low in many countries. As a prereq- uisite for successful post-resuscitation care on the Intensive Care Unit, high-quality CPR must be started as early as pos- sible. As advanced life support (ALS) by emergency medi- cal services (EMS) can only be expected to be commenced several minutes after OHCA, timely initiation of basic life support (BLS) by lay bystanders is crucial to ensure a timely perfusion of the brain and other vital organs. Sufficient per- fusion can only be obtained by high-quality CPR. Hence, it is vital to increase the percentage of bystander-CPR and to improve the quality of both BLS and ALS. According to the German Resuscitation Registry, bystander-CPR is per- formed in less than 20 % of OHCA cases [16], as opposed to 50 % or more in Scandinavian countries. Large-scale programmes raising public awareness of cardiac arrest and CPR as well as providing hands-on training in BLS are a promising tool to improve survival after OHCA. In a national initiative that included CPR-training as early as in primary school, telephone guidance to CPR by EMS- dispatchers, and distribution of automated external defibril- lators (AED) in Denmark [17], the rate of bystander-CPR could be increased from 21 to 45 % and 1-year survival What to perform prior to ICU admission? Percutaneous coronary intervention before hypothermia Hendrik Drinhaus and Bernd Böttiger Mild therapeutic hypothermia (MTH) and percutaneous coronary interven- tion (PCI) are both established components of post-resus- citation care after cardiac arrest, as recommended by the 2008 Statement of the International Liaison Committee on Resuscitation (ILCOR) [25]. Already then, it was suggested that indication for early PCI be extended beyond obvious myocardial infarction (STEMI), given the high probability of significant coronary artery disease in OHCA patients [26]. Since then, several trials have underlined the benefi- cial effects of early (<6 h after the event) PCI after OHCA also without STEMI [4, 27, 28]. Combination of PCI and MTH is feasible and does not necessarily lead to longer Kudenchuk et al. Ann. Intensive Care (2015) 5:22 Page 5 of 24 door-to-balloon times [29]. Its effects on survival and good neurological outcome appear to be synergistic [30, 31]. Hence, modern post-resuscitation care includes hypother- mia and early coronary intervention (unless a non-cardiac origin of cardiac arrest is assumed or confirmed). Induction of MTH is recommended to be started as soon as possible by the ERC guidelines. Meta-analyses of the existing data on very early and prehospital induction of MTH have not shown an improvement in survival [32]. In a recent large randomized controlled trial using large volumes of ice- cold saline to induce MTH before arrival at the hospital, no difference in survival or neurological outcome could be found, but patients who received cold saline had a slightly higher risk of renewed cardiac arrest during transport to the hospital and of transient pulmonary oedema [33]. In the light of the findings presented hitherto, we deem it impor- tant to stress the survival benefit of early PCI after cardiac arrest not only due to definite STEMI and to raise aware- ness among EMS personnel to transport OHCA-patients to hospitals in which early PCI, as well as other components of post-resuscitation care, can and will be performed at any time. Prehospital cooling (at least by infusion of cold saline) on the other hand appears not to convey a benefit, maybe even a risk, and hence, not too much time and effort should be spent on aggressively lowering temperature dur- ing transport using ice-cold saline solution. In any case, induction of MTH must not delay arrival at an appropriate hospital. What to perform prior to ICU admission? Percutaneous coronary intervention before hypothermia It appears worth considering to include into the guidelines on resuscitation and post-cardiac arrest care a recommendation to treat patients after cardiopulmonary in specialized centres that can provide early PCI, MTH and targeted temperature management as well as expert ICU- treatment wherever and whenever possible [34]. What to perform prior to ICU admission: is CT‑scan useful? Alain Cariou Early identification of causes and conse- quences of cardiac arrest is generally considered of impor- tance, in order to prevent recurrence and subsequent clinical deterioration. Apart from coronary angiography, a brain and chest CT-scan can also be performed at admission, when an extra-cardiac cause is suspected and in the absence of an obvious pre-hospital etiology. In a recent study, this strategy was performed in 355 patients and provided a diagnosis in 72 patients (20 %), mainly stroke and pulmonary embolism (PE) [35]. Early identification of brain damages can lead to major changes in therapy such as the use of anticoagulants. In addition, since the rate of subsequent brain death is very high in this subgroup, organ donation can be considered rapidly if an early diagnosis is achieved. Regarding PE, cur- rent guidelines underline the potential benefit of identifying this curable cause of arrest [36]. Based on prodromes and clinical evidence, an algorithm can be proposed in order to manage early imaging after cardiac arrest (Fig. 4). Thrombolysis in the treatment of cardiac arrest Hendrik Drinhaus and Bernd Böttiger In non-traumatic OHCA, acute myocardial infarction (AMI) and pulmo- nary artery embolism (PAE) account for approximately 70–80 % of all cases [37]. Common feature of these aeti- ologies is the obstruction of vital arteries by a blood clot. Dissolving these blood clots by administering fibrinolytic substances to re-establish circulation in the respective ves- sel beds therefore appears to be a logical approach from a pathophysiological point of view. Several case-reports and case-series have shown impressive results in patients with presumed or documented PAE [38, 39]. Improvement of return of spontaneous circulation (ROSC) and survival Fig. 4 Algorithm for early imaging diagnosis after cardiac arrest Fig. 4 Algorithm for early imaging diagnosis after cardiac arrest Fig. 4 Algorithm for early imaging diagnosis after cardiac arrest Kudenchuk et al. Ann. Intensive Care (2015) 5:22 Page 6 of 24 rates in patients who received thrombolysis during CPR has also been observed in non-randomised observational trials [40, 41]. CPR is better without epinephrine in cardiac arrest! Kjetil Sunde Whereas there is clear evidence for improved survival with CPR and defibrillation during cardiac arrest management, there is today lacking evidence that any of the recommended and used drugs leads to any long-term benefit for the patients. Thus, until we have better drugs or combination of drugs, ALS can be performed without the use of drugs, and instead gains all focus on improving the tasks we know improve survival. Good-quality CPR, early defibrillation together with goal-directed post-resuscita- tion care is way more important than giving drugs with no proven benefit [43]. More drug studies are indeed required, and future research needs to incorporate better diagnos- tic tools to test more specific and tailored therapies that account for underlying aetiologies and individual respon- siveness. We should expand our diagnostic capabilities exploring the feasibility of utilizing technologies such as capnography, near-infrared spectrophotometry (NIRS), VF analysis, and ultrasound assessment to allow targeted ther- apy (while maintaining adequate CPR). When good quality of care and improved diagnostics have been ensured, more tailored drug approaches could eventually be tested based on underlying etiologies. What to perform prior to ICU admission? Percutaneous coronary intervention before hypothermia A large randomised, double-blind, placebo- controlled trial—the TROICA-study—was therefore initi- ated to systematically evaluate thrombolysis during OHCA of presumed cardiac origin, not limited to suspected pul- monary artery embolism. In this trial, no difference with regard to ROSC or survival rates in an unselected OHCA- population could be detected. Intracranial haemorrhage occurred more frequently in the tenecteplase-group [37]. Hence, no recommendation to administer fibrinolytics as a standard of care in all OHCA-patients could be gener- ated from this trial. Accordingly, the current guidelines of the European Resuscitation Council recommend not to use fibrinolytics routinely during CPR. However, fibrinolytics should be considered if pulmonary embolism is the proven or suspected cause of cardiac arrest. In this case, cardio- pulmonary resuscitation should be maintained for at least 60–90 min after injection of the fibrinolytic, if necessary [42]. Taken together: if a coronary cause of cardiac arrest is assumed, fibrinolytic drugs should not be used and prompt coronary revascularisation, usually by percutaneous coro- nary intervention, is the treatment of choice—even during ongoing CPR. Fibrinolytics must be considered if pulmo- nary artery embolism is the suspected or proven cause of cardiac arrest. ECMO for cardiac arrest: ECMO is futile ECMO for cardiac arrest: ECMO is futile Martin Dworschak Extended time periods of no and low flow after CA are generally associated with poor out- come. Although return of spontaneous circulation can be achieved after more than 20 min of CPR, only few patients will survive with good functional outcome [44, 45]. By rapid deployment of ECMO in patients with refractory conventional CPR (CCPR) systemic blood flow can be maintained to prevent irreversible organ damage. The best results with extracorporeal CPR (ECPR) have been obtained so far in neonates and children when ECPR was instituted during in-hospital cardiac arrests that had short response times. The benefit of ECPR in adults being resuscitated for in- or OHCA is less clear. One major con- founder is the fact that ECPR is frequently considered for a highly selected patient population (young age, ventricular fibrillation as the initial cardiac rhythm, witnessed arrests with immediate bystander CPR) only [46]. Nevertheless, quoted survival rates after ECPR [47] are either compa- rable with those in CCPR patients [48] or slightly better, but this does not seem to impact on neurological outcome [46, 49]. Accordingly, in 2010 the American Heart Asso- ciation did not recommend the routine use of ECPR [50]. What to perform prior to ICU admission? Percutaneous coronary intervention before hypothermia Yet, it could be taken into consideration when it is readily available and the no-flow duration is brief. Furthermore, the conditions that led to the arrest should either be revers- ible or amenable. Particularly in OHCA, however, most patients are neither young nor hypothermic or intoxicated and they do not present with a shockable rhythm; fewer CAs are witnessed and the quality of BLS/ALS is usually unknown. Another handicap in OHCA appears to be fast deployment of ECMO with quick institution of therapeu- tic hypothermia [51]. Although ECPR seems to improve survival, especially after long-duration CPR, the rate of neurologically intact survivors still remains low. Future research should define criteria for the optimal indication of ECPR and criteria for ECPR as a bridge to LVAD, HTX, or organ procurement to guarantee efficient use of precious and scarce resources. Randomized controlled trials, as well as crucial analysis of uniformly reported CA data from reg- istries would greatly facilitate decision-making. Post res scitation management targeted temperat re How to cool? Fabio Taccone Several cooling methods, both invasive and non-invasive, are currently used to achieve target tem- perature after post-anoxic brain injury. Invasive methods include the administration of intravenous cold fluids or the use of endovascular cooling catheters [52]. The use of Page 7 of 24 Kudenchuk et al. Ann. Intensive Care (2015) 5:22 are quite safe, are not dependent on an external energy source and can be initiated even during cardiopulmonary resuscitation [63]. Alternatives methods for cooling could be continuous renal replacement therapy, peritoneal lav- age and, in case of severe cardiogenic shock, the use of extracorporeal membrane oxygenation (ECMO) devices; however, these methods are particularly invasive and their use has been limited to very selected cases [64–66]. cold (4 °C) fluid infusion is cheap and easy-to-use, even in the pre-hospital setting. This method is generally rec- ommended to initiate cooling in comatose survivors after CA, either alone or in conjunction with other cooling sys- tems [53]. A 2-l bolus given over 30–60 min immediately after ROSC was associated with a mean decrease in core temperature of 1.3 °C [54]. This method is largely used also to induce hypothermia in the fields [33]. However, cold fluids are not effective to maintain target temperature [55] and, because of potential excessive volume-loading and reduced coronary perfusion pressure [56], have been recently associated with an increased risk of re-arrest and pulmonary oedema when given before hospital admission [33]. The endovascular cooling catheter contains a circu- lating cold solution, which is maintained at a controlled temperature; this method can easily achieve a cooling rate of 1.5–4.5 °C/h [52]. Moreover, the use of an endovascular system reduced the variability of body temperature around the target value and increased the proportion of time that patients spent within the therapeutic temperature targets during the maintenance phase [57]. Main limitations are related to the time to insertion into a large vein, the need of a bedside heat exchanger with energy supply (i.e. lim- ited used outside the ICU) and the potential risk of venous thrombosis or infection [58]. Finally, no prospective study has shown a clinical superiority of one method to another, while some retro- spective reports suggest a better survival rate for endo- vascular cooling when compared to surface cooling methods [67, 68]. For how long? l S d Kjetil Sunde The precise description of patients that will benefit from therapeutic hypothermia (TH), or tar- geted temperature management (TTM), the ideal induction technique (alone or in combination), target temperature, optimal maintenance and rewarming times have yet to be established. Independently of the cooling method chosen, TH is easy to perform, and without severe side effects or complications associated with mortality. As for the dura- tion of TH/TTM, the majority of centers use 24 h as their treatment strategy; however, we are lacking of clinical data comparing different durations. Newborn asphyxial arrests are treated with TH for 72 h, with several RCTs proving its benefit compared to normothermia/fever. In a recent ani- mal study, post-cardiac-arrest TH resulted in comparable improvement of survival and survival with good neurologic function when initiated within 4 h after return of sponta- neous circulation. Interestingly, histological assessment of neuronal survival revealed a potentially broader thera- peutic window and greater neuroprotection when TH was maintained for 48 versus 24 h [71]. However, this benefit was not seen in the neurological outcome evaluation. In a clinical registry study among approximately 1000 cooled comatose cardiac arrest patients, factors related to the tim- ing of TH had no apparent association to outcome (Fig. 5). Non-invasive methods include external surface and ice packs or pads. Modern cooling blankets or fluid pads usually operate with a continuous temperature feedback mechanism, which reduced the risk of temperature vari- ability and overcooling, and present a cooling rate around 1.2–2.5 °C/h in CA patients [52, 59]. Unfortunately, these devices also depend on an external energy sup- ply and it remains difficult to use them outside the ICU. Ice packs can be easily applied to different areas of the body, are inexpensive and are not dependent on an exter- nal energy source; however, the cooling rate is extremely poor (<1 °C/h) and could expose patients to overcooling, as there is no feedback temperature control. Ice pads can provide a faster cooling rate (up to 3 °C/h) after CA, but they have been associated with thermal skin damage on the sites of application [60]. An attractive alternative to these methods is to induce brain hypothermia, especially because of the decreased risk of side effects associated with whole-body cooling. How to cool? Importantly, the endovascular catheter and modern surface devices have significant higher costs than intravenous fluids and ice packs [69]; however, their use was associated with an important reduction of nurse workload and improvement in patients’ care [70]. For how long? l S d Cranial cooling cap devices placed around the head and neck can easily decrease tympanic temperature; how- ever, they are mostly effective in children because of their favourable ratio of head-to-body surface area [61]. Nasopharyngeal cooling devices, which use volatile cool- ant fluids via a specific nasal catheter, can rapidly reduce brain temperature with a concomitant reduction of core temperature around 1.0–1.5 °C/h [62]. These systems Any evidence for non‑shockable rhythm patients? Thus the ben- eficial effect observed for in-hospital mortality was no longer significant when analysis was restricted to the two small RCTs available. Another meta-analysis similarly concluded that the group sizes for patients with asys- tole or non-cardiac causes of CA were too small to draw conclusions. At least, several recent non-randomized controlled studies—after adjustment in some studies— reported negative or even harmful TH-effects on out- come in this population [74, 80–82]. Similarly, with a TH implementation in nearly 50 % of patients and a global hospital survival of 2 % in the non-shockable rhythm group, TH was not significantly associated with hospital survival after adjusting for other prognostic factors [75]. The pro-con debate on the use of TH in non-shockable patients has been previously described [74]. Briefly, some argue in favour of the use of TH in non-shockable CA patients: (1) the pathophysiological processes responsi- ble of post-anoxic (brain) damages seem independent of the initial rhythm (i.e. shockable versus non-shockable); (2) most of experimental and animals studies strongly support the use of TH in asphyxia and CA models irre- spective of the initial rhythm; (3) with more than 10 concordant large randomized controlled trials and meta- analyses, TH initiated within 6 h and targeted to 32.5– 35.5 °C for 72 h is unequivocally beneficial on survival and neurological outcome in newborns who sustained asphyxia (with or without cardiopulmonary resuscita- tion: CPR) and exhibit acidosis and/or neonatal hypoxic- ischaemic encephalopathy [73, 74, 78]; (4) TTM with TH is potentially the unique available treatment to date prone to minimize brain damages and long-term dis- abilities, and to increase survival without major sequelae in non-shockable CA patients [74]; (5) some observa- tional, retrospective or prospective, but non-randomized Beyond ethical considerations regarding the TH- related futility/benefit ratio, some major issues remain unsolved regarding the ideal TTM in this specific population. The correct selection of patients that could benefit or not from TTM or TH-implementations seems critical in this heterogeneous group, as well as the underlying co- morbidities and severity of the post-resuscitation syn- drome [74]. Indeed an initial non-shockable rhythm may represent: (1) either the first documented CA rhythm resulting from a severe non-cardiac aetiology with its own prognosis per se; (2) or the consequence of an initial shockable CA with a prolonged time to first CPR leading to prolonged ischaemic times, severe brain damages and/ or multiple organ failures. Any evidence for non‑shockable rhythm patients? Nicolas Deye According to international guidelines, therapeutic hypothermia (TH) might also benefit coma- tose adult CA patients with spontaneous circulation after resuscitation from a non-shockable rhythm [72]. However, there is no large randomized controlled trial evaluating the clinical impact of TH in this situation. Using TH for non- shockable CA patients is supported by only low level of Page 8 of 24 Kudenchuk et al. Ann. Intensive Care (2015) 5:22 Fig. 5 Therapeutic hypothermia side-effects and their categorization clinical studies observed a benefit of TH in non-shocka- ble CA adults patients, with one meta-analysis suggesting a reduced in-hospital mortality without improvement of the neurological outcome in these patients. Conversely, the potential increased TH-related side effects are not in favour of the use of TH in non-shock- able CA patients. Adverse events include the increased delay in time to recovery of consciousness leading to a prolonged delay to evaluate the neurological recovery. This delay seems mainly induced by the use of sedatives and neuromuscular blockers often required during the TH use, leading to a possible prolongation of mechani- cal ventilation and hospitalization durations, and conse- quently of ICU costs. The increased incidence of other main side effects, essentially pneumonia and sepsis, could be another explanation altering the risk/benefit ratio of TH, although neither firmly established nor related to the initial CA rhythm [74, 76, 79]. Fig. 5 Therapeutic hypothermia side-effects and their categorization scientific evidence and extrapolation of data resulting from shockable rhythms. Consequently, the potential impact of TTM including TH-implementation remains controver- sial for CA patients presenting with initial non-shockable rhythm [73, 74]. Treating such patients becomes a major health issue, as the proportion of these patients increases over decades while the proportion of OHCA patients resuscitated from shockable rhythms declines and now represents the minority of OHCA patients [75]. Prognosis of patients experiencing CA still remains poor. However, large registries reported recently for non-shockable CA patients hospitalized in ICU survival up to 26 %, favour- able neurological outcome at 6 months of 22 %, and sur- vival rate at 10 years after hospital discharge alive reaching 43 % [76, 77].h Additionally, several studies describing non-shockable CA patients have not shown significant prognostic effect of TH [74]. Most of the studies included in the first meta- analysis evaluating this issue presented substantial risks of bias and a very low quality of evidence. Hypothermia‑associated complications Alain Cariou Besides infections, several other adverse events such as arrhythmias, seizures, bleeding or thrombo- sis, electrolyte and metabolic disorders, occur commonly in comatose patients treated in critical care units after out- of-hospital cardiac arrest. These events may be related to the cause precipitating the cardiac arrest, the post-cardiac arrest syndrome or the critical care treatment. As it may affect many physiologic processes and responses, hypo- thermia itself is commonly suspected to promote these events (Fig. 2) [87]. However, most of these events are probably not related to TH, as suggested by the results of a recent Cochrane systematic review that revealed no significant difference in reported adverse events between hypothermic and control patients [88]. Regarding meta- bolic disorders, rapid changes in glycemia are possibly the most clinically relevant event that could be worsened by hypothermia [89, 90]. As it may worsen brain damages, it suggests minimizing glycaemic variations during the post- resuscitation period. In addition, TH most often requires sedation, ventilation, and neuromuscular blockade, which may delay the possibility of neurological evaluation. At last, the aetiology and/or mechanisms respon- sible for fever are possibly a key issue to correctly evaluate patients able to receive a TTM or a TH imple- mentation, especially after CA occurrence in non-shock- able patients. In a large population of unselected ICU patients, fever was not always associated with a poor prognosis and control of fever was not always associated with better prognosis, with important differences exist- ing between septic and non-septic patients [84]. Post- CA syndrome often mimics a sepsis-like syndrome. The correct discrimination of patients who may benefit for a strict normothermic control, those who may benefit for a TH implementation targeted to 32–35 °C, versus those who may—or not—benefit for a fever control within the 48–72 h after CA to avoid rebound hyperpyrexia, is the next step to optimize the temperature control after CA in the non-shockable population [74, 85].h The choice of the correct target temperature as the precise achievement of goal temperature seems also crucial in these patients. The recommended treatment for all post-CA patients is at present an early TH-imple- mentation of 12–24 h targeted to 32–34 °C [72]. Hypothermia‑associated complications An adapted scheme of TTM could be of critical importance in non-shockable patients, regarding the optimal dura- tion, speed cooling, level of temperature, therapeutic window, and rewarming rate of TTM, considering that the cerebral damages may be more severe in this popula- tion [72, 74]. Interestingly, in the large recent TTM-trial including 20 % of patients presenting with an initial non- shockable rhythm out of a total 939 enrolled patients, no difference were found between the 2 studied arm, i.e. 33 versus 36 °C applied for 28 h. The TTM-scheme included in both groups a gradual rewarming <0.5 °C/h to reach 37 °C, with tapering or discontinuation of the mandatory sedation at 36 h, and a maintenance of body Any evidence for non‑shockable rhythm patients? A non-prolonged time from collapse to return of spontaneous circulation (i.e. sum of the time to first CPR plus the duration of CPR) could be Page 9 of 24 Kudenchuk et al. Ann. Intensive Care (2015) 5:22 a relevant prognosis factor able to identify patients who may mostly benefit from cooling independently of the initial rhythm. Conversely in witnessed-OHCA patients, the more prolonged is the time from collapse to first CPR the better could be the TH-benefit after adjustment on other factors including the initial rhythm. The target pop- ulation in non-shockable patient probably results from a complex balance between patients that can benefit from TH (i.e., those with TH-accessible brain damages with adequate durations of time to first CPR and CPR dura- tions) versus those who do not (i.e., those with multiple organ failure or too severe prognosis that could lead to early death or early treatments withdrawal). Addition- ally, the TH-implementation in non-shockable patients is often left at the bedside physician’s discretion in most studies evaluating this issue, and important bias should be introduced regarding the correct selection of patients [74, 83]. temperature for unconscious patients <37.5 °C until 72 h after CA [86]. Similar results were herein observed in all pre-planned subgroups including analyses performed according to initial rhythm (i.e. non-shockable versus shockable), suggesting that “strict normothermia” tar- geted to 36 °C should be an alternative in non-shockable rhythm CA patients. This ongoing debate clearly underlines the need for a large multicentre study evaluating the effects of different scheme of TTM and TH in non-shockable CA patients after careful patients’ selection, including insights in sub- groups according to the pathophysiology of the CA. To date, the indication for TH in non-shockable patients should be based using a case-by-case approach, whereas a TTM approach targeting strict normothermia remains reasonable for these patients. What is a real targeted temperature management: TTM at 36 °C Hans Friberg In 2002, two landmark trials were pub- lished showing that lowering body temperature to 33 °C improved neurological outcome and saved lives in coma- tose survivors of out-of-hospital cardiac arrest [52, 91]. This therapy was rapidly adopted by international guide- lines and not questioned until a systematic review using the GRADE methodology and trial sequential analysis showed that the quality of the evidence for treating cardiac arrest patients at 33 °C was low [86]. Criticism included the low number of enrolled patients and the fact that both trials had Kudenchuk et al. Ann. Intensive Care (2015) 5:22 Page 10 of 24 Fig. 7 The TTM-trial included and randomized 950 comatose patients to either 33 or 36 °C. Probability of survival through the end of the trial and the number of patients at risk at each time point is presented. From Nielsen et al. [86]. Copyright © (2013) Massachusetts Medical Society. Reprinted with permission weaknesses, including quasi-randomization, lack of power analysis and early stopping which was not adjusted for. Furthermore, patients in the control groups were treated according to standard care of that time which allowed fever [92]. This led to the design of a new trial, randomizing patients to two controlled temperatures, 33 and 36 °C [93]. The Target Temperature Management after Out- of-hospital Cardiac Arrest trial (TTM-trial) included and randomized 950 patients in 27 months, almost three times the number of the two previous trials combined. Thirty-two sites enrolled patients in nine countries in Europe plus Australia. The TTM-trial differs from pre- vious trials in several aspects, the most important one being that all patients received controlled temperature management avoiding fever in both intervention arms (Fig. 6). Other differences include a contemporary setting with improved pre-hospital care with a high bystander CPR rate, and improved hospital care in patients with ROSC. For example, two of three patients in the TTM- trial received an early angiography (<24 h). Other qual- ity criteria that were acknowledged in the TTM-trial, as opposed to previous trials, were that the design and the statistical analysis plan were published in advance. [93, 94] In addition, prognostication of comatose patients was performed according to a strict and predefined protocol and the rules for decisions on withdrawal of life-sustain- ing therapy (WLST) were transparent and published in advance [90]. The primary outcome was survival until the end of trial. What is a real targeted temperature management: TTM at 36 °C A neurological assessment, using both the Cerebral Performance Category scale (CPC) and the modified Rankin Scale (mRS), was performed at 6 months and was blinded. Fig. 7 The TTM-trial included and randomized 950 comatose patients to either 33 or 36 °C. Probability of survival through the end of the trial and the number of patients at risk at each time point is presented. From Nielsen et al. [86]. Copyright © (2013) Massachusetts Medical Society. Reprinted with permission Fig. 7 The TTM-trial included and randomized 950 comatose comatose patients after cardiac arrest were treated at either 33 or 36 °C [86]. We can therefore conclude that a real target temperature management after cardiac arrest is a controlled temperature at either of the two tempera- tures, both avoiding fever. This should be part of a bun- dle of care, including treatment of acute coronary disease and contemporary and active intensive care. Our rationale for changing to 36 °C is that this tem- perature is closer to normal and less invasive, reducing known and unknown risks. The number and severity of adverse events in the two intervention arms did not dif- fer in the TTM-trial, although there was a tendency towards less events in the 36°-arm (p = 0.086). Treat- ing the patients closer to normal temperatures allows for predictable drug effects, including those of sedatives and antithrombotic therapy. In a recently published post hoc analysis of the TTM-trial of the subgroup of patients in circulatory shock at admission, a higher mortality at ICU discharge was suggested in the 33 °C-arm (66 ver- sus 44 %, adjusted p value 0.03) [95]. We cannot exclude that other subgroups may benefit from treatment at 36 °C or for that matter, from treatment at 33 °C. The optimal temperature, duration of temperature management and target population are yet to be defined. In summary, the TTM-trial has sent a clear message to the medical community that no difference in survival (Fig. 7) or neurological outcome could be detected when Fig. 6 Mean bladder temperature in the 33 and 36 °C intervention groups of the Target Temperature Management after Out-of-hospital Cardiac Arrest Trial (TTM-trial), during the 36 h of temperature intervention. Temperature values are presented with 95 % confidence intervals. In the original publication, temperature curves displayed the means ± 2SD. From Wise MP et al. [179]. Reprinted with permis‑ sion Fig. What is a real targeted temperature management: TTM at 36 °C 6 Mean bladder temperature in the 33 and 36 °C intervention groups of the Target Temperature Management after Out-of-hospital Cardiac Arrest Trial (TTM-trial), during the 36 h of temperature intervention. Temperature values are presented with 95 % confidence intervals. In the original publication, temperature curves displayed the means ± 2SD. From Wise MP et al. [179]. Reprinted with permis‑ sion Fig. 6 Mean bladder temperature in the 33 and 36 °C intervention groups of the Target Temperature Management after Out-of-hospital Cardiac Arrest Trial (TTM-trial), during the 36 h of temperature intervention. Temperature values are presented with 95 % confidence intervals. In the original publication, temperature curves displayed the means ± 2SD. From Wise MP et al. [179]. Reprinted with permis‑ sion Post‑resuscitation management: optimizing organ perfusion and metabolic parameters Targeted tempera- ture management (TTM), including therapeutic hypother- mia (TH) targeted to 32–34 °C, is one of the therapeutic measure improving prognosis and neurological outcome after CA. Induced hypothermia could minimize the CA- related injuries by decreasing O2 free-radical production, mitochondrial dysfunction, brain O2 consumption [96]. Thus TH could minimize neuronal death and improve neu- rological outcome and survival. However, hypothermia is associated with several arterial blood gas (ABG) modifica- tions, mainly induced by the leftward shift of the hemo- globin dissociation curve, increased O2 and CO2 solubility, modification of the pH regulation, with hypoventilation and hypometabolism, leading per se to hypoxia and hypocap- nia. Iatrogenic dyscarbia incidence (hypo- or hyper-carbia) has been reported up to 69 % [97], hypoxia up to 63 % [98], and hyperoxia up to 41 % [99]. International guide- lines mainly focused on potential hypocapnia and hypoxia/ hyperoxia harmful effects for CA patients [72, 100]. They advocate initial resuscitation with 100 % O2 ventilation to avoid hypoxia followed by a titration of O2 therapy target- ing arterial O2 saturation levels between 94 and 96(−98) % especially during the initial post-CA period to avoid hyper- oxia. y Most clinical studies depicting the O2 effects after ROSC from CA are methodologically of low level quality. The only small randomized clinical trial evaluating this issue found a significant decreased value of the Neuron Specific Enolase biomarker in favor of a normal level of PaO2 versus a high level PaO2 in the subgroup of patients without TH [105]. However, this result was not observed for the S100B Protein biomarker and in the whole cohort including the 28 patients regardless TH implementa- tion. Hypoxia and hyperoxia harmful effects regarding in-hospital mortality and functional status were initially described in a large American registry in 2010 [98]. Sev- eral issues have been pointed out for these studies: (1) the statistical methods (registries and databases versus scarce randomized studies, retrospective versus prospective studies, single center versus multicentre studies, adjust- ment not usually performed to control for other poten- tial confounders); (2) the definitions regarding hyperoxia thresholds (i.e. what precise level of PaO2 to choose?); (3) the different time-point measurements and the period of data collection (i.e. Post‑resuscitation management: optimizing organ perfusion and metabolic parameters Oxygen and carbon dioxide after cardiac arrest: friend or foe? Nicolas Deye In CA patients, neurological injury is a major cause of mortality [72]. A first hypoxo-anoxia phe- Page 11 of 24 Kudenchuk et al. Ann. Intensive Care (2015) 5:22 nomenon occurs initially during CA before and during CPR maneuvers. The ischemia–reperfusion syndrome occurring in the post-resuscitation phase after obtaining an ROSC can lead to secondary insults including oxidative stress with free radical formation or mitochondrial dys- function [72, 96]. Oxygen (O2) and carbon dioxide (CO2) abnormalities can promote these insults. Targeted tempera- ture management (TTM), including therapeutic hypother- mia (TH) targeted to 32–34 °C, is one of the therapeutic measure improving prognosis and neurological outcome after CA. Induced hypothermia could minimize the CA- related injuries by decreasing O2 free-radical production, mitochondrial dysfunction, brain O2 consumption [96]. Thus TH could minimize neuronal death and improve neu- rological outcome and survival. However, hypothermia is associated with several arterial blood gas (ABG) modifica- tions, mainly induced by the leftward shift of the hemo- globin dissociation curve, increased O2 and CO2 solubility, modification of the pH regulation, with hypoventilation and hypometabolism, leading per se to hypoxia and hypocap- nia. Iatrogenic dyscarbia incidence (hypo- or hyper-carbia) has been reported up to 69 % [97], hypoxia up to 63 % [98], and hyperoxia up to 41 % [99]. International guide- lines mainly focused on potential hypocapnia and hypoxia/ hyperoxia harmful effects for CA patients [72, 100]. They advocate initial resuscitation with 100 % O2 ventilation to avoid hypoxia followed by a titration of O2 therapy target- ing arterial O2 saturation levels between 94 and 96(−98) % especially during the initial post-CA period to avoid hyper- oxia. alive in multivariate analysis [104]. Incidence of hyper- oxia defined as PaO2 >300 mmHg reached 14 %, whereas hypoxia was defined as PaO2 <60 mmHg. The potential deleterious effect of hypoxia during the resuscitative efforts has been also found in another recent retrospec- tive study [100]. nomenon occurs initially during CA before and during CPR maneuvers. The ischemia–reperfusion syndrome occurring in the post-resuscitation phase after obtaining an ROSC can lead to secondary insults including oxidative stress with free radical formation or mitochondrial dys- function [72, 96]. Oxygen (O2) and carbon dioxide (CO2) abnormalities can promote these insults. Post‑resuscitation management: optimizing organ perfusion and metabolic parameters A “U-shaped” relationship between the mean PaCO2 and the in-hospital mortality was found with the best survival observed for the normocapnia group (35–45 mmHg). No association between hypo- and hyper-capnia with poor neurologic outcome were observed. Another recent multicenter and prospective study applying TH in 71 % defined PaCO2 as “low” when <30 mmHg, “middle” when 30–37.5 mmHg, “intermedi- ate” when 37.5–45 mmHg, and “high” when >45 mmHg [107]. Patients with poor versus good outcome had simi- lar highest, mean and lowest PaCO2. The mean 24-h PaCO2 and the time spent in PaCO2 >45 mmHg regard- less TH-implementation were independently associated with better 1-year good outcome. However, same criti- cisms can be made for all these studies than those made above regarding statistics, threshold, data collection, or treatments. To date mainly because of paucity of data on this issue, no clear thresholds have been found regard- ing the harmful impact of hypo- or hyper-capnia after g ABG measurements performed at 37 °C are sec- ondarily expressed either as temperature-corrected or uncorrected according to biochemical centers [108]. Substantial discrepancies can be related to these meth- ods: PaO2 = 100 mmHg at 37 °C becomes 79 mmHg if corrected at 33 °C, when PaCO2 = 36 mmHg becomes 30 mmHg at 33 °C. There are no clear recommendations regarding the ventilation strategy to be used in resusci- tated CA patients despite such differences. Normocap- nia in hypothermic patients can be achieved according to two different mechanical ventilation strategies: α-stat versus pH-stat. In the α-stat strategy ventilation is set to achieve physiological arterial CO2 tension measured at 37°, unadjusted to the patient’s temperature, whereas in the pH-stat strategy ventilation is set to achieve physi- ological arterial CO2 tension measured at the patient’s actual temperature. The latter strategy leads to a relative hypoventilation compared to the α-stat strategy. Using either α-stat or pH-stat strategy to guide normocapnia after CA remain open to discussion. In the recent mul- ticenter Finnish study focusing on ABG abnormalities after CA, 13 ICUs used temperature-correction; whereas, eight did not [107]. Two recent exploratory studies in TH-treated CA patients compared either a “lower versus upper threshold normocapnia” (32 versus 45 mmHg) or the α-stat versus the pH-stat strategies while maintaining a PaCO2 target level between 36 and 42 mmHg. Post‑resuscitation management: optimizing organ perfusion and metabolic parameters In a retrospective cohort including 145 adult OHCA patients with available arterial O2 pressure (PaO2) on ABG sample during CPR attempts, an increased PaO2 was associated with improved rate of hospital admission Page 12 of 24 Page 12 of 24 Kudenchuk et al. Ann. Intensive Care (2015) 5:22 with worse outcome, hypercapnia is not [100]. The man- agement of PaCO2 after CA by using specific mechanical ventilation strategy could influence the outcome of these patients especially during TH. In a recent retrospective study focusing on initial post-CPR mechanical ventila- tion settings minute ventilation was weakly correlated with the initial PaCO2 [110]. Normocapnia alone (31– 49 mmHg) was again associated with a better favorable neurological outcome. Cerebral autoregulation physiologically maintain a con- stant CBF within a large range of mean arterial pressure [100, 108]. This relationship is modified by dyscarbia as cerebral perfusion depends on CO2. Hypercapnia leads to cerebral vasodilation and potentially increased intracra- nial pressure, whereas hypocapnia leads to cerebral vaso- constriction and potentially ischemia (decreasing PaCO2 of 1 mmHg can decrease CBF up to 3 %). Ensuring physi- ological CO2 tension in CA patients seems important to prevent worsening of the neurological status. Further- more, impaired autoregulation has been described in some brain-injured areas, in some TH-treated patients, and inconstantly in CA patients. Recommendations in resuscitated CA patients suggest a PaCO2 target of 40–45 mmHg during the post-ROSC period in this popu- lation of brain-injured patients regardless of TH use [72, 100]. In a retrospective single-center study implementing TH in 41 %, hypocapnia (defined by PaCO2 ≤30 mmHg), hypercapnia (PaCO2 ≥50 mmHg), and association of both dyscarbia occurring within the first 24 h after ROSC were all independently associated with poor neu- rologic outcome at hospital discharge [97]. An observa- tional multicenter registry recently enrolled 16.542 adult CA patients TH-treated in 39 %. Increased in-hospital mortality and rate of poor outcome were observed in the hypocapnia group compared to normocapnia after adjustment for illness severity and propensity score [109]. Conversely, hypercapnia (PaCO2 ≥45 mmHg) within the first 24 h after admission was independently associated with similar in-hospital mortality and a higher rate of dis- charge home among survivors. In a smaller study of TH- treated patients, hypocapnia but not hypercapnia was independently associated with an increased risk of in- hospital death [106]. Post‑resuscitation management: optimizing organ perfusion and metabolic parameters what value to consider between the first ABG, the ABG on admission, the ABG within the first hours or the first day after ROSC or CA, between the mean or median versus the maximal or minimal PaO2 values or the worst PaO2 using worst (A-a) DO2 or the PaO2/FiO2 ratio?); (4) the associated treatments (i.e. TH versus the absence of TTM); (6) the best endpoint (i.e. in- hospital mortality versus neurological outcome). Beyond general detrimental effects such as decreased stroke volume, cardiac output and coronary blood flow and increased systemic vascular resistances, poten- tial neurological harm of O2 therapy have been largely described [100, 101]. Hyperoxia can exacerbate cellular oxidative stress injury and mitochondrial dysfunction to key mitochondrial enzymes or mitochondrial lipids, leads to cerebral O2-related vasoconstriction with a decreased cerebral blood flow (CBF) and cerebral energy metabolism impairment. It can increase the neuronal lipid peroxida- tion and protein oxidation, enhance O2 free radical for- mation and reactive O2 species, or react with nitrite oxide to produce toxic metabolites (peroxynitrite, superoxide ion, hydrogen peroxide). All of this will finally participate to cell death. Despite several limitations or controversial results in specific experimental models [100–102], most animal studies have suggested that hyperoxia after CA could worsen neurological outcome [103]. p y g A recent meta-analysis concluded that hyperoxia in the post-resuscitation phase after ROSC was significantly associated with an increased in-hospital mortality [99]. Conversely, the poor neurologic outcome at hospital dis- charge did not reach significance suggesting a possible lack of association or a review underpowered. However, these results need further confirmation because of its sig- nificant heterogeneity (results were inconsistent in sub- group and sensitivity analyses) and the limited number of studies analyzed (3 abstracts out of the 10 pooled studies were finally included). Since this review, two other stud- ies describing a low hyperoxia incidence rate (3 and 6 %) have been published [106, 107]. The potential harmful effect of hyperoxia regarding mortality was not signifi- cant. In only one of these 2 studies [106], hyperoxia was significantly associated with poor neurological outcome in the multivariate analysis, depicting a “V-shaped” rela- tionship between probability of unfavorable outcome and the mean PaO2 value obtained from ROSC to rewarming. Other means of cardio‑ and neuro‑protection Fabio Taccone Heart and brain protection strategies aim to prevent or attenuate disease progression and secondary injuries by halting or at least slowing the loss of cardio- myocytes and/or neurons [111]. Although many pathologi- cal mechanisms, including endothelial damage and tissue hypoperfusion, inflammation, impaired mitochondrial res- piration with induction of reactive oxygen species, calcium overload or excitotoxicity, are common in both cardiac and neurological injury following CA [112], some drugs may present a more specific organ protection or have a lower brain penetration, thus acting especially on the cardiac tis- sue. Many therapeutic agents have been shown to be effec- tive in protecting the heart and/or the brain in animal mod- els of global or local ischemia [113]; nevertheless, these findings were flawed because of the differences between the experimental setting and the clinical scenario (i.e. absence of co-morbid diseases and need for anesthetics in animal studies), the low mortality rates or the administra- tion of the specific drug before the development of injury (i.e. pre-treatment approach). g Reperfusion injury leads to mitochondrial dysfunc- tion also through the opening of a nonspecific pore in the inner mitochondrial membrane, known as the mito- chondrial permeability transition pore (MPTP) [127]. This phenomenon causes the loss of ionic homeostasis and ultimately cell swelling and death. The inhibition of the MPTP opening may provide some protection against reperfusion injury; importantly, this may be mediated by a direct interaction of cyclosporine A (CsA) with a pro- tein located on the MPTP, called cyclophilin-D [127]. In a murine model of CA, CsA was effective in reducing myocardial dysfunction when given at the onset of resus- citation but not after ROSC [128]. In one human study conducted in patients suffering from acute myocardial infarction, the administration of a 2.5 mg/kg bolus of CsA before percutaneous coronary intervention was associ- ated with a significant reduction in biomarkers of myo- cardial injury (i.e. troponin I) and the extent of ischemic areas on cardiac magnetic resonance imaging [129]. Nowadays, no clinical studies have evaluated the effects of CsA on the neurological recovery of CA survivors yet. A promising approach to reduce brain excitotoxic- ity (i.e. excessive extracellular glutamate levels) could be the administration of intravenous magnesium or inhaled noble gases (i.e. xenon or argon). In two studies, magne- sium administration did not result in a better survival or neurological outcome for CA patients [114, 115]. Post‑resuscitation management: optimizing organ perfusion and metabolic parameters The first study showed that the “lower threshold normocapnia” induced decreased internal jugular vein O2 saturation and CBF mean velocity suggesting an increased risk of cerebral ischemia. The second study found that the alpha- stat strategy increased jugular vein desaturation and cer- ebral O2 extraction and decreased transcranial Doppler cerebral velocities in survivors but not in non-survivors. In summary, dyscarbia especially hypocapnia should be associated with an increased harm after CA leading to increased unfavorable outcome or in-hospital mortality. Conversely O2 seems a two halves phenomenon with: (1) a former deleterious effect of hypoxia during resuscita- tion efforts, the potential benefit of hyperoxia implying more evaluations; (2) followed by a deleterious effect of both hypoxia and hyperoxia after ROSC in the post-CA phase. To date because O2 and CO2 derangements have no obvious benefit, aiming at normoxia and normocapnia Page 13 of 24 Page 13 of 24 Kudenchuk et al. Ann. Intensive Care (2015) 5:22 hematopoietic hormone regulating erythropoiesis, which shows also anti-apoptotic, anti-inflammatory and anti-oxi- dant properties [122]. In a swine model of VF, high-dose EPO administration during CPR reduced post-resuscita- tion myocardial dysfunction and improved cardiac func- tion [123]. However, post-ischemic EPO administration in rats exposed to CA exerted no protective effect on hip- pocampal neurons [124]. One human study has compared the effects of 90.000 UI of EPO given during CPR to an historical matched control group [125]. The EPO group had higher rates of ROSC (92 versus 53 %, p = 0.006) and hospital survival (54 versus 20 %, p = 0.011) when com- pared to the control group. In another study [126], EPO therapy was associated with a trend towards higher full neurological recovery (55 versus 38 %) when compared to an historical cohort. Unfortunately, a recent unpublished randomized clinical trial (RCT) found no benefits of EPO administration on comatose survivors after CA when compared to placebo (Cariou et al.—presented at the 27th ESICM congress—Barcelona, October 2014). after CA are potentially two very easy targets to achieve in post-resuscitation care bundles. Meanwhile all stud- ies describing ABG parameters in TTM- or TH-treated CA patients should actually emphasize the strategy used, either a temperature-corrected or a non-corrected strat- egy to more precisely evaluate potential thresholds. Neurological assessment of brain damages Minimal consciousness: classification and prognosis in CA patients Steven Laureys and Didier Ledoux Successful resuscita- tion after cardiac arrest or cerebral hypoxia can have an extremely wide variety of functional and cognitive conse- quences, ranging from transient cognitive or motor dys- function lasting several weeks or months; to awakening after coma in a state of unawareness or inability to com- municate lasting months to years or decades (e.g. as seen in vegetative or minimally conscious states) [137]. At pre- sent, the medical and economic impacts of differences in clinical management (including therapeutic hypothermia), care pathways and disparate health policies within Europe remain unclear. wakefulness syndrome” (PVS/UWS) [139] or without recovery of functional communication or functional object use, a condition referred to as “minimally con- scious state” (MCS) [140]. In contrast to coma, such chronic disorders of consciousness can last for many months to years and at present there are no reliable epi- demiological data regarding these challenging patients. The heterogeneous MCS group is subcategorized in MCS—when patients only show non-reflexive behav- iour such as eye tracking, orientation to pain or contin- gent behaviour to specific stimuli (e.g. smiling exclusively in the presence of a family member) and MCS+ when a reproducible (albeit often inconsistent) response to com- mand can be observed [141]. Figure 8 illustrates the timeline of events that may occur after anoxic coma. When cerebral hypoxia leads to a prolonged loss of consciousness (lasting >2 h to differ- entiate from syncope), the patient is considered to be in coma. Patients in coma will never open the eyes, even if stimulated by a loud noise or intense noxious stimulus, and will only show reflex movements. Coma will not last longer than a couple of days to weeks. Most patients who show good recovery will rapidly show signs of conscious- ness and functional communication. Some patients, however, will evolve to brain death (i.e., irreversible coma with absent brainstem reflexes) and organ dona- tion should be discussed [138]. Many patients will remain with some brainstem function (i.e., will not be brain death), but clinical and complimentary testing shows that there is no reasonable chance for a meaningful recovery and the decision is made to withhold or withdraw treat- ment. In these cases, organ donation after cardiac death can be discussed. Other means of cardio‑ and neuro‑protection How- ever, hypothermia was not used in these studies, while the combination of magnesium with cooling procedures has been shown to result in the highest neuroprotective effects [116]. Importantly, many questions remain unan- swered on the optimal timing to initiate magnesium per- fusion, optimal dosing and circulating levels and potential side-effects, which may explain the negative results of magnesium therapy after subarachnoid hemorrhage and traumatic brain injury [117, 118]. Noble gases have been proved to reduce the extent of neurological damage after ischemia in animal models of CA [119, 120]. The admin- istration of Xenon was also feasible in the human setting, reporting both no adverse events and an improvement of cardiovascular function after CA [121]. Unfortunately, we still do not have any human data describing the potential neurological benefits of such treatment. Also, mitochondrial dysfunction can be modulated through the nitric oxide (NO)-related pathways; NO inhibits ROS-producing enzymes and directly scavenges ROS production [130]. Other potential beneficial effects are the direct vasodilation of coronary arteries, which could improve cardiac function in this setting. Experi- mental models have suggested a protective role of intra- venous NO-donors or inhaled NO both on cardiac and neurological function after CA [131, 132]. Unfortunately, only one pilot study showed the feasibility and safety of low-dose nitrite infusion in cardiac arrest survivors, but did not report any improvement in outcome [133]. i Mitochondrial dysfunction could be attenuated by the administration of erythropoietin (EPO), the principal Kudenchuk et al. Ann. Intensive Care (2015) 5:22 Page 14 of 24 Finally, recent data suggest that the early administration of corticosteroids after in-hospital CA was associated with an increased survival rate and reduced occurrence of extra-cerebral organ failures [134]; whether these effects were secondary to an anti-inflammatory effect or the treatment of a relative adrenal insufficiency [135], it remains to be further evaluated. Similarly, abnormalities of peripheral microcirculation, similar to those found in septic patients, have been described after CA [136]; the pathophysiology of such alterations as well as their role on patients’ outcome and the various therapeutic options to manipulate the microvascular flow have to be better characterized in future studies. Fig. 8 Timeline of events that may occur after anoxic coma Clinical tools Mauro Oddo The ideal tool for coma prognostication is the one yielding the lowest false-positive rate (FPR) for poor outcome. The definition of FPR is 1—specificity, whereby the “perfect” predictor would allow 100 % (i.e. 1.0) speci- ficity and an FPR for poor outcome of 0 and limit as far as possible the risk of false predictions. Clinical exami- nation comprises tools for the assessment of neurological responses/reflexes, including the Glasgow Coma Scale (GCS), brainstem reflexes and the comprehensive Full Out- line of Unresponsiveness (FOUR) score. Clinical examina- tion is an essential step for coma prognostication after CA [153]. However, clinical examination may be misleading, partly because of the effect of mild induced hypothermia and sedation on neurological responses. In particular, using the motor component of the GCS, it was found that motor response to pain may be delayed up to 5–6 days following CA [154]. Motor reaction to pain no better than extension indeed yields an unacceptably high FPR (10–20 %) for poor prognosis [155]. Apart from GCS, the assessment of brain- stem reflexes (pupillary and corneal reflexes in particular) is crucial. Corneal and pupillary reflexes have very high speci- ficity: bilaterally absent pupillary/corneal reflexes yield an FPR <1–2 % for poor prognosis, while their FPR for good prognosis is relatively high (40–50 %) [155].h EEG-patterns that are strongly associated with a poor outcome after cardiac arrest and considered “malignant patterns” include a generalized suppression to <20 µV, burst-suppression pattern with generalized epilepti- form activity, or generalized periodic complexes on a flat background [163]. In addition, a non-reactive EEG back- ground pattern after rewarming from hypothermia treat- ment has been found to be a predictor of poor outcome [164]. A reactive EEG-pattern on the other hand, and a return of a continuous EEG background pattern after cardiac arrest are both strongly associated with recovery and a good outcome [163, 164]. Continuous EEG (cEEG) provides dynamic information and can be used to monitor evolution of EEG-patterns and to detect seizures in the postischemic brain [165]. A traditional multichannel montage is commonly used in adult ICUs, while a simplified EEG-montage with few channels and trend analysis is more common in neonatal ICUs [166]. In order to reach general use, cEEG needs to be simple, possible to apply bedside and cost-effective. The predictive value of pupillary reactivity is under- lined by recent systematic meta-analyses [155, 156]. Neurological assessment of brain damages Minimal consciousness: classification and prognosis in CA patients When patients remain in PVS/UWS for over 3 months after the cardiac arrest, the condition is considered irre- versible and life-sustaining treatment (i.e., artificial hydration and nutrition) may be considered as futile and hence be withdrawn. At present, the chances of recovery after post-anoxic MCS are considered to be better than for PVS/UWS but remain ill defined. Many patients who recover from coma or related disorders of consciousness will show cognitive dysfunction and may remain insti- tutionalized or dependent of others for many activities of daily living, but few reliable data exist regarding their remaining quality of life. Recent advances in automated clinical EEG analysis [142], combined EEG transcranial magnetic stimulation studies [143] and structural and functional neuroimaging have permitted to better document the clinical diagnosis [144] and levels of consciousness in patients with severe post-anoxic encephalopathy [145]. Several studies also show their value in predicting the chances of recovery after cardiac arrest (e.g., the assessment of EEG reactiv- ity [142]; the quantification of white matter damage using Patients who survive their coma may awaken (i.e., open the eyes) without any behavioural sign of consciousness (i.e., only show reflex or automatic movements), a condi- tion coined “persistent vegetative state” or “unresponsive Page 15 of 24 Kudenchuk et al. Ann. Intensive Care (2015) 5:22 MRI diffusion tensor imaging [146] or PET imaging of residual cortical metabolism [147]. prognostication [158]. Clinical tools should be inte- grated into a multimodal approach, which ideally also include electrophysiology (EEG reactivity and/or SSEP) and blood biomarkers (e.g. NSE). Using this multimodal approach, prediction of 3-month survival and neurologi- cal outcome might approach 90 % accuracy [159]. Appli- cation of such approach is supported by several recent independent studies and can be recommended in ICU practice [160]. At present, there are no evidence-based guidelines regarding the treatment of patients with chronic anoxic disorders of consciousness [148]. In terms of noninvasive intervention, transcranial direct current stimulation has recently been shown to be of potential interest [149]. A better understanding of the temporal dynamics of pos- sible residual neural plasticity following anoxic coma and related conditions will permit to improve their clini- cal management (including treatment for possible pain perception [150]) and to rationalize our medical inter- ventions [151] acute and chronic care pathways and end- of-life decisions [152]. New electrophysiological tools: continuous and simplified electroencephalography (aEEG) Hans Friberg Electroencephalography (EEG) is a com- monly used method for identification of seizures and for estimation of prognosis in comatose survivors after car- diac arrest [161]. Limitations with an EEG-investigation include its sensitivity to sedatives and the fact that a single conventional EEG depicts the actual status during a limited time, commonly 20–30 min. Another problem has been the lack of consensus regarding EEG terminology and the absence of a uniform classification system, a problem that may be resolved by implementation of a standardized criti- cal care EEG-terminology as recently proposed [162]. Clinical tools In this setting, newly available automated infrared pupil- lometers hold great promise, because they allow quan- titative measurement of the pupillary response. One single-center study recently showed quantitative pupil- lometry is superior to standard pupillary examination for post-CA coma prognostication and performs comparably to electro-physiological exams (including EEG and SSEP) [157]. f We applied aEEG in consecutive cardiac arrest patients [167]. The aEEG curve facilitates rapid surveillance of extended time periods, but interpretation of patterns is done on the original EEG-tracings, one from each hemi- sphere. The relative simplicity of cEEG with a reduced montage and aEEG trend analysis makes it attractive to generalists in the ICU. It is applied bedside by the ICU- staff (registered nurse) and data are linked to the neu- rophysiologists, who will assess the collected data on a In summary, careful and complete neurological examination is the first and still valid tool for coma Page 16 of 24 Page 16 of 24 Kudenchuk et al. Ann. Intensive Care (2015) 5:22 regular basis and on demand. In order to facilitate for clinicians, four dominating EEG-patterns after cardiac arrest were identified and described (Fig. 9) [167]. Our experience is that cEEG with a simplified montage and aEEG trend analysis is well suited to follow transitions in background patterns and has acceptable sensitivity to detect clinically relevant electrographic seizures. A return of a continuous EEG-pattern at normothermia is an early and strong prognostic indicator with a positive-predictive value for a good outcome of 0.87 (95 % CI 0.76–0.94) while other patterns at normothermia indicate a poor prognosis with a negative predictive value of 0.91 (95 % CI 0.76–0.98) [167]. The clinical benefit of treating electrographic seizure activity after cardiac arrest is yet to be proven, but simpli- fied cEEG is a reasonable routine method to diagnose sei- zure activity and to guide treatment. erentially memory and executive functioning, anxiety, depression, and finally quality of life. Surprisingly, there is no prospective study with extensive neuropsychological assessment and longitudinal follow-up allowing to confirm these retrospective data [169]. Moreover, the era of thera- peutic hypothermia may have changed not only long-term outcomes, but also its early prognosis assessment [170]. Thus, three important questions can be raised. p q What tools do we use to describe the long-term out- come and are these tools sensitive? Clinical tools Whereas a recent review demonstrated a broad heterogeneity in the out- come measures utilized in clinical trials, the Cerebral Performance Category (CPC) score is the recommended measurement of long-term outcome after CA [170]. The question of reliability of the CPC score has been evalu- ated in several studies. There was a strong association between the CPC score at hospital discharge and long- term outcome. Moreover, the CPC score demonstrated good correlation with various neuropsychological evalu- ations in extensive cognitive battery tests, memory tests, adaptative behavior and quality-of-life evaluations. Thus the CPC could be considered as a gross indicator of long- term functional outcome after CA. In conclusion, simplified continuous EEG (cEEG) with trend analysis (aEEG) is a feasible and probably cost- effective method to improve care after cardiac arrest [168]. Collaboration with neurologists and neurophysiol- ogists and education of staff are keys to success. We fore- see that simplified cEEG will be part of routine bedside monitoring of comatose survivors after cardiac arrest in the near future. How could we evaluate the outcome during the long- term follow up? According to the World Health Organi- zation guidelines, evaluation of health after CA should associate body functions and structures, with physical examination and cognitive functions, activity and partici- pation, personal factors such as anxiety and depression, Neurological assessment in the long‑term follow‑up after cardiac arrest Stephane Legriel Half the survivors after CA have long-term follow-up cognitive impairment affecting pref- Fig. 9 A simplified electroencephalogram (EEG) with two original EEG curves (lower panel), in combination with an amplitude integrated EEG (aEEG) trend curve (upper panel). Four dominating EEG patterns after cardiac arrest are shown: a flat, b suppression-burst, c continuous and d elec‑ trographic status epilepticus. From Friberg et al. [161]. Reprinted with permission Fig. 9 A simplified electroencephalogram (EEG) with two original EEG curves (lower panel), in combination with an amplitude integrated EEG (aEEG) trend curve (upper panel). Four dominating EEG patterns after cardiac arrest are shown: a flat, b suppression-burst, c continuous and d elec‑ trographic status epilepticus. From Friberg et al. [161]. Reprinted with permission oencephalogram (EEG) with two original EEG curves (lower panel), in combination with an amplitude integrated EEG panel). Four dominating EEG patterns after cardiac arrest are shown: a flat, b suppression-burst, c continuous and d elec‑ us. From Friberg et al. [161]. Reprinted with permission Page 17 of 24 Kudenchuk et al. Ann. Intensive Care (2015) 5:22 and so quality of life (Fig. 10) [171, 172]. The American Academy of Clinical Neuropsychology recently rec- ommended evaluation of the following items during a neuropsychological evaluation: intellectual function- ing, attention and executive functioning, memory and learning, language and communication, visual–spatial cognition and visual–motor praxis, motor and sensory function, mood, conduct, personality, quality of life, adaptative behavior (activities of daily living), social– emotional awareness and responsivity, psychopathology (psychotic thinking or somatization), motivation and effort [173]. Thereby, a formal neuropsychological evalu- ation covers the whole spectrum of objectives of health evaluation. [174, 175], but survivors with good long-term functional outcome have been described in 6 % of cases [175]. Con- tinuous background pattern and reactivity during TH or normothermia has been associated with recovery and EEG plays now an increasingly important role in prog- nostication of coma after CA. Thus, continuous EEG has been suggested to assist with prognostication of coma after CA [176]. Finally, literature data clearly favour a multimodal approach of prognostication after CA. In conclusion, there is a broad variability in tools used for evaluation during the long-term follow and a lack of standardization. The cerebral performance category scale is an efficient but imperfect surrogate tool to describe long-term outcome after CA. Neurological assessment in the long‑term follow‑up after cardiac arrest The neuropsychological evaluation seems able to cover the whole spectrum of objectives of health evaluation needed to evaluate the long-term outcome. Early prognosis of long-term out- come at the era of therapeutic hypothermia should be based on a multimodal approach in order to minimize false predictions. What approach should we use to make an early prog- nosis of long-term outcome? All the parameters provided in 2006 by the American Academy of Neurology for pre- diction of outcome in comatose survivors after cardio- pulmonary resuscitation have been challenged since the application of therapeutic hypothermia. Predictors based on clinical examination within 72 h after CA have dem- onstrated higher false-positive rates [164]. Exceptional survivors have been described despite bilateral absence of N20 waves in median nerve somatosensory evoked potentials. Neuron Specific Enolase demonstrated vari- able cut-off values associated with poor outcome ranging from 25 to 49.5 ng/mL on day 2 after cardiac arrest, with one survivor with an NSE value of 110 ng/mL. On day 3, variability was greater ranging from 10.6 to 97 ng/mL. Protein S100B was also associated with variability on day 1 after CA [156]. Postanoxic status epilepticus has been demonstrated as an independent factor associated with poor outcome in retrospective and prospective studies Author details 1 University of Washington, Seattle, USA. 2 Department of Anaesthesiol‑ ogy and Intensive Care, Catholic University School of Medicine, Rome, Italy. 3 Department of Anaesthesiology and Intensive Care Medicine, University of Koeln, Cologne, Germany. 4 Medical Intensive Care Unit, AP-HP, Cochin Hos‑ pital, Paris, France. 5 Paris Descartes University and Sorbonne Paris Cité-Medical School and INSERM U970 (Team 4), Cardiovascular Research Center, European Georges Pompidou Hospital, Paris, France. 6 Division of Emergencies and Criti‑ cal Care, Department of Anaesthesiology, Surgical Intensive Care Unit Ullevål, Oslo University Hospital, Oslo, Norway. 7 Division of Cardiothoracic and Vascu‑ lar Anesthesia and Intensive Care Medicine, Vienna General Hospital, Medical University Vienna, Vienna, Austria. 8 Department of Intensive Care, Laboratoire de Recherche Experimentale, Erasme Hospital, Brussels, Belgium. 9 Medical Intensive Care Unit, AP‑HP, Lariboisière University Hospital, Inserm U942, Paris, France. 10 Anaesthesiology and Intensive Care Medicine, Skåne University Hospital, Lund University, Lund, Sweden. 11 Coma Science Group, Cyclotron Research Centre, University of Liège and Liège 2 Department of Neurology, University Hospital of Liège, Liège, Belgium. 12 Coma Science Group, Cyclotron Research Centre, University of Liège and Department of Intensive Care Medi‑ cine, University Hospital of Liège, Liège, Belgium. 13 Department of Intensive Care Medicine, Faculty of Biology and Medicine, CHUV-University Hospital, Lausanne, Switzerland. 14 Intensive Care Unit, Centre Hospitalier de Versailles, Le Chesnay, France. 15 Department of Intensive Care, Cliniques Universitaires Saint‑Luc, Université Catholique de Louvain, Brussels, Belgium. 16 Medical Intensive Care Unit, AP‑HP, European Georges Pompidou Hospital, Paris Descartes University and Sorbonne Paris Cité-Medical School, Paris, France. 17 Department of Intensive Care, Cliniques Universitaires Saint‑Luc, Université Catholique de Louvain Brussels, Brussels, Belgium. Finally, the wishes expressed by the patient (antici- patively) or by the relatives have clearly to be taken into account. The treatment-withdrawal modalities will clearly be also be influenced by these opinions. When the decision is made to stop the cares in the operating room, it is with the objective that cardiocirculatory death would occur in a delay shorter than one hour and that the pro- cedure of donation would be successful. Abbreviations G aEEG: continuous and simplified EEG-montage with two channels in com‑ bination with amplitude integrated trend analysis; AED: automated external defibrillators; ALS: advanced life support; BLS: basic life support; CA: cardiac arrest; CCPR: conventional CPR; cEEG: continuous electroencephalography; CPR: cardio-pulmonary resuscitation; DBD: donation after brain death; DCD: donation after cardiocirculatory death; DNAR: do-not-attempt-resuscitation; ECPR: extracorporeal CPR; EMS: emergency medical services; ETI: endotra‑ cheal intubation; FOUR: full outline of unresponsiveness; GCS: Glasgow coma score; ICU: intensive care unit; IHCA: in-hospital cardiac arrest; MCS: minimally conscious state; MET: medical emergency team; MTH: mild therapeutic hypo‑ thermia; NIRS: near-infrared spectrophotometry; OHCA: out-hospital-cardiac arrest; PCI: percutaneous coronary intervention; PE: pulmonary embolism; PEA: pulseless electrical activity; PVS: persistent vegetative state; ROSC: return of spontaneous circulation; RRS: rapid response systems; TH: therapeutic hypothermia; TTM: targeted temperature management; UWS: unresponsive wakefulness syndrome; VF: ventricular fibrillation; WLST: withdrawal of life- sustaining therapy. Another difficult ethical issue is the dying process itself. It remains difficult to predict the time to death by circu- latory arrest after the withdrawal of the life-sustaining treatments. When organ donation is not considered, the patient is dying in the ICU, while in case of DCD, death will occur in the operating room, with ideally a short period of hypotension in order to prevent organ damages. But in fact, the dying process has already started several hours or days before the final decision in the ICU. The vision that the DCD procedure could hasten the patient’s death is not totally true, but there are still different per- ceptions among the ICU physicians. Some of them feel that when comfort therapy with potent sedatives and analgesics (“palliative sedation”) is started in the ICU, the priority is to give a lot of time to the family to accompany the dying person. Other ICU physicians or even scientific societies consider that shortening the dying process with use of medications may sometimes be appropriate, even in the absence of discomfort, and can actually improve the quality of dying. Most of the families express the opinion that the priority should be that the patient would die peacefully, the duration of the dying process being of secondary importance. This is a key issue in the debate “taking care of the patient or taking care of the organs?” [178]. Authors’ contributions PFL and JLD were responsible for the organization of the meeting, for the collection of the summaries, and for the redaction of the Introduction and Conclusion sections, as well as for structuring the Review. PJK, CS, HRD, BWB, AC, KS, MD, FST, ND, HF, SL, DL, MO, SL, and PH were responsible for individual presentation and for redaction of the corresponding summaries. All authors read and approved the final manuscript. ub a t a Stopping cares leads to organ donation? Philippe Hantson There is still currently a shortage of organs for transplantation and accordingly, different strat- egies have been developed, including the possibility of organ donation after cardiocirculatory death (DCD) as opposed to donation after brain death (DBD). Following the Maastricht workshop on non-heart beating donation in 1995, several categories of donors have been identified, but the acceptance of such procedures still varies largely among the different European countries. There is no doubt that the ratio DCD/DBD will continue to increase over time. The ICUs are mainly concerned by categories II (unsuc- cessful resuscitation) and III (awaiting cardiac arrest). Category III is a controllable situation that could be man- aged either in the ICU or in the operating room. The pos- sibility of organ donation under category III is also linked to ethical discussions regarding the management of the “end of life”, with a clear distinction between the concepts of euthanasia and withdrawal/withholding of some spe- cific cares [177].i Fig. 10 Assessment of health condition in the long-term follow-up after cardiac arrest (adapted from World Health Organization [171]) i In contrast to DBD where specific protocols are now well accepted, there is a great variability for the DCD protocols regarding the criteria for the determination of death, but also for the definition of the “no-touch” period. This latter corresponds to the time period between deter- mination of death and initiation of organ procurement, and should be theoretically kept as short as possible in order to prevent warm ischemia. Page 18 of 24 Kudenchuk et al. Ann. Intensive Care (2015) 5:22 cares orientated to the patient and the cares given to the organs. The main ethical difficulties are related to the perceived possibility of conflict of interest for the ICU physician who in one hand could take the decision to withdraw or withhold some specific care, and in a next step could also be authorized to take the decision of organ donation in the same patient. The separation (in time and space) between the two processes is absolutely mandatory. There is a need of full confidence between all the mem- bers of the ICU staff taking care of the patient, so that each individual could be convinced that the treatment- withdrawal decision is always made independently from any considerations about the potential of subsequent organ donation. Compliance with ethical guidelines In conclusion, organ procurement under Maastricht III category will remain a sensitive issue as no protocol is widely accepted. Each centre has the responsibility to initiate the debate between all the participants involved in critical care and organ transplantation. 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https://openalex.org/W2897724477	https://link.springer.com/content/pdf/10.1007/s00429-018-1771-5.pdf	English	null	The orbitofrontal cortex projects to the parvafox nucleus of the ventrolateral hypothalamus and to its targets in the ventromedial periaqueductal grey matter	Brain structure & function	2,018	cc-by	18,169	Abstract Although connections between the orbitofrontal cortex (OFC)—the seat of high cognitive functions—the lateral hypothala- mus and the periaqueductal grey (PAG) have been recognized in the past, the precise targets of the descending fibres have not been identified. In the present study, viral tracer-transport experiments revealed neurons of the lateral (LO) and the ven- trolateral (VLO) OFC (homologous to part of Area 13 in primates) to project to a circumscribed region in the ventrolateral hypothalamus, namely, the horizontally oriented, cylindrical parvalbumin- and Foxb1-expressing (parvafox) nucleus. The fine collaterals stem from coarse axons in the internal capsule and form excitatory synapses specifically with neurons of the parvafox nucleus, avoiding the rest of the hypothalamus. In its further caudal course, this contingent of LO/VLO-axons projects collaterals to the Su3- and the PV2 nuclei, which lie ventral to the aqueduct in the (PAG), where the terminals fields overlap those deriving from the parvafox nucleus itself. The targeting of the parvafox nucleus by the LO/VLO-projections, and the overlapping of their terminal fields within the PAG, suggest that the two cerebral sites interact closely. An involve- ment of this LO/VLO-driven circuit in the somatic manifestation of behavioural events is conceivable. Keywords PV1 · Viral tracers · Su3 · PV2 · Somatic marker Keywords PV1 · Viral tracers · Su3 · PV2 · Somatic marker 1 Keywords PV1 · Viral tracers · Su3 · PV2 · Somatic marker Abbreviations 3N Oculomotor nucleus 3PC Parvicellular part oculomotor nucleus AAV Adeno associated virus AcbSh Accumbens nucleus shell AID Agranular insular cortex, dorsal part AIP Agranular insular cortex, posterior part AIV Agranular insular cortex, ventral part Amb Ambiguus nucleus BDA Biotinylated dextrane BLA Basolateral amygdaloid nucleus anterior part Cb Cerebellum Cl Claustrum CM Central thalamic nucleus cpd Cerebral peduncle CPu Cudatoputamen DA8 Dopamine cells DH Dorsal horn spinal cord DI Dysgranular insular cortex DLO Dorsolateral orbital cortex dlPAG Dorsolateral PAG DTG Dorsal tegmental nucleus DR Dorsal raphe nucleus Eth Ethmoid thalamic nucleus fmi Forceps minor corpus callosum FrA Frontal association cortex fv Flat vesicles Gem Gemini hypothalamic nucleus Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00429-018-1771-5) contains supplementary material, which is available to authorized users. * Marco R. Celio marco.celio@unifr.ch 1 Anatomy and Programme in Neuroscience, Faculty of Science and Medicine, University of Fribourg, Rte. A. The orbitofrontal cortex projects to the parvafox nucleus of the ventrolateral hypothalamus and to its targets in the ventromedial periaqueductal grey matter Alexandre Babalian1 · Simone Eichenberger1 · Alessandro Bilella1 · Franck Girard1 · Viktoria Szabolcsi1 · Diana Roccaro1 · Gonzalo Alvarez‑Bolado2 · Chun Xu3 · Marco R. Celio1 Received: 14 February 2018 / Accepted: 5 October 2018 / Published online: 12 October 2018 © The Author(s) 2018 Brain Structure and Function (2019) 224:293–314 https://doi.org/10.1007/s00429-018-1771-5 Brain Structure and Function (2019) 224:293–314 https://doi.org/10.1007/s00429-018-1771-5 ORIGINAL ARTICLE ORIGINAL ARTICLE Abstract Gockel 1, 1700 Fribourg, Switzerland 2 Institute of Anatomy and Cell Biology, University of Heidelberg, im Neuenheimer Feld 307, 69120 Heidelberg, Germany 3 Friedrich Miescher Institute, Maulbeerstrasse 66, 4058 Basel, Switzerland Brain Structure and Function (2019) 224:293–314 294 GFP Green fluorescence protein Gi Gigantocellular reticular nucleus GI Granular insular cortex GiA Giggantocellular reticular nucleus alpha part HDB Nucleus of the horizontal limb of the diago- nal band IC Inferior colliculus IL Infralimbic cortex InG Intermediate gray layer of the superior colliculus InWh Intermediate white layer of the superior colliculus IRt Intermediate reticular nucleus isRT Isthmic reticular formation LDTg Laterodorsal tegmental nucleus scp Superior cerebellar peduncle Su3 Supraoculomotor periaqueductal grey Sub Submedius thalamic nucleus TH Thalamus TO Optic tract Tu Olfactory tubercle VH Ventral horn spinal cord VM Ventromedial thalamic nucleus V2 Secondary visual cortex VLO Ventrolateral orbitofrontal cortex VM Ventromedial prefrontal cortex VO Ventral orbitofrontal cortex VP Ventral pallidum VTA Ventral tegmental area GFP Green fluorescence protein Gi Gigantocellular reticular nucleus GI Granular insular cortex GiA Giggantocellular reticular nucleus alpha par HDB Nucleus of the horizontal limb of the diago nal band IC Inferior colliculus IL Infralimbic cortex InG Intermediate gray layer of the superior colliculus InWh Intermediate white layer of the superior colliculus IRt Intermediate reticular nucleus isRT Isthmic reticular formation LDTg Laterodorsal tegmental nucleus LH Lateral horn spinal cord LO Lateral orbital cortex LPBreL Lateral parabrachial nucleus M1 Primary motor cortex M2 Secondary motor cortex MCPO Magnocellular preoptic nucleus MDC Mediodorsal thalamic nucleus central part MdD Medullary reticular nucleus dorsal part MdV Medullary reticular nucleus ventral part MO Medial orbitofrontal cortex mRt Mesencephalic reticular formation NRth Reticular thalamic nucleus OFC Orbitofrontal cortex OL Olfactory lobe PAG Periaqueductal gray parvafox Parvafox nucleus PBP (VTA) Parabrachial pigmented nucleus PcRtA Parvicellular reticular nucleus (anterior part) PDR Posterodorsal raphe nucleus PF Parafascicular nucleus PIF (VTA) Parainterfascicular nucleus Pir Piriform cortex PN (VTA) Paranigral nucleus of the VTA Pn Pontine nuclei PrEW Pre-Edinger Westphal nucleus Prh Perirhinal cortex PrL Prelimbic area PT Parataenial thalamic nucleus PV2 PV2 nucleus (in LDTg) py Pyramidal tract pyx Pyramidal decussation RFP Red fluorescent protein RhF Rhinal fissure rsv Round synaptic vesicles RtTg Reticulotegmental nucleus S1 Primary somatosensory cortex SC Superior colliculus Materials and methods This study was conducted in accordance with the regulations of the Swiss Federal Animal Protection Law and under the supervision of the Veterinary Authority of the Canton of Fri- bourg (permissions 2013-04-Fr; 2013-05-FR, 2016-36-Fr). Experiments were performed on 36 adult C57/Bl6 mice and 13 Wistar albino rats (Janvier, Lyon, France) of both genders, weighing 26–39 g and 240–325 g, respec- tively (Table 1). Other strains of mice that were used were either homozygous for the Pvalb-Cre genotype [129P2-Pvalb < tm1(cre)Arbr>/J] (Hippenmeyer et al. 2005) or heterozygous for the Foxb1-Cre one [Foxb1tm1cre−EGFPGabo] (Alvarez-Bolado et al. 2000) (Table 1). TVA-floxed mice [B6;129P2-Gt(ROSA)26Sortm1(CAG−RABVgp4,−TVA)Arenk/J, strain 024708] were used in the rabies experiments and VGAT-Cre mice [Slc32a1tm2(cre)Low l, strain 016962 (Jackson Labora- tory, Bar Harbor, Maine, USA)] for anterograde tracing with Cre-dependent constructs. The mice that were used Using viral constructs (Chamberlin et al. 1998; Wick- ersham et al. 2007a) and genetically modified mice that express Cre-recombinase in parvalbumin- (Parv) (Hippen- meyer et al. 2005) and/or Foxb1-expressing neurons (Zhao et al. 2007), we demonstrate that the LO- and the VLO-corti- ces heavily project collaterals to three as yet less well-known structures, namely, the parvafox nucleus (Bilella et al. 2014; Celio 1990; Celio et al. 2013; Girard et al. 2011; Meszar et al. 2012), the supraoculomotor nucleus (Su3) (Carrive and Paxinos 1994) and the parvalbumin 2 nucleus (PV2) (Celio et al. 2013) of the PAG. The parvafox nucleus [formerly Table 1 Strains of rats and mice that were utilized to study the projections from the OFC Table 1 Strains of rats and mice that were utilized to study the projections from the OFC To check for the presence of projecting parvalbumin-expressing neurons, Cre-dependent tracers were also injected into the OFC of Pvalb-Cre mice. The Pvalb-Cre and the Foxb1-Cre mice were used in the co-labelling experiments: the non-Cre-dependent tracers were injected into the OFC and the Cre-dependent ones, labelled with another fluorescence-dye, into the parvafox nucleus. The TVA-floxed mice, and the Pvalb- Cre/Foxb1-Cre ones with which they were bred, were employed for the injection of Cre-dependent rabies-tracers into the parvafox nucleus, with a view to studying the trans-synaptic location of the retrogradely labelled neurons in the OFC Name Line Strain Nr. (Jackson) Source Wild type Wistar Janvier, Lyon, France Wild type C57/Bl6 Janvier, Lyon, France Pvalb-Cre 129P2-Pvalbtm1(cre)Arbr/J 008069 Dr. Silvia Arber, Basel (Switzerland) and Jackson Laboratory Foxb1-Cre Foxb1tm1cre−EGFPGabo Dr. Introduction 1996), the parafascicular nucleus (Jones and Leavitt 1974), the claus- trum (Zhang et al. 2001), the lateral hypothalamus (Gab- bott et al. 2005; Hardy 1994; Hurley et al. 1991; Price et al. 1991), the PAG (Hardy 1986; Wyss and Sripanidkulchai 1984), the ventrolateral PAG (Beckstead 1979; Craig et al. 1982; Leonard 1969) and the oculomotor complex (Leich- netz and Gonzalo-Ruiz 1987b; Leichnetz et al. 1987a). The VLO-cortex projects additionally to the visual cortex (Reep et al. 1996). The targets of OFC projections are distributed throughout the entire brain—the hippocampus and the cer- ebellum exempted. Although the lateral hypothalamus and the periaqueductal grey matter (PAG) have been shown to receive terminals, their localization has not been identified with precision. called PV1 nucleus (Celio 1990)], is located amongst the fibres of the medial forebrain bundle in the ventrolateral hypothalamus. The Su3 and the PV2 nuclei are two longi- tudinally oriented columns of neurons, which are located ventral to the aqueduct at the border of the PAG in the mes- encephalic tegmentum. Although the largest contingent of axon terminals derives from pyramidal cells in layer V–VI of the LO/VLO-cor- tex and is excitatory, parvalbumin-expressing and a few GABAergic neurons also contribute to the projections. By virtue of its dual inhibitory and excitatory projections, the LO/VLO-cortex may modulate the activity of the parvafox nucleus and its PAG targets. Introduction In a series of magisterial papers, Joseph Price and his col- leagues characterized the frontal cortex and its projections in various species (Bacon and Smith 1993; Carmichael and Price 1994, 1996; Ongur et al. 2003). In the rat, the OFC was subdivided—medially-to-laterally—into ventral (VO), ventrolateral (VLO) and lateral (LO) regions (Krettek and Price 1977), which are analogous to the multimodal areas 14, 13a and 13 m/l, respectively, in the monkey (Price 2007). Area 13a, the so-called “visceromotor” region, is a constit- uent of the medial network (Carmichael and Price 1996), receiving inputs from limbic structures and projecting to the hypothalamus and the PAG. Area 13 m/l forms a part of the orbital network (Carmichael and Price 1996). Receiving multimodal information, it is consequently referred to as the “viscerosensory” region. It projects almost no descending axons to either the amygdala, the hypothalamus or the PAG (Ongur et al. 1998). The LO/VLO cortex in rodents is therefore a homologue of part of the reward network region in the ventromedial prefrontal cortex (VM) (Ongur and Price 2000) that is involved in decision-making and emotional handling in pri- mates (Bechara et al. 2000). In humans, lesioning of the VM interferes with the processing of somatic or emotional sig- nals, thereby leading to an impairment in decision-making (Bechara et al. 2000). An influential hypothesis (“somatic marker”) postulates the VM to be a key link between emo- tional regions (amygdala) and the autonomic nervous sys- tem (Damasio 1996), of which the hypothalamus is the key organizer. The projections from the OFC in rodents have been exhaustively investigated by various authors by means of antero- and retrograde tracing, The findings have revealed the OFC to be connected with the primary olfactory cor- tex (Price 1985), the piriform cortex (Illig 2005), the caud- atoputamen (Beckstead 1979; Berendse et al. 1992; Gabbott Brain Structure and Function (2019) 224:293–314 295 et al. 2005; Groenewegen et al. 1990; Leonard 1969; Schil- man et al. 2008), the amygdala (Groenewegen et al. 1990; McDonald et al. 1996), the extended amygdala (Groenewe- gen et al. 1990; Reynolds and Zahm 2005), the submedial (Coffield et al. 1992; Craig et al. 1982; Price and Slotnick 1983; Yoshida et al. 1992) and the mediodorsal thalamic nuclei (Beckstead 1979; Gabbott et al. 2005; Groenewe- gen 1988; Guldin et al. 1981; Leonard 1969; Price and Slotnick 1983; Ray and Price 1993; Reep et al. Materials and methods Gonzalo Alvarez-Bolado Heidelberg, Germany TVA-floxed mice B6;129P2- Gt(ROSA)26Sortm1(CAG−RABVgp4,−TVA)Arenk/J 024708 Jackson Laboratory VGAT-Cre Slc32a1 tm2(cre)Low l 016962 Jackson Laboratory TVA-Pvalb-Cre/Foxb1-Cre TVA-Pvalb-Cre/Foxb1-Cre Bred in house To check for the presence of projecting parvalbumin-expressing neurons, Cre-dependent tracers were also injected into the OFC of Pvalb-Cre mice. The Pvalb-Cre and the Foxb1-Cre mice were used in the co-labelling experiments: the non-Cre-dependent tracers were injected into the OFC and the Cre-dependent ones, labelled with another fluorescence-dye, into the parvafox nucleus. The TVA-floxed mice, and the Pvalb- Cre/Foxb1-Cre ones with which they were bred, were employed for the injection of Cre-dependent rabies-tracers into the parvafox nucleus, with a view to studying the trans-synaptic location of the retrogradely labelled neurons in the OFC To check for the presence of projecting parvalbumin-expressing neurons, Cre-dependent tracers were also injected into the OFC of Pvalb-Cre mice. The Pvalb-Cre and the Foxb1-Cre mice were used in the co-labelling experiments: the non-Cre-dependent tracers were injected into the OFC and the Cre-dependent ones, labelled with another fluorescence-dye, into the parvafox nucleus. The TVA-floxed mice, and the Pvalb- Cre/Foxb1-Cre ones with which they were bred, were employed for the injection of Cre-dependent rabies-tracers into the parvafox nucleus, with a view to studying the trans-synaptic location of the retrogradely labelled neurons in the OFC 1 3 Brain Structure and Function (2019) 224:293–314 296 for the trans-synaptic rabies injections were of the TVA- Pvalb/Foxb1 genotype; they were bred in house (Table 1). microinjection unit (Kopf, model 5000). In rats, the injec- tions were made at different sites of the LO around cen- tral coordinates of anteroposterior (AP): + 4.2, medi- olateral (ML): ± 2.4, dorsoventral (DV): − 3.5 (in mm, with respect to the bregma level and the brain surface). If not otherwise specified elsewhere, the injections in mice were usually made around the stereotaxic coordinates of AP: + 2.8, ML: ± 1.3, DV: − 1.8. 20–80 nl of the tracer (Table 2a) was injected into rats and 15–20 nl into mice during an interval of 0.5–1 min. After the injection, the needle was left in place for 3–5 min to allow the tracer to diffuse at the injection site. The needle was then with- drawn, the skin over the skull was sutured and the animals were left to recover. In most of the experiments with rats, bilateral injections of the same or different tracers were made at various AP and ML coordinates. Materials and methods The position of the needle was varied such that deposits of label involved in entirety the medial, lateral and insular portions of the dorsal sulcal cortex (MO, VO, VLO, LO, DLO, AIV). Due to the intricacy and the small size of the various orbito- frontal regions, injections were almost never confined to one region alone and tracers often suffused adjacent corti- cal areas. for the trans-synaptic rabies injections were of the TVA- Pvalb/Foxb1 genotype; they were bred in house (Table 1). The animals were anaesthetized with a mixture of ketamine (40–60 mg/kg of body weight) and xylazine (10–15 mg/kg of body weight) which was diluted with physiological (0.9%) saline. If necessary, supplementary, lower (1/4–1/3) doses of the anaesthetic were administered during the stereotactic procedure, if any signs of awakening became manifest. Anterograde tracing experiments (Table 2a) The head of the animal was secured in the stereotaxic apparatus (Kopf Model 5000) and a craniotomy was per- formed over the target region in the orbital cortex. Tracers 1–6 in Table 2a were used as anterograde tracers in these experiments. Injections in the OFC The tracers were injected via a fine-bored needle (external diameter: 0.14 mm, GA: 34), which was connected to a 2.5-µl Hamilton syringe that was mounted on a manual Table 2 List of the antero- and retrograde tracers that were used for the experiments. The anterograde, non-Cre-dependent tracers were injected primarily into the OFC, and the Cre-dependent ones into the parvafox nucleus of Pvalb-Cre, Foxb1-Cre or Pvalb-Cre/Foxb1-Cre mice. The classical retrograde tracers (BDA, Fluorogold) were injected into the targets of the OFC projection, namely, into the parvafox nucleus and the PAG, to reveal the presence of double-stained neurons in the OFC a: Anterograde tracers Species Source Viral, non-Cre-dependent 1 AAV2/1.hSynapsin.EGFP.WPRE.bGH Rats and mice Vector Core, University of North Carolina, USA 2 AAV1.hSynapsin.TurboRFP.WPRE.rBG Rat Vector Core, University of North Carolina, USA 3 AAV9.hSynapsin.TurboRFP.WPRE.rBG Rat Vector Core, University of Pennsylvania, USA Viral, Cre-dependent 4 AAV2/1.CAG.FLEX.EGFP.WPRE.bGH Mouse Vector Core, University of Pennsylvania, USA 5 AAV1.CAG.flex.tdTomato.WPRE.bGH Vector Core, University of Pennsylvania, USA Non-viral 6 Biotinylated dextran (MW 10′000) anterograde tracer Mouse Invitrogen (D1956 Lot 1148353) b: Retrograde tracers Source Trans-synaptic transport (rabies) 7 rAAV8/CA-Flex-RG.ape Vector Core, University of North Carolina (USA) 8 rAAV5/EF1-Flex-TVA-Cherry.ape Vector Core, University of North Carolina (USA) Env-A Δ-G rabies-EGFP Friedrich Miescher Institute-Basel (Switzerland) Others 10 Biotinylated dextran (BDA, MW 3000) retrograde tracer Invitrogen (D7135), Waltham, USA 11 Fluorogold Anti-fluorogold antiserum Fluorogold, Denver, USA Millipore AB153 Lot 2161122, USA Table 2 List of the antero- and retrograde tracers that were used for the experiments. The anterograde, non-Cre-dependent tracers were injected primarily into the OFC, and the Cre-dependent ones into the parvafox nucleus of Pvalb-Cre, Foxb1-Cre or Pvalb-Cre/Foxb1-Cre mice. Injections in the OFC The classical retrograde tracers (BDA, Fluorogold) were injected into the targets of the OFC projection, namely, into the parvafox nucleus and the PAG, to reveal the presence of double-stained neurons in the OFC a: Anterograde tracers Species Source Viral, non-Cre-dependent 1 AAV2/1.hSynapsin.EGFP.WPRE.bGH Rats and mice Vector Core, University of North Carolina, USA 2 AAV1.hSynapsin.TurboRFP.WPRE.rBG Rat Vector Core, University of North Carolina, USA 3 AAV9.hSynapsin.TurboRFP.WPRE.rBG Rat Vector Core, University of Pennsylvania, USA Viral, Cre-dependent 4 AAV2/1.CAG.FLEX.EGFP.WPRE.bGH Mouse Vector Core, University of Pennsylvania, USA 5 AAV1.CAG.flex.tdTomato.WPRE.bGH Vector Core, University of Pennsylvania, USA Non-viral 6 Biotinylated dextran (MW 10′000) anterograde tracer Mouse Invitrogen (D1956 Lot 1148353) b: Retrograde tracers Source Trans-synaptic transport (rabies) 7 rAAV8/CA-Flex-RG.ape Vector Core, University of North Carolina (USA) 8 rAAV5/EF1-Flex-TVA-Cherry.ape Vector Core, University of North Carolina (USA) Env-A Δ-G rabies-EGFP Friedrich Miescher Institute-Basel (Switzerland) Others 10 Biotinylated dextran (BDA, MW 3000) retrograde tracer Invitrogen (D7135), Waltham, USA 11 Fluorogold Anti-fluorogold antiserum Fluorogold, Denver, USA Millipore AB153 Lot 2161122, USA Table 2 List of the antero- and retrograde tracers that were used for the experiments. The anterograde, non-Cre-dependent tracers were injected primarily into the OFC, and the Cre-dependent ones into the parvafox nucleus of Pvalb-Cre, Foxb1-Cre or Pvalb-Cre/Foxb1-Cre mice. The classical retrograde tracers (BDA, Fluorogold) were injected into the targets of the OFC projection, namely, into the parvafox nucleus and the PAG, to reveal the presence of double-stained neurons in the OFC 3 A note on the OFC tracing data found in the Allen Brain Atlas (ABA) After its first appearance in 2013, subsequent editions of the Allen Brain Atlas (ABA) have reported an ever- increasing body of data that have been garnered from the stereotactic injection of viral tracers into the OFC of murine brains (suppl. Table. 1). We drew on some of the self-same viral tracers in our own experiments. The data that are presented in the ABA are based upon the imple- mentation of an iontophoretic technique, which involves the injection of a viral tracer at two levels in the cortex, whence it attains all layers. The volume of the injected tissue is then calculated. In our experiments, a defined volume of the viral tracer was delivered in a single injec- tion via a Hamilton syringe to the targeted site. Since our data accord well with those that are presented in the ABA, we presume that the different modes of delivery of the viral tracers (iontophoresis versus microinjection) had no impact on their accessibility to nerve cells. Retrograde tracing experiments (Table 2b) A red anterograde tracer was injected in the OFC and a green one in the parvafox nucleus (or vice-versa). In these speci- mens, we investigated the spatial relationship between the OFC endings and the endings from the parvafox nucleus in the Su3 and the PV2 regions of the PAG. A Cre-dependent red-construct was injected into the parvafox nucleus and a Cre-dependent (or non-Cre-dependent) GFP-tracer into the LO/-VLO-cortex. Eight PV-Cre (552-12, 553-12, 138-13, 222-13, 223-13, 357-14, 394-14, 395-14), three Foxb1-Cre (209-14, 223-14, 390-14) mice, and one PV-Cre/Foxb1-Cre (223-14) mouse were investigated. To confirm the existence of the projections that were observed in the anterograde tracing experiments and to ascertain whether the projections to the different sites origi- nate from the same or different populations of neurons, retrograde (including double) labelling experiments were performed (tracers 7–11 in Table 2b). These included also some double-labelling experiments targeting in the same animal the PAG and the parvafox (both targets of the OFC as the present study reveals). Two classical retrograde trac- ers, viz. Fluorogold [2% in physiological (0.9%) saline; 12–30 nl/1930s] and biotinylated dextran amine [(BDA) 10% in physiological (0.9%) saline; 40–100 nl/1–2 min] were injected into targeted regions of the LO/VLO-projec- tions in mice, namely, into the PAG (coordinates of AP: − 4.1, ML:± 0.5, DV: − 2.7) and/or the parvafox nucleus (coordinates of AP: − 1.5, ML: ± 1.3, DV: − 4.9) using the same tools and procedures that are described above for the injection of the anterograde tracers. 3 Brain Structure and Function (2019) 224:293–314 297 394-14; 306-15; 307-15; 308-15) and three VGAT-Cre mice (234-16, 235-16, 236-16). 394-14; 306-15; 307-15; 308-15) and three VGAT-Cre mice (234-16, 235-16, 236-16). Injections in the parvafox nucleus For the injections into the parvafox nucleus of mice, the nee- dle was positioned at bregma level: − 1.5 mm, ML: 1.3 mm, DV: 4.9–5.1 mm (Bilella et al. 2016). In a few experiments, the injection needles pierced the OFC and penetrated the olfactory bulb (394-13, 395-13), the anterior olfactory nucleus (132-15; 309-15), the olfac- tory tract (386-13) and the olfactory tuberculum (386-13). Do experiments with classical tracers confirm the results found with viral tracers? In eight mice (250-13, 399-15, 400-15, 401-15, 402-15; 28-16, 29-16), biotinylated dextran (BDA 10,000 MW, Invit- rogen, USA) was injected at various medio-lateral coordinates into the OFC. The distribution of the terminals in the parvafox nucleus and in the PAG corresponded exactly to the picture that was revealed after the injection of the viral tracers. Only injections that involved the LO/VLO-cortex disclosed the typical “pony-tail-like” terminal field in the parvafox nucleus. yp p yi p The cortices at the medial and lateral boundaries of the orbital cortex were selectively targeted in various experi- ments (126-13 [prelimbic (PrL), medial orbital (MO)]; 185- 13 [MO]; 186-13 [MO]; 130-15 [PrL]; 132-15 [infralimbic (IL)]; 552-12 [DLO]; 553-12 [DLO]; 556-12 [DLO]). None revealed targeted terminals in the parvafox- or in the Su3 and PV2-nuclei, only a diffuse innervation of the lateral hypothal- amus and of other columns in the PAG. As demonstrated in the ABA-database, tracer injection in the AID, AIV and AIP did not show selective innervation neither of the parvafox nor of Su3 and PV2-nuclei (http://connectivity.brain-map.org/). In a few experiments, the injection needles pierced the OFC and penetrated the olfactory bulb (394-13, 395-13), the anterior olfactory nucleus (132-15; 309-15), the olfac- tory tract (386-13) and the olfactory tuberculum (386-13). i The cortices at the medial and lateral boundaries of the orbital cortex were selectively targeted in various experi- ments (126-13 [prelimbic (PrL), medial orbital (MO)]; 185- 13 [MO]; 186-13 [MO]; 130-15 [PrL]; 132-15 [infralimbic (IL)]; 552-12 [DLO]; 553-12 [DLO]; 556-12 [DLO]). None revealed targeted terminals in the parvafox- or in the Su3 and PV2-nuclei, only a diffuse innervation of the lateral hypothal- amus and of other columns in the PAG. As demonstrated in the ABA-database, tracer injection in the AID, AIV and AIP did not show selective innervation neither of the parvafox nor of Su3 and PV2-nuclei (http://connectivity.brain-map.org/). Histological procedures After 6–8 days for retrograde, and 3–4 weeks for antero- grade tracing experiments, deep anaesthesia was induced in the animals by the administration of a lethal dose of pento- barbital (150–200 mg/kg of body weight). They were then transcardially perfused, first with physiological (0.9%) saline and then with paraformaldehyde (4% in 0.1 M phosphate buffer, pH 7.4) the brains were excised and postfixed over- night in 4% paraformaldehyde. They were then submerged in a 30% solution of sucrose for cryo-protection. Using a cryomobile (Reichert-Jung), the brains were serially sliced into 40/80-µm-thick sections, which were collected in 0.1 M phosphate buffer containing 0.01% sodium azide. The sec- tions were usually cut in the coronal (frontal) plane. However, in four brains (393-13, 142-14, 132-15, 307-15) the sections were prepared in the sagittal plane, and in three (386-13, 394-13, 141-14) they were cut in the horizontal direction. The sections were subsequently incubated with the appropri- ate antibodies to confirm the precision of the injection and the presence of neuronal endings in the region of interest, particularly around the cells of the parvafox nucleus. Series of sections that were derived from untreated mice and rats were stained with Nissl and exposed to an antibody against non-phosphorylated neurofilaments (SMI-32), which served as a neuronal marker (Franklin and Chudasama 2012) to Are subpopulations of the OFC‑cells projecting to the parvafox nucleus inhibitory or Parv‑expressing (Lee et al. 2014)? A Cre-dependent tracer was injected in the OFC of VGAT -Cre or PValb-Cre mice to detect terminals on neurons of the parvafox nucleus and in the PAG. The animals used for these experiments were six Pvalb-Cre mice (356-14; 357-14; In addition, trans-synaptic labelling of the parvafox nucleus was executed in Pvalb-Cre/Foxb1-Cre mice that had 1 3 Brain Structure and Function (2019) 224:293–314 298 define the subdivision of the prefrontal cortex. The sections were mounted on glass slides for histological inspection in either a Leica 6000 epifluorescence microscope [equipped with a Hamamatsu C4742-95 camera], a digital slide-scanner (Nanozoomer, Hamamatsu), or a Leica TCS SP5 confocal laser microscope. Staining with GFP, RFP, Tomato or Fluoro- gold was detected by virtue of the intrinsic fluorescence; that with BDA was revealed after exposure to streptavidin-Cy3. been bred with TVA-floxed (Pvalb-Foxb1-TVA) mice. To this end, a Cre-dependent glycoprotein-deleted mutant strain of the SAD B19 rabies vaccine strain bearing an EGFP- insert (Env-A Δ-G rabies-EGFP) was concomitantly injected with AAV-Flex-G into the parvafox nucleus (Table 2b) (Wickersham et al. 2007a). Immunohistochemistry After 6–8 days for retrograde, and 3–4 weeks for antero- grade tracing experiments, deep anaesthesia was induced in the animals by the administration of a lethal dose of pento- barbital (150–200 mg/kg of body weight). They were then transcardially perfused, first with physiological (0.9%) saline and then with paraformaldehyde (4% in 0.1 M phosphate buffer, pH 7.4) the brains were excised and postfixed over- night in 4% paraformaldehyde. They were then submerged in a 30% solution of sucrose for cryo-protection. Using a cryomobile (Reichert-Jung), the brains were serially sliced into 40/80-µm-thick sections, which were collected in 0.1 M phosphate buffer containing 0.01% sodium azide. The sec- tions were usually cut in the coronal (frontal) plane. However, in four brains (393-13, 142-14, 132-15, 307-15) the sections were prepared in the sagittal plane, and in three (386-13, 394-13, 141-14) they were cut in the horizontal direction. The immunofluorescence technique and the immunoper- oxidase reaction were performed as previously described (Gerig and Celio 2007; Meszar et al. 2012). In short, float- ing sections were incubated in 24-well plates with the pri- mary antisera or antibodies, which were diluted in the range 1:1000–1: 5000 (Table 3). The efficacy of the antisera and the antibodies against Parv had been hitherto established in antigen-pre-adsorption experiments, by immunoblotting and by the absence of immunoreactivity in knock-out mice (see Table 3). Incubation with the biotinylated secondary anti- body was followed by exposure to either streptavidin-Cy2 (Alexa460), -Cy3 (Alexa 550) or -Cy5 (Alexa 650). The antibodies against Parv were utilized primarily to confirm that the endings from the OFC did indeed impinge on parvalbumin-immunore- active neurons in the parvafox nucleus. The serotonin- and TH-antisera helped to define the borders of the raphe and the coeruleus nuclei. GFP- antisera served to enhance the fluorescence in the terminals of the brainstem and the spinal cord Electron microscopy The sections were subsequently incubated with the appropri- ate antibodies to confirm the precision of the injection and the presence of neuronal endings in the region of interest, particularly around the cells of the parvafox nucleus. Series of sections that were derived from untreated mice and rats were stained with Nissl and exposed to an antibody against non-phosphorylated neurofilaments (SMI-32), which served as a neuronal marker (Franklin and Chudasama 2012) to In one case (127-13) five coronal sections of the region of the hypothalamus in which positive terminals were observed around the parvafox nucleus were incubated with antibod- ies against GFP during 5 days (without Triton-X100). The immunostaining was continued with a biotinylated antiserum against rabbit-IgG (1 day) and followed by avidin-peroxidase (1 day). After washing, the enzymatic activity was revealed Table 3 List of the antibodies and the antisera that were used in the experiments The antibodies against Parv were utilized primarily to confirm that the endings from the OFC did indeed impinge on parvalbumin-immunore- active neurons in the parvafox nucleus. The serotonin- and TH-antisera helped to define the borders of the raphe and the coeruleus nuclei. GFP- antisera served to enhance the fluorescence in the terminals of the brainstem and the spinal cord Antibody Antigen Source Species Dilution PV235 Purified carp parvalbumin Swant Inc., Marly, Switzerland Mouse monoclonal Lot 10-11F 1:1000–5000 PV25 Recombinant rat parvalbumin Swant Inc., Marly, Switzerland Rabbit polyclonal Lot 5.10 1:1000–1:5000 GP72 Recombinant mouse parvalbumin Swant Inc., Marly, Switzerland Guinea pig polyclonal 1:1000–1:5000 PVG213/214 Recombinant rat parvalbumin Swant Inc., Marly, Switzerland Goat polyclonal 1:1000 GFP Recombinant peptide Molecular Probes, Waltham, (USA) Rabbit polyclonal 1:3000 5-HT Serotonin Immunonuclear Rabbit polyclonal 1:2000 TH Tyrosine-hydroxylase Immunostar, Stillwater (USA) Rabbit polyclonal 1:10,000 SMI-32 Non-phosphorylated filaments Millipore, USA Mouse monoclonal 1:1000 VGlut 1 Recombinant peptide Synaptic System, Germany Rabbit or mouse 1:5000/1:20,000 VGlut 2 Recombinant peptide Synaptic System, Germany Rabbit 1:10,000 GAD Recombinant peptide Millipore, USA Mouse 1:2000 Table 3 List of the antibodies and the antisera that were used in the experiments The antibodies against Parv were utilized primarily to confirm that the endings from the OFC did indeed impinge on parvalbumin-immunore- active neurons in the parvafox nucleus. The serotonin- and TH-antisera helped to define the borders of the raphe and the coeruleus nuclei. Electron microscopy GFP- antisera served to enhance the fluorescence in the terminals of the brainstem and the spinal cord The antibodies against Parv were utilized primarily to confirm that the endings from the OFC did indeed impinge on parvalbumin-immunore- active neurons in the parvafox nucleus. The serotonin- and TH-antisera helped to define the borders of the raphe and the coeruleus nuclei. GFP- antisera served to enhance the fluorescence in the terminals of the brainstem and the spinal cord The antibodies against Parv were utilized primarily to confirm that the endings from the OFC did indeed impinge on parvalbumin-immunore- active neurons in the parvafox nucleus. The serotonin- and TH-antisera helped to define the borders of the raphe and the coeruleus nuclei. GFP- antisera served to enhance the fluorescence in the terminals of the brainstem and the spinal cord 1 Brain Structure and Function (2019) 224:293–314 299 by incubating the sections with DAB-H2O2. The sections were further post-fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3 and after washing, were exposed to 1% OsO4 (osmium tetroxide) in phosphate buffer for 2 h. Embedding took place in Epon. Semi- (0.5 µm) and ultrathin sections (60 nm, grey interference colour) were cut with a Reichert Ultramicrotome and mounted on one-hole grids. Uranyl acetate and lead citrate were employed for contrast- ing purposes. The region of the parvafox was searched for the presence of synapses between immunoreactive terminals and dendrites or cell bodies using a Jeol microscope. between the LO and the VO in mice is debated (Franklin and Chudasama 2012). The extension of the LO-cortex is revealed after exposure to an antibody against SMI-32, which is a marker of a non-phosphorylated neurofilament subunit in rats (Franklin and Chudasama 2012) and mice (not shown). On the basis of immunoreactivity for SMI-32, we tentatively identified and delimited the VLO-region in our specimens (Figs. 1, 2 ,3) [see also (Dong 2008)]. By adopting the subdivision of the prefrontal cortex in rats that was introduced by the groups of Price (Price 2007) and Reep (Reep et al. 1996), most of the successful injections that revealed the presence of terminals in the parvafox nucleus were located in the LO- and the VLO-cortices. After the injection of viral tracers into the mid portion of the LO- and the VLO-cortex in rats (Figs. 1, 2, 3E, F), and in mice (Fig. Electron microscopy 1, 2, 3A–D, G–J), enriched terminal staining was revealed in the parvafox nucleus (Fig. 4A–G). Injections in the most rostral and caudal position of the LO/VLO cortex often gave negative results (Fig. 1; see also specimen 180673746 in the ABA-database, Suppl Table 1). The injected tracer tended to Delimitation of the OFC and the injection sites Although the structural organization of the murine prefrontal cortex is assumed to be similar to that in the rat (Frank- lin and Chudasama 2012), the presence of a VLO-cortex awings of the positions nto the OFC of 26 mice wings depict the basal y bulb and the piriform tudinal regions: medial d dorsolateral (DLO), ections into mice were nto rats in various other e positions of injections us of the ventrolateral d the PV2 nuclei of the the positions of injec- ucleus of the ventrolat- The grey circles indicate the positions of injections that revealed ter- minals in the Su3- and the PV2 nuclei of the PAG, not those in the parvafox nucleus of the ventrolateral hypothalamus. The white cir- cles indicate the positions of injections that did not reveal terminals either in the parvafox nucleus or in the Su3- and the PV2 nuclei of the PAG. The squares indicate the positions of injections into the OFC of Pvalb-Cre- or Foxb1-Cre-mice. The colour-coding of the squares corresponds to that of the circles. The OFC-subdivision of the mouse brain was based upon information that was derived from cresyl violet- stained and SMI-31 immunostained stained serial sections through the brains of wild-type mice (C57/B16). The right-hand picture is a modi- fied version of a published figure (Groenewegen 1988) Fig. 1 Map of the injections sites. Schematic drawings of the positions of the needle tip in the stereotactic injections into the OFC of 26 mice (left) and 12 rats (right, 4 bilateral). The drawings depict the basal surface of the OFC after removing the olfactory bulb and the piriform region. The OFC is subdivided into four longitudinal regions: medial (MO), ventrolateral (VLO), lateral (LO) and dorsolateral (DLO), according to (Dong 2008; Price 2007). The injections into mice were located in the central LO/VLO portion, those into rats in various other parts of the OFC. The black circles indicate the positions of injections that revealed terminals in the parvafox nucleus of the ventrolateral hypothalamus as well as those in the Su3- and the PV2 nuclei of the PAG. The circles with a central point indicate the positions of injec- tions that revealed terminals in the parvafox nucleus of the ventrolat- eral hypothalamus alone, not those in the Su3- and the PV2 nuclei. Delimitation of the OFC and the injection sites The grey circles indicate the positions of injections that revealed ter- minals in the Su3- and the PV2 nuclei of the PAG, not those in the parvafox nucleus of the ventrolateral hypothalamus. The white cir- cles indicate the positions of injections that did not reveal terminals either in the parvafox nucleus or in the Su3- and the PV2 nuclei of the PAG. The squares indicate the positions of injections into the OFC of Pvalb-Cre- or Foxb1-Cre-mice. The colour-coding of the squares corresponds to that of the circles. The OFC-subdivision of the mouse brain was based upon information that was derived from cresyl violet- stained and SMI-31 immunostained stained serial sections through the brains of wild-type mice (C57/B16). The right-hand picture is a modi- fied version of a published figure (Groenewegen 1988) Fig. 1 Map of the injections sites. Schematic drawings of the positions of the needle tip in the stereotactic injections into the OFC of 26 mice (left) and 12 rats (right, 4 bilateral). The drawings depict the basal surface of the OFC after removing the olfactory bulb and the piriform region. The OFC is subdivided into four longitudinal regions: medial (MO), ventrolateral (VLO), lateral (LO) and dorsolateral (DLO), according to (Dong 2008; Price 2007). The injections into mice were located in the central LO/VLO portion, those into rats in various other parts of the OFC. The black circles indicate the positions of injections that revealed terminals in the parvafox nucleus of the ventrolateral hypothalamus as well as those in the Su3- and the PV2 nuclei of the PAG. The circles with a central point indicate the positions of injec- tions that revealed terminals in the parvafox nucleus of the ventrolat- eral hypothalamus alone, not those in the Su3- and the PV2 nuclei. Fig. 1 Map of the injections sites. Schematic drawings of the positions of the needle tip in the stereotactic injections into the OFC of 26 mice (left) and 12 rats (right, 4 bilateral). The drawings depict the basal surface of the OFC after removing the olfactory bulb and the piriform region. The OFC is subdivided into four longitudinal regions: medial (MO), ventrolateral (VLO), lateral (LO) and dorsolateral (DLO), according to (Dong 2008; Price 2007). The injections into mice were located in the central LO/VLO portion, those into rats in various other parts of the OFC. Delimitation of the OFC and the injection sites The drawings are not to scale Fig. 2 Extent of the injections. Schematic drawings of coronal sec- tions of the right hemisphere depicting the extent of the injection sites through the brains of four rats (557-12, 127-13, 158-13 and 23-16) and five mice (138-13, 222-13, 223-13, 250-13 and 400-15), repro- duced from (Franklin and Paxinos 2008). The medial boundary of the LO-cortex has been slightly modified, and the VLO-cortex has been introduced according to (Dong 2008; Krettek and Price 1977). In most cases, an adeno-associated virus tracer was injected stereo- tactically. In cases 250-13 and 400-15, BDA was applied. Although the tracers were injected into the LO-cortex, also dorsal (Fr, Cl and M2) or adjacent regions (DLO and AIV) were sometimes co-labelled. In rats, the injections 557-12 and 127-13 were located mainly in the LO-cortex. In the murine OFC, the injections in the LO-cortex trans- gressed the border to surrounding areas. In both rats and mice, the tracer injections revealed clearly visible terminal fields in the parva- fox-, the Su3- and the PV2 nuclei. The drawings were prepared based on the intrinsic fluorescence of the tracers, in the absence of amplifi- cation with antibodies. The drawings are not to scale spread in a rostrocaudal, longitudinal direction (not shown), as if the intrinsic texture of the OFC facilitated this path of diffusion. A strong and selective projection to the parvafox nucleus was disclosed only if the viral tracer impregnated the deep cortical layers (V and VI). Injections that targeted the superficial layers (I–IV) or the neighbouring regions of the upper lip of the sulcal cortex, namely, the VO-, the DLO- or the frontal association (FrA)-cortex, revealed scattered, dif- fuse terminal fields in the lateral hypothalamus (Fig. 4D). In the PAG, terminals were apparent not only after infections in the LO and VLO (Fig. 4A–D, H–J), but also after injections into the DLO-cortex (507-12; 552-12), although these were located more dorsally and were less strong than when the injection hit the LO-cortex. In recent years, the Allen Institute has recorded data appertaining to random tracer-injections that have targeted the OFC (http://connectivity.brain-map.org/) (Oh et al. 2014). The data that are presented in the ABA are inter- esting also because gleaned from transgenic mice that had been genetically engineered to permit targeting of specific cell types. Delimitation of the OFC and the injection sites The black circles indicate the positions of injections that revealed terminals in the parvafox nucleus of the ventrolateral hypothalamus as well as those in the Su3- and the PV2 nuclei of the PAG. The circles with a central point indicate the positions of injec- tions that revealed terminals in the parvafox nucleus of the ventrolat- eral hypothalamus alone, not those in the Su3- and the PV2 nuclei. The grey circles indicate the positions of injections that revealed ter- minals in the Su3- and the PV2 nuclei of the PAG, not those in the parvafox nucleus of the ventrolateral hypothalamus. The white cir- cles indicate the positions of injections that did not reveal terminals either in the parvafox nucleus or in the Su3- and the PV2 nuclei of the PAG. The squares indicate the positions of injections into the OFC of Pvalb-Cre- or Foxb1-Cre-mice. The colour-coding of the squares corresponds to that of the circles. The OFC-subdivision of the mouse brain was based upon information that was derived from cresyl violet- stained and SMI-31 immunostained stained serial sections through the brains of wild-type mice (C57/B16). The right-hand picture is a modi- fied version of a published figure (Groenewegen 1988) 1 3 3 Brain Structure and Function (2019) 224:293–314 300 Fig. 2 Extent of the injections. Schematic drawings of coronal sec- tions of the right hemisphere depicting the extent of the injection sites through the brains of four rats (557-12, 127-13, 158-13 and 23-16) and five mice (138-13, 222-13, 223-13, 250-13 and 400-15), repro- duced from (Franklin and Paxinos 2008). The medial boundary of the LO-cortex has been slightly modified, and the VLO-cortex has been introduced according to (Dong 2008; Krettek and Price 1977). In most cases, an adeno-associated virus tracer was injected stereo- tactically. In cases 250-13 and 400-15, BDA was applied. Although the tracers were injected into the LO-cortex, also dorsal (Fr, Cl and M2) or adjacent regions (DLO and AIV) were sometimes co-labelled. In rats, the injections 557-12 and 127-13 were located mainly in the LO-cortex. In the murine OFC, the injections in the LO-cortex trans- gressed the border to surrounding areas. In both rats and mice, the tracer injections revealed clearly visible terminal fields in the parva- fox-, the Su3- and the PV2 nuclei. The drawings were prepared based on the intrinsic fluorescence of the tracers, in the absence of amplifi- cation with antibodies. The LO/VLO‑cortex‑derived projection in general The projection from the LO/VLO-cortex extended as a straight, compact bundle of descending axons directed towards the hypothalamus (Fig. 7A). It has a length of 7.5 mm in rats and one of 4.5 mm in mice. From the injection site, it coursed through the caudatoputamen and the internal capsule to the level of the posterior lateral hypothalamus (Figs. 3G, 7). The angle between the axonal bundle and the upper surface of the brain is 55°; that between the bundle and the medial surface of the brain is 12° (as revealed on 3D-images in the ABA; Fig. 7A, B). From the posterolateral hypothalamus, two separate bundles left the cerebral peduncle coursing in an upward direction at an angle of 95° towards the rostral PAG, and at an angle of 115 towards the distal PAG (Fig. 7A). A third bundle left the cerebral peduncle caudally, one branch extending to the medial pontine nuclei (Pn) and a second to the reticulotegmental nucleus (RtTg) of the pons (Fig. 7B). A third branch continued distally in the cerebral peduncle and distributed axons bilaterally at various levels of the brainstem, with contralateral targets predominating (Fig. 7A, C). In cross sections of the brain, the fan-shaped axons were distributed perpendicular to the surface of the brain and gave rise to rich terminal fields in all parts of the reticular formation in the pons and the medulla oblongata (schematic in Fig. 7C). The terminal fields were most patent after immunostaining of the transported GFP-tracer with antibodies that were directed against GFP (222-13, 250-13, 556-12). When tracers were injected into the prelimbic cortex (PrL), terminals were widespread in the lateral hypothalamus, but were rare in the region of the parvafox nucleus (Sesack et al. 1989; Takagishi and Chiba 1991; Vertes 2004). They were notably absent from the region that was occupied by the parv-immunoreactive neurons in the parvafox nucleus, as revealed by immunofluorescence with another fluorochrome on the same section (Injection 130-13, not shown). Injections into the insula (AIV and AID) (e.g. 507-12), gave rise to diffuse terminal fields in the parvafox nucleus but endings targeted the PAG, albeit in other quadrants.i To attain the superior lip of the rhinal fissure, where the orbital cortex is located, the injection needle traversed corti- cal areas on the dorsal (FrA, M2) and the dorsolateral (M1) aspects of the prefrontal cortex. The LO/VLO‑cortex‑derived projection in general Leakage of the tracer along the needle track sometimes involved these dorsal cortical areas in the injection. When area M2 was involved, it may have contributed to some of the terminal labelling seen in the brainstem. However, area FrA does not appear to pro- ject substantially to the hypothalamus or brainstem, because in two experiments in which the injection was accidentally made into area FrA by itself (553-12; 83-13), only a few terminals were observed in the hypothalamus or PAG. The projections from the LO- and the VLO-cortices were always bilateral, but were more abundant ipsilaterally until the end of the midbrain (Fig. 7C), with one exception: the projection from the VLO-cortex to the dorsal parabrachial leaflet (LPBreL) was more apparent contralaterally than ipsilaterally (Figs. 4j, 7C). In the pons and the medulla the contralateral projections were always stronger, except in the reticulotegmental (RtTg), and the pontine (Pn) nuclei. Caudal to the decussation of the pyramidal tract, they were more pro- nounced on the ipsilateral side of the spinal cord (Fig. 7C). Delimitation of the OFC and the injection sites As soon as ABA-images appertaining to injec- tions into the OFC were published, these were evaluated in parallel with the data that arose from our own injec- tion experiments (Suppl. Table 1). The outcomes of our own injections corresponded well with the data that are recorded in the ABA. However, the mapping of data in the ABA ends at the level of the spine–medullary junction, spread in a rostrocaudal, longitudinal direction (not shown), as if the intrinsic texture of the OFC facilitated this path of diffusion. A strong and selective projection to the parvafox nucleus was disclosed only if the viral tracer impregnated the deep cortical layers (V and VI). Injections that targeted the superficial layers (I–IV) or the neighbouring regions of the upper lip of the sulcal cortex, namely, the VO-, the DLO- or the frontal association (FrA)-cortex, revealed scattered, dif- fuse terminal fields in the lateral hypothalamus (Fig. 4D). In the PAG, terminals were apparent not only after infections in the LO and VLO (Fig. 4A–D, H–J), but also after injections into the DLO-cortex (507-12; 552-12), although these were located more dorsally and were less strong than when the injection hit the LO-cortex. 1 3 3 Brain Structure and Function (2019) 224:293–314 301 whereas axons projecting from the OFC continue distally, terminating in the lateral horn of the spinal cord (Fig. 7C). nucleus of the amygdala (BLA). The bundles of axons were likewise located more medially in the caudatoputamen and the terminals in the Su3 nucleus were restricted dorsomedi- ally, over the oculomotor nucleus. When the injection hit the LO-cortex, the density of labelled terminals was greater in the parvafox and Su3-nuclei, and endings were also observed in the basolateral nucleus of the amygdala (BLA), the ethmoid thalamic nucleus (Eth) and the mediodorsal tha- lamic nucleus (MDC). Detailed course of the axons and the location of their terminal fields The description is limited to the regions in which novel projections arising from the VLO/LO cortex were observed (e.g. parvafox, Su3 and PV2). Observations that confirm already known terminal fields of the LO/VLO cortex— respectively, differences between already known projections of the LO-VLO cortex—are reported in the schematic draw- ing of Fig. 7C. After injections into the OFC of Pvalb-Cre- (Fig. 4G) or VGAT-Cre mice (not shown), terminals were revealed at the level of the thalamus and that of the parvafox nucleus (Fig. 4G) in the hypothalamus, but only rarely more distally in either the PAG or the brainstem. Hypothalamic targets of the OFC Scale bars A–D, E, H, I, L, N: 0.5 mm; J, K 0.05 mm; F, G 1 mm; M 0.1 mm ◂ Hypothalamic targets of the OFC The axonal bundles from the CPu merged to form the medial- most tip of the internal capsule at the level of the tuberal hypothalamus (Fig. 4A–D, G). Ventromedially, fine collater- als detached like a “pony-tail” from these axons (Figs. 3G, inset, 4A, C) and proceeded ventromedially to the region that is occupied by the parvafox nucleus (Figs. 3G, 4A–G). These fine axons branched profusely and terminated as The range of possible targets of the LO- and the VLO- cortices varied somewhat: when the injections hit primarily the VLO-cortex, the PV2 and the LDTg in particular were strongly stained, whereas the parvafox- and the Su3 nuclei received fewer terminals. The primary and the secondary visual cortices were also stained, as was the perirhinal area; likewise the parabrachial (LPBreL), but not the basolateral 1 3 3 Brain Structure and Function (2019) 224:293–314 302 302 Brain Structure and Function (2019) 224:293 314 1 3 synaptic “boutons” in an horizontal column—with a length and a breadth of 1 mm and 0.5 µm, respectively, in rats, and of 0.5 µm and 0.2 µm, respectively, in mice—which was sandwiched between the optic tract and the fornix. The fine axons sent out collaterals along their entire hypothalamic course. They were oriented perpendicular to the parental sandwiched between the optic tract and the fornix. The fine axons sent out collaterals along their entire hypothalamic course. They were oriented perpendicular to the parental synaptic “boutons” in an horizontal column—with a length and a breadth of 1 mm and 0.5 µm, respectively, in rats, and of 0.5 µm and 0.2 µm, respectively, in mice—which was 1 3 3 3 Brain Structure and Function (2019) 224:293–314 303 traversed the most distal portion of the parvafox nucleus (bregma level: − 2.2) and innervated the Gemini nuclei (Gem), which are located dorsomedially (Fig. 6E). The ter- minal field innervating the Gemini nuclei (ipsilateral > than contralateral) had a larger diameter than the one depicted in a standard mouse brain atlas (Paxinos and Franklin 2013). Fig. 3 Injection sites in the OFC and terminal fields in lateral hypo- thalamus and PAG. A–F Nanozoomer scans of coronal sections through six specimens (4 mice: A–D; 2 rats: E, F), revealing the course of the Hamilton-syringe needle (vertical arrows) and the site of deposition of the tracer at its tip (white dot). Hypothalamic targets of the OFC In three cases (A 222-13; E 127-13; F 164-15; G 557-12), the six layers of the OFC- cortex were imbibed with the tracer, whereas in cases B (223-13) and c (250-13), only the deeper layers (V and VI) of the LO-cortex were labelled. In each of the depicted cases, axonal terminals were revealed in the parvafox nucleus, as well as in the Su3- and the PV2 nuclei. G: Para-sagittal section through the brain of a mouse in which the tracer had been injected into the LO-cortex [with spreading to the VLO- and the VO- cortices (142-14)]. The bundles of axons passing through the CPu and converging on the cerebral peduncle (cp) of the posterior hypothalamus are clearly visible (see also Fig. 4a). The ter- minal field to the parvafox nucleus is indicated with an arrow. The inset shows the almost vertical projection of thin collateral axons (bracket), deriving from the cp and generating the rich terminal field of the parvafox nucleus. H Horizontal section through a mouse brain (rostral side up), injected into the LO-with spreading to the VLO- cortex (141-14). I Coronal section through a mouse brain showing an injection limited to the MO-cortex (129-15), with no projections to parvafox, Su3 and PV2. J, K Sections through the LO-cortex in a Pvalb-Cre mouse (394-14) which had been injected with a Cre- dependent AAV-virus (J). The section was then incubated with an antibody against Parv (K). The cells that took up the tracer are Parv- immunoreactive (arrows). Blood vessel. L Transsynaptic retrograde visualization of neurons in the prefrontal cortex after Cre-dependent rabies injection in the parvafox nucleus of a Pvalb-Cre/Foxb1-Cre mouse (183-16). Positive neurons are mainly detected in layers V–VI of the LO/VLO cortex. The inset shows an image stack taken with the confocal microscope in the area of the VLO-LO-cortex (frame): the perikarya and the apical dendrites of the pyramidal cells are well visible. Lat lateral, Med medial. M Overlapping terminal fields in the ventrolateral portion of the Su3-region of the PAG (arrow). The green (tracer injected in the LO-cortex) and the red (tracer injected in the parvafox of a Pvalb-Cre/Foxb1-Cre mouse) terminals intermingle and generate the orange tonality (arrow) in Su3. Pvalb-immunoreactivity (magenta) highlights the oculomotor nucleus (3N). 3PC: parvicellular part of the oculomotor nucleus. N Presence of terminals coming from the LO/VLO cortex in the reticular thalamic nucleus (NRth). Mesencephalon At a slightly more distal level, namely, at the location in which the substantia nigra appears in the midbrain (bregma level: − 2.7), another bundle of coarse axons emitted from the cerebral peduncle in the same dorsomedial direction heavily innervated an oval volume that occupied a large por- tion of the ventral tegmental area (VTA) (Fig. 6F). It also gave rise to terminals in the PBP, the PN and PiF (Fig. 6F). This pattern of endings was observed after injections into both the LO- and the VLO-cortices, but the staining was more intense in the former than in the latter case. A projec- tion from the LO-cortex to the VTA-region has indeed been reported in a trans-synaptic retrograde study using rabies viruses as a tracer (Ogawa et al. 2014; Watabe-Uchida et al. 2012). Axons of the cerebral peduncle subdivided into two formations. One was located medially and extended to the mid-portion of the PAG (Fig. 7A, C). It sent out collaterals, which ended in a column of terminals in the ventrolateral part of the Su3 nucleus (Fig. 5A–C), as has been indicated by others (Beckstead 1979). This terminal field corre- sponded in location to the terminal field of axons deriving from both the Parv as well as the Foxb1-subpopulation of neurons in the parvafox nucleus of the hypothalamus (Bilella et al. 2016; Celio et al. 2013). Injections preponderantly in the VLO-cortex lead to terminals in the dorsolateral part of the Su3 (Fig. 5C′) and additionally to endings located in the dorsolateral PAG (not shown). The second termination field was located slightly more caudally, in the region of the PV2 nucleus (Figs. 4i, 5B′) (Celio et al. 2013). It extended to the laterodorsal tegmental nucleus (LDtg) of the PAG (Fig. 7A, C), as has been already reported (Leichnetz et al. 1987b). The existence of connections between the OFC and the ocu- lomotor region was confirmed earlier by retrograde tracing techniques (Leichnetz and Gonzalo-Ruiz 1987a; Leichnetz et al. 1987a).i shafts and often ran in parallel with the dendrites of the Parv-expressing neurons (Celio et al. 2013). Most Parv- and Foxb1-expressing neurons in the parvafox nucleus received a substantial input from a few collaterals via repetitive axo- dendritic (and axo-somatic) synapses, and also sampled activity via non-repetitive boutons en passant. Mesencephalon That the ter- minals from the OFC impinged on neurons in the parvafox nucleus was confirmed by injecting a Cre-dependent AAV- tomato tracer into its Parv and Foxb1-expressing neurons. The OFC-derived terminal endings abutted on the surfaces of the cell bodies and the dendrites, which were revealed with the tomato tracer (Fig. 4E, F). Hence, the presence of synaptic contacts is conceivable, and has been definitively proved by electron microscopy (see suppl. Figure 1). shafts and often ran in parallel with the dendrites of the Parv-expressing neurons (Celio et al. 2013). Most Parv- and Foxb1-expressing neurons in the parvafox nucleus received a substantial input from a few collaterals via repetitive axo- dendritic (and axo-somatic) synapses, and also sampled activity via non-repetitive boutons en passant. That the ter- minals from the OFC impinged on neurons in the parvafox nucleus was confirmed by injecting a Cre-dependent AAV- tomato tracer into its Parv and Foxb1-expressing neurons. The OFC-derived terminal endings abutted on the surfaces of the cell bodies and the dendrites, which were revealed with the tomato tracer (Fig. 4E, F). Hence, the presence of synaptic contacts is conceivable, and has been definitively proved by electron microscopy (see suppl. Figure 1). A lateral formation of fibres dissociated in the Crus cerebri from the one that was destined to innervate the region of the Su3-nucleus. These fibres projected to, and terminated in the tectum of the midbrain, specifically, in the lateral edge of the intermediate grey (InG) and white (InWh) layers of the superior colliculus, where they formed a spherical (223-13, LO-Injection) or a honeycomb or “fence”-like structure (Figs. 5D, E, 7A, C), as has been before described (Beckstead 1979). These terminals were The coarse parental axons of the cerebral peduncle curved upwards dorsomedially to form the periventricular system. A slender bundle of axons arising from the cerebral peduncle 1 3 304 Brain Structure and Function (2019) 224:293–314 identified both in LO- as well as in VLO-cortex injected specimen. Those deriving from the VLO-cortex were located more dorso-medially in respect to those from the LO-cortex. Terminals were observed also in the medial pontine nuclei (Fig. 5F) and also in the reticulotegmental nucleus (Fig. 7). Metencephalon The OFC-derived terminals around the neurons in the PV2 nucleus continued distally to the dorsal portion of the LDTg and to the sublaterodorsal nucleus. Mesencephalon A slender comma-shaped field of terminals was evident contralateral to the injection site in the region of the lateral parabrachial nucleus (LPB) 304 Brain Structure and Function (2019) 224:293–314 identified both in LO- as well as in VLO-cortex injected specimen. Those deriving from the VLO-cortex were located more dorso-medially in respect to those from the LO-cortex. Terminals were observed also in the medial pontine nuclei (Fig. 5F) and also in the reticulotegmental nucleus (Fig. 7). The OFC-derived terminals around the neurons in the PV2 nucleus continued distally to the dorsal portion of the LDTg and to the sublaterodorsal nucleus. A slender comma-shaped field of terminals was evident contralateral to the injection site in the region of the lateral parabrachial nucleus (LPB) 1 3 Brain Structure and Function (2019) 224:293–314 305 the compact portion of the nucleus ambiguus (Fig. 7C) in the parvicellular reticular nucleus (PcRtA). On the ipsilateral side, the field of projection of the OFC-derived axons and that of the terminals were identical to those on the contralat- eral one, but were less dense and less intensely stained. Fig. 4 Axonal endings in the ventrolateral hypothalamus and the ven- trolateral PAG. Terminals of LO/VLO-cortex-derived axons in the region of the parvafox nucleus of the ventrolateral hypothalamus and in the Su3 and PV2 nucleus of the PAG. A, B Low- and higher mag- nification views of the endings in the parvafox nucleus of rat speci- men 557-12, taken at two different coronal levels. A group of thin axons (small white arrows) derives from thicker ones that are located in the cerebral peduncle (cp) and converge towards the parvalbumin- immunoreactive neurons (red, in B) which define the axis of the parvafox nucleus. The terminal field embraces a larger area than is occupied by the parvalbumin-expressing neurons (B), since the axons project also to the co-axially located Foxb1-expressing ones, which are located peripherally in the parvafox nucleus (Bilella et al. 2014). The parvafox nucleus is rich in terminals, some of which lie adjacent to the cell bodies and the dendrites of the parvalbumin-expressing neurons (B). The rest of the lateral hypothalamus receives almost no inputs. TO: optic tract Scale bars: A 0.1 mm; B 0.05 mm. C, D Coro- nal views of the parvafox nucleus and the surrounding lateral hypo- thalamus on both sides of the same specimen (557-12 right and left). Mesencephalon C (557-12r), terminals are apparent almost exclusively in the parva- fox nucleus (traced in white). D (557-12 l), the entire lateral hypothal- amus is richly supplied with terminals. The injection (C) was located in the LO, the injections (D) were located more laterally (DLO). TO: optic tract. Scale bars: 0.2 mm. E LO-cortex-derived axonal endings around Parv-neurons of the parvafox nucleus in a Pvalb-Cre mouse that was injected stereotactically with the AAV-tomato tracer (223- 13). Scale bar: 0.5 mm. F LO-cortex derived axonal endings around Foxb1 neurons of a Foxb1 mouse that was injected stereotactically with the tomato tracer (specimen 390-14). Scale bar: 0.5 mm. G LO- cortex-derived terminals in the parvafox nucleus (arrow) of a Pvalb- Cre mouse (specimen 356-14). The terminals in the parvafox nucleus stem from thin axons that emanate from thicker ones in the cerebral peduncle (cpd). TO: optic tract. Scale bar: 0.2 mm. H Topographic relationship of the OFC endings in the Su3 region and catecholamin- ergic neurons revealed by TH-immunofluorescence. DR dorsal raphe, CLi caudal linear raphe nucleus, 3N oculomotor nucleus, DA8 DA8 dopamine cells. Scale bar: 0.4 mm. I LO-cortex derived axonal end- ings around the Parv-positive neurons of the PV2-nucleus in the pos- terodorsal raphe nucleus. Scale bar: 0.3 mm. J VLO-cortex derived axons terminate in the lateral parabrachial nucleus (LPBreL) of the contralateral side. DTg, VTG: dorsal, resp. ventral tegmental nucleus. LDTg laterodorsal tegmental nucleus, scp superior cerebellar pedun- cle. Scale bar: 0.3 mm ◂ Medulla oblongata Rostrally, the contralateral IRT and the GIA received most of the terminals, and the Amb none (Fig. 7C). More distally, the upper portions of the contralateral IRt, MdV and MdD, received terminals from the LO-cortex. Ipsilaterally, the den- sity of the terminals was much lower. Cre‑dependent tracers Injections of Cre-dependent tracers into the OFC of Parv- Cre mice lead to the impregnation of neurons in various lay- ers (Fig. 3J, K). A small contingent of axons left the cortex above the external capsule, passing through the caudatopu- tamen and occupying the internal capsule. Their position therein corresponded to that of the projections of the pyrami- dal neurons in layers V–VI, and they distributed terminals to the same sites [whole extent of the parvafox (Fig. 4G)], Su3 nucleus, PV2 nucleus (a few). In the thalamus, the endings were detected in the mediodorsal central nucleus (306-15; 394-14), in the submediodorsal and the ventral nuclei (309- 15, 394-14) and in the Gemini nuclei (394-14). They were also observed in the laterodorsal tegmental nucleus (LDTg) and in the reticulotegmental nucleus (RtTG; 307-15) of the brainstem; they ended in the central grey-alpha region of the pons. Retrograde tracing experiments 4i). Aq aqueduct, DRD, DRV, DRL dorsal, ventral and lateral parts of the dorsal raphe nucleus, PDR posterodorsal raphe nucleus. Scale bars: A 0.2 mm. C After the injection of a tracer into the LO-cortex of a mouse (specimen 222-13, depicted in Fig. 3a), labelled terminals were observed ventro-laterally in the Su3 nucleus (Su3 vl, arrow). Scale bar: 0.2 mm. C′ Injecting the tracer in the VLO-cortex (speci- men 164-15, depicted in Fig. 3d) revealed the presence of termi- nals in the dorsomedial part of the Su3 (Su3 dm, arrow). Scale bar 0.2 mm. D In addition to the Su3-region, LO-derived axons ascend from the cerebral peduncle to terminate in the most lateral edge of the intermediate grey (InG) and white layers (InWh) of the superior col- liculus (SC). Terminals from the VLO-cortex are located more dorso- medially. mRT mesencephalic reticular formation, DLPAG dorsolat- eral PAG. Scale bar: 0.5 mm. E In horizontal sections, this terminal field in the superior colliculus (SC) had a honeycomb- () or “fence”- like appearance (141-14), see also Beckstead (1979). Ant anterior, Cb cerebellum, IC inferior colliculus, SC superior colliculus. Scale bar: 0.15 mm. F Terminals were observed also in the medial pontine nuclei (Pn) and in the reticulotegmental nucleus (RtTg; Figs. 6f, 7b, c). Scale bar: 0.1 mm Fig. 5 Terminal endings in the midbrain. Fluorescence images of LO- cortex-derived terminals in the Su3- and the PV2 nuclei of the PAG. A, B, B′′ Endings of the axonal collaterals of OFC-derived projec- tions in two rats (specimen 557-12 and 127-13, injections depicted in Fig. 3f, g), which form a cap over the ventrolateral part of the oculomotor nucleus (3N; A, B compare also Fig. 3m) referred to as the Su3 nucleus (Carrive and Paxinos 1994). Slightly distally, end- ings terminate in the PV2 nucleus (B′ compare also with Fig. 4i). Aq aqueduct, DRD, DRV, DRL dorsal, ventral and lateral parts of the dorsal raphe nucleus, PDR posterodorsal raphe nucleus. Scale bars: A 0.2 mm. C After the injection of a tracer into the LO-cortex of a mouse (specimen 222-13, depicted in Fig. 3a), labelled terminals were observed ventro-laterally in the Su3 nucleus (Su3 vl, arrow). Scale bar: 0.2 mm. C′ Injecting the tracer in the VLO-cortex (speci- men 164-15, depicted in Fig. 3d) revealed the presence of termi- nals in the dorsomedial part of the Su3 (Su3 dm, arrow). Scale bar 0.2 mm. Retrograde tracing experiments Concomitant retrograde tracing with fluorogold from the ventrolateral region of the hypothalamus that harboured the parvafox nucleus and with BDA (MW: 3000) from the ventrolateral PAG revealed layer-V pyramidal cells in the OFC to contain both tags (Fig. 6A–D). Hence, collaterals emanating from the axons of some of the pyramidal cells in layer V–VI innervated the parvafox nucleus whilst the main fibres continued distally to innervate the PAG. Similar findings have been documented for neurons of the VLO- and the LO-cortices, which collateralize first to the caudatoputa- men and then to the core of the accumbens (Reynolds and Zahm 2005). (Figs. 4J, 7C), which is sandwiched between the LPB and the MPB. This region may be occupied by one of the CCK- expressing group of neurons that relay thermosensitive information to the lateral hypothalamus (Geerling et al. 2016). This projection was observed only when the injec- tions involved the VLO-cortex. The parental axons continued caudally to the cerebral peduncle of the pons, wherefrom they spread out in a fan- like manner to both of its halves (Fig. 7C). After the dis- tance of a few micrometres in the dorsal direction, the axons branched into collaterals of unequal thickness. The thicker ones innervated the contralateral reticular formation (Gi, IRt and PcRt). The terminal fields were confined by the sharp boundaries of the prepositus hypoglossi nucleus upwards, by those of the spinal trigeminal nucleus sidewards and by those of the lateral paragigantocellular nucleus downwards. A field of higher-density terminals was observed just laterodorsal to Retrograde, trans-synaptic labelling with Cre-depend- ent rabies-virus constructs, which were injected into the region that was occupied by the parvafox nucleus in Pvalb-Cre/Foxb1-Cre mice, confirmed the presence of tagged neurons in the LO- and the VLO-cortices (Fig. 3L) but also in the medial prefrontal cortex (MO). 1 3 Brain Structure and Function (2019) 224:293–314 306 ( ) Fig. 5 Terminal endings in the midbrain. Fluorescence images of LO- cortex-derived terminals in the Su3- and the PV2 nuclei of the PAG. A, B, B′′ Endings of the axonal collaterals of OFC-derived projec- tions in two rats (specimen 557-12 and 127-13, injections depicted in Fig. 3f, g), which form a cap over the ventrolateral part of the oculomotor nucleus (3N; A, B compare also Fig. 3m) referred to as the Su3 nucleus (Carrive and Paxinos 1994). Slightly distally, end- ings terminate in the PV2 nucleus (B′ compare also with Fig. Retrograde tracing experiments D In addition to the Su3-region, LO-derived axons ascend from the cerebral peduncle to terminate in the most lateral edge of the intermediate grey (InG) and white layers (InWh) of the superior col- liculus (SC). Terminals from the VLO-cortex are located more dorso- medially. mRT mesencephalic reticular formation, DLPAG dorsolat- eral PAG. Scale bar: 0.5 mm. E In horizontal sections, this terminal field in the superior colliculus (SC) had a honeycomb- (*) or “fence”- like appearance (141-14), see also Beckstead (1979). Ant anterior, Cb cerebellum, IC inferior colliculus, SC superior colliculus. Scale bar: 0.15 mm. F Terminals were observed also in the medial pontine nuclei (Pn) and in the reticulotegmental nucleus (RtTg; Figs. 6f, 7b, c). Scale bar: 0.1 mm This pattern of distribution indicates that all these OFC regions target the Foxb1-expressing sub-population of the parvafox nucleus. The MO-neurons probably label single Foxb1-expressing neurons located in the region surrounding the parvafox. In the Pvalb-Cre mice, the label was observed in layers V–VI of the LO-cortex, at the location of the upper lip of the rhinal sulcus. In the Foxb1-Cre mice, the label was detected also in the adjoining VLO- and the VO-cortices. 1 3 Brain Structure and Function (2019) 224:293–314 307 VLO pyramidal neurons send collaterals to both hypo- nd PAG. Terminals in the Gemini (Gem) and reticuloteg- leus (RtTg). Axons terminating in the parvafox nucleus are als of fibres that continue their course distally to innervate ateral PAG. Transverse sections through the rat OFC after (A, C) and BDA (B, D), had been simultaneously injected ntrolateral region of the hypothalamus that harbours the ucleus (fluorogold) and into the ventrolateral region of the hich the Su3- and the PV2 nuclei reside (BDA). Pyrami- layers V–VI of the LO/VLO-cortex were double-labelled ows in C, D) with two different retrogradely transported the medial portions of the prefrontal cortex than did BDA injected in the ventrolateral PAG. OL olfactory lobe, RhF rhinal fissure, I–VI cortical layers. Scale bars: A, B 0.3 mm; C, D 0.03 mm. E The ipsi- lateral nucleus Gemini (Gem), receives a strong innervation, particu- larly from the LO-cortex, visible also on the contralateral side. Notice the tendril-like bundle of axons (arrowheads) joining the nucleus Gemini from the ventromedial part of the cerebral peduncle. Scale bar 0.5 mm. Retrograde tracing experiments F In this parasagittal section (rostral to the right), in addition to PBP and PN, also the rich innervation of the reticuloteg- mental nucleus (RtTg) is visible. A large number of axons perpen- dicular to the brain surface course in the isthmic reticular formation Fig. 6 LO/VLO pyramidal neurons send collaterals to both hypo- thalamus and PAG. Terminals in the Gemini (Gem) and reticuloteg- mental nucleus (RtTg). Axons terminating in the parvafox nucleus are the collaterals of fibres that continue their course distally to innervate the ventrolateral PAG. Transverse sections through the rat OFC after fluorogold (A, C) and BDA (B, D), had been simultaneously injected into the ventrolateral region of the hypothalamus that harbours the parvafox nucleus (fluorogold) and into the ventrolateral region of the PAG in which the Su3- and the PV2 nuclei reside (BDA). Pyrami- dal cells in layers V–VI of the LO/VLO-cortex were double-labelled (white arrows in C, D) with two different retrogradely transported dyes. Fluorogold injected in the ventrolateral hypothalamus revealed the presence of a larger number of labelled neurons in the orbital and the medial portions of the prefrontal cortex than did BDA injected in the ventrolateral PAG. OL olfactory lobe, RhF rhinal fissure, I–VI cortical layers. Scale bars: A, B 0.3 mm; C, D 0.03 mm. E The ipsi- lateral nucleus Gemini (Gem), receives a strong innervation, particu- larly from the LO-cortex, visible also on the contralateral side. Notice the tendril-like bundle of axons (arrowheads) joining the nucleus Gemini from the ventromedial part of the cerebral peduncle. Scale bar 0.5 mm. F In this parasagittal section (rostral to the right), in addition to PBP and PN, also the rich innervation of the reticuloteg- mental nucleus (RtTg) is visible. A large number of axons perpen- dicular to the brain surface course in the isthmic reticular formation (isRt) to innervate the laterodorsal tegmental nucleus (LDTg). Scale bar: 0.5 mm 1 3 3 308 Brain Structure and Function (2019) 224:293–314 Relationship between terminals in the PAG At the level of the PAG, the relationship between endings deriving from neurons in the LO-VLO-cortices and those emanating from Parv- or Foxb1-expressing neurons were studied in the confocal scanning microscope after a double injection of fluorescence tracers Axons from the OFC were Ultrastructure Axons from the OFC ended primarily with asymmetric syn- apses, characterized by round synaptic vesicles in the pre- terminal endings and a thick postsynaptic density (suppl. Retrograde tracing experiments Figure 1A). In the region of the parvafox, axons of the OFC themselves received both excitatory (suppl Figure 1B) as well Relationship between terminals in the PAG At the level of the PAG, the relationship between endings deriving from neurons in the LO-VLO-cortices and those emanating from Parv- or Foxb1-expressing neurons were studied in the confocal scanning microscope after a double injection of fluorescence tracers. Axons from the OFC were revealed with a Cre-independent EGF-tracer and those in the parvafox nucleus with a Cre-dependent tomato tracer (Fig. 3M). Although most of the terminals impinged on resi- dent neurons in the PAG, contacts between the two popu- lations of terminals were common (not shown). Terminals deriving from the parvafox nucleus were sometimes pre- synaptic and sometimes post-synaptic relative to those that Ultrastructure Axons from the OFC ended primarily with asymmetric syn- apses, characterized by round synaptic vesicles in the pre- terminal endings and a thick postsynaptic density (suppl. Figure 1A). In the region of the parvafox, axons of the OFC themselves received both excitatory (suppl. Figure 1B) as well as inhibitory, symmetric synapses, characterized by oval syn- aptic vesicles (suppl. Figure 1C). Relationship between terminals in the PAG Ultrastructure At the level of the PAG, the relationship between endings deriving from neurons in the LO-VLO-cortices and those emanating from Parv- or Foxb1-expressing neurons were studied in the confocal scanning microscope after a double injection of fluorescence tracers. Axons from the OFC were revealed with a Cre-independent EGF-tracer and those in the parvafox nucleus with a Cre-dependent tomato tracer (Fig. 3M). Although most of the terminals impinged on resi- dent neurons in the PAG, contacts between the two popu- lations of terminals were common (not shown). Terminals deriving from the parvafox nucleus were sometimes pre- synaptic and sometimes post-synaptic relative to those that emanated from the LO-cortex. Axons from the OFC ended primarily with asymmetric syn- apses, characterized by round synaptic vesicles in the pre- terminal endings and a thick postsynaptic density (suppl. Figure 1A). In the region of the parvafox, axons of the OFC themselves received both excitatory (suppl. Figure 1B) as well as inhibitory, symmetric synapses, characterized by oval syn- aptic vesicles (suppl. Figure 1C). 1 3 Brain Structure and Function (2019) 224:293–314 309 arising in the LO-cortex. This medial location of the VLO- projection is also observed in other part of the brain like the caudatoputamen, and superior colliculi. In the ventral tegmental area, the projection from the LO-cortex is more important than the one deriving from the VLO-cortex. There are also specific projections for each one of these two sub- divisions of the OFC. The VLO-cortex for example, inner- vates parts of the visual cortex (Reep et al. 1996), the dorsal raphe (DR), the dorsolateral column of the PAG, a region that is also target of the Foxb1-subpopulation of neurons of the parvafox nucleus (Bilella et al. 2016) and has a specific projection to the contralateral, dorsal parabrachial nucleus (LPBrel). The LO-cortex innervates the ethmoid thalamic nucleus, the basolateral amygdala (Groenewegen et al. 1990; McDonald et al. 1996) and the ventral part of the submedius nucleus (Craig et al. 1982). In addition, terminals of the LO/ LO cortex were also observed in nuclei not mentioned in the previous literature, namely the Gemini nuclei, the lateral parabrachial nucleus, the pontine nuclei, the reticulotegmen- tal nucleus, the reticular substance of the brainstem and the lateral horn of the spinal cord. No major differences in the projection patterns were detected between rats and mice and in both species the projections to parvafox, Su3 and PV2 were of comparable intensity. Ultrastructure Fig. 7 Schematic representation of the projections from the LO/ VLO cortex to the rest of the brain. The courses of the major axonal branches deriving from the LO-cortex in mice are here superim- posed to reconstructions from the Allen Brain Atlas (experiment no. 112306316). A In this lateral view, the bulk of the axons descend from the LO-cortex to the lateral hypothalamus at an angle of ~ 120° relative to the surface of the brain (see also Fig. 3e). During their course, axons send out branches to various parts of the brain: to the olfactory tubercle (Tu), to the piriform cortex (Pir), the caudate– putamen (Cpu), various nuclei of the thalamus (TH), the nucleus of the horizontal limb of the diagonal band (HDB), the magnocellular hypothalamic nucleus (MCPO), the parvafox nucleus, the Gemini nucleus (Gem), the ventral tegmental area (VTA), the superior col- liculus (SC), the Su3 nucleus and the PV2/LTDg-region. The main track of parent axons arches and sends out projections to the pontine nuclei (Pn) and the reticulotegmental nucleus (RtTg). It continues its course more distally to innervate the entire dorsal part of the reticular substance PcRt, IRt, mRT, MdD, MdV, and ends in the lateral horn of the cervical spinal cord. B In this frontal view the LO-cortex-derived projection follows a low-angled course relative to the sagittal plane. It sends out branches to the motoric and the sensory cortices, the baso- lateral nucleus of the amygdala (BLA), the thalamus [central medi- odorsal (MDC) and submedius-(Sub)], the pontine nuclei (Pn) and the reticulotegmental nucleus (RtTg). C Flat view of the projections from the LO- and the VLO-cortices to various parts of the brain, as represented in Swanson’s atlas (Swanson 2004). The axons deriving from the LO/VLO cortices send out branches and collaterals at vari- ous levels, mainly ipsilaterally, until the end of the midbrain. In the medulla oblongata, the thicker collaterals project contralaterally and the thinner ones ipsilaterally (to simplify the drawing, the collater- als are drawn only on the side of the brain where they are more pro- nounced). The axons cross to the contralateral side via the pyramidal decussation (pyx). Many of the OFC projections to individual targets depicted here, have been described by earlier authors: we additionally observed substantial innervation of the parvafox nucleus in the vent- rolateral hypothalamus and of the Su3- and the PV2 nuclei in the ven- trolateral PAG. Ultrastructure Furthermore, we recognized terminals in the Gemini nucleus (Gem), in the lateral parabrachial nucleus LPBreL (all indi- cated in red) and in the reticular formation. We could also distinguish between projections that emanated from either the VLO- (green) or the LO-cortex (light-blue). Each of the indicated structures is men- tioned in the list of abbreviations. LH lateral horn, DH dorsal horn, SC superior colliculus, VH ventral horn of the spinal cord. Scale bars in A, B: 0.6 mm ◂ Using a Cre-dependent rabies virus (Wall et al. 2010; Wickersham et al. 2007b) the afferences to the Parv- and the Foxb1-expressing neurons could be studied separately and selectively in the corresponding Cre-mice. The input to the Parv-expressing neurons that represent the core of the parva- fox nucleus originates mainly in the LO-cortex, whereas the inputs to the Foxb1-expressing neurons derive from the medial (IL, PrL), the orbitofrontal (MO, VO, VLO, LO) and even the lateral prefrontal cortices. These projection pat- terns correspond well with those that have been revealed by retrograde tracing with peroxidase from the rat ventrolateral hypothalamus [(Allen and Cechetto 1993) their Fig. 5a]. Our study is the second of its kind in which the anter- ograde projections of the LO-VLO cortex throughout the entire brain have been mapped. In the other investigation, which appeared four decades ago, autoradiographic tech- niques were implemented to map the staining profiles of OFC-derived projections after the injection of tritiated amino acids (Beckstead 1979). In parallel to our study, a large body of data appertaining to stereotactic injections into the OFC have been published during the past 3 years in the ABA (http://www.brain-map.org), some of which reveal patterns of labelling of the terminal fields that are almost identical to those that we observed. Our data and those that are recorded in the ABA largely confirm the previous, pre- cise observations of Leonard (1969), Beckstead (1979) and Reep et al. (1996) in the rat as well as for VLO in the cat (Craig et al. 1982). To the list of known targets, we append three chemically defined sites (the parvafox-, the Su3- and Discussion The distribution of the terminal fields accorded with our own observations, although those in the lateral hypothalamic parvafox nucleus were more sparse (their Fig. 3F). The ter- minals that were detected in the “ventrolateral wings of the dorsal raphe nucleus” probably correspond to those in the region of the Su3-region (their Fig. 3G). The Su3 nucleus is known to project to the contralateral rostral- and the caudal ventrolateral medulla, which together constitute the sympathetic cardiovascular control centre (Chen and Aston-Jones 1996; Van Bockstaele et al. 1989). The Su3 nucleus receives inputs from the medial cerebel- lar nucleus (fastigium) (Gonzalo-Ruiz and Leichnetz 1987; Gonzalo-Ruiz et al. 1990) and from the parvafox nucleus (Bilella et al. 2016; Celio et al. 2013) and is activated dur- ing predation [of insects as well as by cats (Comoli et al. 2012)]. The connections and functions of the PV2-nucleus are yet unknown. The hypothalamus and the PAG, two regions of the brain that harbour the parvafox-, and the Su3- and the PV2 nuclei, are themselves interconnected. This is a general organiza- tion, which has been described for the lateral hypothalamus broadly and for the ventrolateral PAG in particular (Floyd et al. 2000, 2001). The parvafox nucleus, does not recipro- cate the projections from the OFC (Bilella et al. 2016; Celio et al. 2013), either directly or indirectly, via an innervation of the mediodorsal thalamic nucleus. The findings of the double retrograde tracing experiments that were conducted by Gabbott and his colleagues (Gabbott et al. 2005), as well as by ourselves, indicate that the projection from the LO/ VLO-orbitofrontal cortex is—at least in part—serial, with the same axon successively innervating multiple subcortical targets via collaterals. Using the mouse connectivity programme of the Allen Database (ABA; http://www.brain-map.org) and entering as source structures various areas of the prefrontal cortex without any filter for mouse line or tracer type, the results corresponded well with our own data. For the purpose of our study, the most relevant injections in the ABA were the numbers 112423392 and 112306316, since they were performed in mice of the same wild-type strain (C57/ BI6). But also injections into the genetically modified lines A930038C07Rik-Tg1-Cr (Cre-recombinase confined to layer V; no. 168164972) and Rbp4-Cre_KL100 (Cre-recombinase confined to layer V; no. 287769286) revealed patterns of projections that accorded with the observations in our own. Discussion Beckstead observed the projection to terminate in the laterodorsal tegmental nucleus, probably in the region that we refer to as the PV2 nucleus, owing to its composition of parvalbumin-expressing neurons (Celio et al. 2013). In the publication by Jasmin (Jasmin et al. 2004), tracers were injected into a region which, according to Price’s group is probably still the LO-cortex but which the former authors defined as the AIV [images 1a and b in Jasmin et al. (2004)]. The distribution of the terminal fields accorded with our own observations, although those in the lateral hypothalamic parvafox nucleus were more sparse (their Fig. 3F). The ter- minals that were detected in the “ventrolateral wings of the dorsal raphe nucleus” probably correspond to those in the region of the Su3-region (their Fig. 3G). the PV2 nuclei). Our knowledge of its existence (Bilella et al. 2016; Celio et al. 2013; Meszar et al. 2012), guided our attention to recognize the parvafox, a small elongated neuronal aggregates as targets of the projections of the LO- and the VLO-cortices [“Chance favours the prepared mind” (citation of a statement by Louis Pasteur)]. In the aforemen- tioned autoradiographic study, the presence of terminals in a tiny oval region that lay along the optic tract was actu- ally documented pictorially [injection 8, Fig. 6 (Beckstead 1979)], but not mentioned in the text. From its position, we presume it to be the parvafox nucleus. The cortical projec- tion proceeded caudally to the ventrolateral PAG, namely, to a region that lay dorsal to the nucleus of the third nerve, which was referred to by Beckstead as a “subaqueductal portion”, and which approximately corresponds to the ter- minal field that we observed in the supraoculomotor region (the Su3 nucleus). A terminal field at precisely the same location has been described in feline brains after the injec- tion of tracers into the VLOβ-region [Fig. 15 in Craig et al. (1982)]. Beckstead observed the projection to terminate in the laterodorsal tegmental nucleus, probably in the region that we refer to as the PV2 nucleus, owing to its composition of parvalbumin-expressing neurons (Celio et al. 2013). In the publication by Jasmin (Jasmin et al. 2004), tracers were injected into a region which, according to Price’s group is probably still the LO-cortex but which the former authors defined as the AIV [images 1a and b in Jasmin et al. (2004)]. Discussion The hypothalamic target of the murine LO/VLO cortical regions—corresponding to the Area 13a and 13 m/l in pri- mates—is the newly described parvafox nucleus and its tar- gets Su3 and PV2 in the PAG. By drawing on self-replicating virus-based tracing tools, which enhance the sensitivity of connectivity studies, we have defined three targets of the LO/VLO cortices that have been hitherto overlooked, namely, the parvafox nucleus which is located in the ventrolateral hypothalamus, and the Su3- and the PV2 nuclei which are serially located ventral to the aqueduct in the periaqueductal grey matter (PAG). Both LO and VLO innervate these three targets, although partly in different portions: in the SU3-nucleus, for example, the VLO-projection is located more medially than the one 1 3 3 Brain Structure and Function (2019) 224:293–314 310 frontal eye field, Leichnetz (Leichnetz et al. 1987b) injected retrograde tracers into the oculomotor nucleus, to which end, the needle was oriented slightly obliquely (their experiments OMR2 and OMR3), thereby perforating the supraoculo- motor region (harbouring the Su3 nucleus). Retrogradely labelled neurons occurred primarily in the cortex of the dor- somedial shoulder (the putative rodent homologue of the primate frontal eye field), scatterings of stained cells were nevertheless observed throughout the entire OFC (Leichnetz et al. 1987b). the PV2 nuclei). Our knowledge of its existence (Bilella et al. 2016; Celio et al. 2013; Meszar et al. 2012), guided our attention to recognize the parvafox, a small elongated neuronal aggregates as targets of the projections of the LO- and the VLO-cortices [“Chance favours the prepared mind” (citation of a statement by Louis Pasteur)]. In the aforemen- tioned autoradiographic study, the presence of terminals in a tiny oval region that lay along the optic tract was actu- ally documented pictorially [injection 8, Fig. 6 (Beckstead 1979)], but not mentioned in the text. From its position, we presume it to be the parvafox nucleus. The cortical projec- tion proceeded caudally to the ventrolateral PAG, namely, to a region that lay dorsal to the nucleus of the third nerve, which was referred to by Beckstead as a “subaqueductal portion”, and which approximately corresponds to the ter- minal field that we observed in the supraoculomotor region (the Su3 nucleus). A terminal field at precisely the same location has been described in feline brains after the injec- tion of tracers into the VLOβ-region [Fig. 15 in Craig et al. (1982)]. Discussion In addition to its role in olfactory processing and in vis- ceromotor activity, the OFC is best known for its involve- ment in the expression of emotion and in reward-driven decision-making (Bechara et al. 2000; Rolls 2000; Schultz et al. 2000). This co-habitation of sensory, autonomic and behavioural networks in the OFC permits the “integration of primitive autonomic mechanisms (such as are associated with instinctive urges or emotional reactions) with neural activities at the highest functional level of the brain” (Clark Le Gros and Meyer 1950).i The lateral hypothalamic region in which the parvafox nucleus is located receives inputs from various olfactory regions and from the amygdala (Price et al. 1991). Each of these areas has reciprocal connections with the OFC, which the findings of our study have revealed to target, non-recip- rocally, the neurons of the parvafox nucleus. Furthermore, the olfactory tubercle projects to the Gemini nuclei, which are also targeted by the LO/VLO-cortex-derived projection and by the parvafox. It remains to be established whether the parvafox- or the Gemini nuclei have any distinct olfac- tory functions. The olfactory projection to the ventrolateral hypothalamus may regulate autonomic or neuroendocrine functions or related behaviours (Price et al. 1991), or it may simply contribute olfactory information to be integrated with other influences. Patients with lesions in the OFC [specifically of the VM], manifest an impaired ability to generate anticipatory “skin conductance responses” (SCR) to a conceived outcome of an action (Bechara et al. 2005; Damasio 1996). SCRs are emotional signals (somatic marker) that are generated by the activity of the autonomic nervous system, of which the hypothalamus is the main organizer (Hess 1947, 1957). Future studies will reveal whether the OFC → parvafox → PAG network that we have delineated in our study is the scaffold on which the first-named region engages the auto- nomic nervous system. In analogy to the SCRs, the OFC → parvafox → PAG network could also affect the physiology of the cardiovascular and the respiratory systems (Rainville et al. 2006) and be involved in pathologies thereof that relate to disturbances in high mental activities (Pickering et al. 1996). l In addition to its involvement in the processing of olfac- tory and gustatory information, the OFC also controls the cardiovascular and the respiratory systems (Fuster 2008). Discussion Part of the projection from the LO/VLO-cortex stems from parvalbumin-expressing neurons, which represent a sub-population of cortical GABAergic cells (Celio 1986). Long-range-projecting neurons expressing NO-synthase have been observed to connect cortical areas (Tamamaki and Tomioka 2010) and parvalbumin-expressing GABAergic ones are known to project from the medial prefrontal cortex to the nucleus accumbens (Lee et al. 2014). These parval- bumin-expressing neurons in the OFC are well positioned for a “top-down” inhibitory control of subcortical processes (Fuster 2008). In our study, their GABAergic nature was suggested by the presence of GAD-immunoreactive axonal endings on neurons in the parvafox nucleus. However, injec- tions of Cre-dependent tracers into VGAT-ires-cre mice (in which Cre-recombinase is expressed in the bodies of GABAergic neurons), revealed the presence of only a few projections outside the cortex (not shown). In the ABA, the The precise targets of the OFC-derived projection in the PAG are, with a high degree of axial and radial specificity, the Su3 nucleus, which is located slightly dorsolateral to the oculomotor nucleus, and the parvalbumin-expressing ones in the PV2 nucleus, which is located in the posterodorsal raphe nucleus (PDR) and rostral portion of the dorsolateral tegmental nucleus (LDTg). With a view to mapping the 1 3 1 3 311 Brain Structure and Function (2019) 224:293–314 injection of a tracer into the VLO of a Slc32a1-IRES-Cre mouse (VGAT) revealed no projecting axons (injection no. 309580102). Sweet 1940; Chapman et al. 1948; Delgado et al. 1960; Fer- nandez De Molina and; Hunsperger 1962, 1956; Gelsema et al. 1989; Hall and Cornish 1977; Hess 1957; Kaada et al. 1953; Loewy 1991; Ruggiero et al. 1987; Sachs et al. 1949; Verberne 1996; Verberne and Owens 1998; Yasui et al. 1991). The rat LO-cortex has been hitherto regarded as a con- stituent of the orbital network (Krettek and Price 1977) and to be more of a “sensory” than a visceromotor region. In addition to many other cerebral sites that have been reported by various authors, we have demonstrated the LO-cortex to project to circumscribed horizontal columns of neurons in the parvafox nucleus of the ventrolateral hypothalamus in the SU3 and the PV2 nuclei of the PAG. These findings suggest that the subdivision of the OCF-cortex into sensory and visceromotoric regions may not be as absolute in rodents as it is in monkeys (Price 2007). Discussion The pioneering work of electrophysiologists revealed the most prominent consequences of stimulating Area 13 of the OFC in primates to be manifested in the cardiovascular and the respiratory systems (Fuster 2008). The effects include changes in blood pressure, heart rate, cardiac dynamics, res- piratory rate and skin temperature (Bailey and Sweet 1940; Chapman et al. 1948; Delgado et al. 1960; Hall and Cornish 1977; Kaada et al. 1953; Sachs et al. 1949; Spencer 1894), which can even lead to cardiac histopathology (Hall and Cornish 1977). An exploration of various parts of the brain with electrodes whilst stimulating the OFC with strychnine (neuronography) has permitted a mapping of the connec- tions of Area 13 with the lateral hypothalamus, particularly with the ventrolateral region in which the median forebrain bundle resides (Sachs et al. 1949; Ward and McCulloch 1947). Interestingly, some of the autonomic effects that are evoked by stimulation of the OFC may be likewise elicited by stimulating the lateral hypothalamic region (where the parvafox is located) and the ventrolateral PAG (where the Su3 and PV2 nuclei are found), which receive these corti- cal afferences. (Allen and Cechetto 1992, 1993; Bailey and Acknowledgements We thank Laurence Clément, Christiane Marti and Rachel Ververidis for technical and administrative assistance. Funding This work was supported by the Canton of Fribourg and by a grant of the Swiss National Foundation (Grant number 31003A_160325). Compliance with ethical standards Ethical approval All procedures performed in studies involving ani- mals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. Conflict of interest The authors declare that they have no conflict of interest. Conflict of interest The authors declare that they have no conflict of interest. References J Physiol 160:200–213 Beckstead RM (1979) An autoradiographic examination of corticocor- tical and subcortical projections of the mediodorsal-projection (prefrontal) cortex in the rat. 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https://openalex.org/W2956526359	https://europepmc.org/articles/pmc6735767?pdf=render	English	null	TERC promotes cellular inflammatory response independent of telomerase	Nucleic acids research	2,019	cc-by	11,027	ABSTRACT positive cells, including human stem cells and most cancer cells (2,3). However, TERC is also ubiquitously expressed in terminally differentiated human somatic cells, which do not express TERT and therefore have no telomerase activ- ity (4). The function of TERC in these cells remains largely unknown. In telomerase positive cells, the level of TERC is often higher than that required to form telomerase with TERT (5,6). For telomerase negative cancers, termed alter- native lengthening of telomeres (ALT) cancers, TERC is of- ten detected, but TERT is lacking (7). TERC has been con- sidered for a long time as a nonfunctional RNA waiting for TERT to form active telomerase. However, recent dis- coveries hypothesized that TERC may play a role beyond telomerase. For instance, Gazzaniga et al. found that TERC has telomerase independent anti-apoptotic functions in hu- man T cells (8). In addition, it has been reported that TERC is involved in regulating ATR-mediated DNA damage sig- nals and in activation of DNA-PKcs that phosphorylates hnRNP A1 in a telomerase independent manner (9,10). TERC is an RNA component of telomerase. However, TERC is also ubiquitously expressed in most hu- man terminally differentiated cells, which don’t have telomerase activity. The function of TERC in these cells is largely unknown. Here, we report that TERC enhances the expression and secretion of inﬂam- matory cytokines by stimulating NK-B pathway in a telomerase-independent manner. The ectopic ex- pression of TERC in telomerase-negative cells alters the expression of 431 genes with high enrichment of those involved in cellular immunity. We perform genome-wide screening using a previously identiﬁed ‘binding motif’ of TERC and identify 14 genes that are transcriptionally regulated by TERC. Among them, four genes (LIN37, TPRG1L, TYROBP and USP16) are demonstrated to stimulate the activation of NK- B pathway. Mechanistically, TERC associates with the promoter of these genes through forming RNA– DNA triplexes, thereby enhancing their transcription. In vivo, expression levels of TERC and TERC target genes (TYROBP, TPRG1L and USP16) are upregu- lated in patients with inﬂammation-related diseases such as type II diabetes and multiple sclerosis. Col- lectively, these results reveal an unknown function of TERC on stimulating inﬂammatory response and highlight a new mechanism by which TERC modu- lates gene transcription. TERC may be a new target for the development of anti-inﬂammation therapeu- tics. p ( , ) LncRNAs may regulate gene expression in different ways. C⃝The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT For instance, lncRNAs can modulate the epigenetic status of target genes, influencing their transcription (11). In addi- tion, lncRNA may up- or downregulate gene transcription in cis by associating with their promoters (12). In this sce- nario, recent studies found that lncRNA-chromatin interac- tion is highly sequence dependent with many ‘binding mo- tifs’ exiting in lncRNA (13). It is thus proposed that using its binding motif, lncRNA may hybridize with targeted genes and regulate their transcription. Indeed, it has been demon- strated that many lncRNA–DNA interactions are mediated by formation of RNA–DNA triplexes (14–16). Although the ‘binding motif’ of TERC was identified years ago and it is hypothesized that as a typical lncRNA, TERC may par- ticipate in the regulation of gene transcription, whether and how many genes are transcriptionally regulated by TERC still remained elusive. TERC promotes cellular inﬂammatory response independent of telomerase Haiying Liu1, Yiding Yang1, Yuanlong Ge1, Juanhong Liu2 and Yong Zhao1,* 1MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Sun Yat-sen University 510006, P.R. China and 2Department of Endocrinology, Tongji Hospital, Tongji Medical College, Huazhon of Science and Technology, Wuhan, Hubei 430030, P.R. China eceived March 27, 2019; Revised June 09, 2019; Editorial Decision June 23, 2019; Accepted July 05, 2019 Published online 11 July 2019 Published online 11 July 2019 8084–8095 Nucleic Acids Research, 2019, Vol. 47, No. 15 doi: 10.1093/nar/gkz584 To whom correspondence should be addressed. Tel: +86 20399 43401; Fax: +86 20399 43778. Email: zhaoy82@mail.sysu.edu.cn C⃝The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://crea permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Melting profiles The ds-promoter segments of LIN37, TPRG1L, TYROBP and USP16 (in Table 1) were formed and mixed with or without TERC or TERC1–49nt at the ratio of 1:1 (mole). The melting profile was obtained in q-PCR working buffer containing SYSB green I in LightCycler 480 (Roche). Sam- ples were incubated at 60◦C for 1 min, and then increased to 95◦C at the rate of 2.5◦C/s. Fluorescence intensity was detected at 0.2◦C interval. ChIRP-PCR assay The ChIRP pull down assay was performed exactly as previously reported (13,21) using odd probes of TERC and lacZ. The product was detected by PCR with primers tar- geting promoters containing potential TERC binding sites. The potential target genes and PCR primer sequences are as follows: Lin37: F 5’-TTGGTCAGGATGCGAGATT- 3’, R 5’-TCCTCCGCCTTTGGTTGT-3’; TPRG1L: F 5’-GCAAGGCGGAGCCAATCG-3’, R 5’- ACCCCTTACCGACCCCGAC-3’; TYROBP: F 5’-CAAGTGAAGGAGGAAGTCTGA-3’, R 5’- CCTGATTCTTTCTTGGGTTTT -3’; USP16: F 5’- TCAGAGCCGATGGTCCCG -3’, R 5’- CTCCGTCTTC- CTCCTGGTGA -3’. Electrophoretic mobility shift assay Oligonucleotides were synthesized (Generay, China) and annealed into dsDNA in binding buffer (10 mM Tris–HCl pH7.4, 125 mM NaCl, 6 mM MgCl2). Oligonucleotides were heated at 95◦C for 3 min and cooled down to 50◦C with every 5◦C interval (90, 85, 80◦C...) helding for 3 min. In vitro TERC transcription was performed using High Yield Transcription Kit (Ambion), 450 nt Scramble sequence was used as a control. 1pmol of dsDNA was incubated with dif- ferent amounts of TERC in binding buffer for 2 h at 42◦C. The samples were analyzed on 2% agarose gel at 4◦C. p SiRNAs were used to knock down genes. SiRNA trans- fection was carried out with Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. Experiments were performed 72 h after siRNA transfection. The sequences of siRNAs are as follows: TERC si1: GUCU AACCCUAACUGAGAAGG; TERC si2: CCGUUCAU UCUAGAGCAAAC; Lin37 si1: GCAGCGAUCCAACA CAUAU; Lin37 si2: CCAACACAUAUGUGAUCAA; SL C26A1 si1: GCAACACCCAUGGCAAUUA; SLC26A1 si2: GCCUCUAUACGUCCUUCUU; TPRG1L si1: CC AUUUCCUACGGAGAAUU; TPRG1L si2: GGAAUC CCUGGUCUACCAA; TYROBP si1: GGUGCUGACA GUGCUCAUU; TYROBP si2: UCCUUCACUUGCCU GGACG; USP16 si1: CCAUGAGCCAGUUUCUUAA; USP16 si2: GCAGAUGCUAAUUUCUCUU; Potential TERC binding site search in the promoter database Promoter sequences of Homo sapiens were downloaded from Eukaryotic Promoter Database (https://epd.vital-it. ch/index.php) (20). Promoters containing at least 10 con- tinuous nucleotides of ‘GGCCACCACCCC’ or its reverse complement sequence were defined as potential binding sites of TERC. Reagents Antibodies to phosphorylated STAT3, STAT3, phosphory- lated p65 and p65 were purchased from Beyotime (Shang- hai, China). The AKT inhibitor perifosine, NF-B cofac- tor IB inhibitor bay11-7082, and STAT3 inhibitor Stat- tic were from EFEbio (Shanghai, China). LPS was from Sigma. TNF- was from PeproTech. ELISA kits for cy- tokines were purchased from 4A Biotech (Beijing China). ELISA and Western blotting For detection of cytokine secretion, U2OS cells were treated with 10 ng/ml TNF-, whereas B2-17 cells were treated with 1 g/ml LPS for 6 h. The culture medium was collected and cytokines were detected using corresponding ELISA kits. For Western blotting, U2OS cells were treated with or without TNF- for indicated times. To inhibit NF-B Cell culture, vectors, and transfections Cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) with 10% fetal bovine serum, 100 U/ml penicillin and 100 g/ml streptomycin. To generate TERC stably overexpressing cell line, 293T cells were transfected with pBabe-TERC or empty pBabe plasmid and the retrovi- ral packaging plasmids pCMV-VSV.G and pCMV-Gag-Pol (Addgene) using calcium phosphate precipitation. The vi- ral supernatants were collected 72 h after transfection, ul- tracentrifuged at 40 000 rpm for 2h at 4◦C, and then used to infect U2OS cells. Forty-eight hours later, cells were selected with 2 g/ml puromycin for 3 days. The retained cells were cultured in 1 g/ml puromycin to produce a polyclonal cell population. The NF-B luciferase plasmid was a gift from Prof. Jun Cui at Sun Yat-sen University. Plasmid contains a firefly luciferase gene driven by minimal TATA promoter with NF-B response elements. INTRODUCTION Here, we reported a new function of TERC as an lncRNA. It stimulates the NF-B pathway and increases the expression and secretion of inflammation cytokines. By screening the genome for potential TERC binding sites in promoters, we identified 30 genes, of which four (LIN37, Human telomerase RNA component (TERC) is a 451 nt long, noncoding RNA (lncRNA) that is an essential com- ponent of telomerase. TERC serves as a template for re- verse transcriptase TERT, which adds GGTTAG repeats to chromosome ends (1). TERC is expressed in all telomerase Nucleic Acids Research, 2019, Vol. 47, No. 15 8085 TPRG1L, TYROBP and USP16) were validated to be tran- scriptionally regulated by TERC. Interestingly, these four genes, including three first defined, were related to activa- tion of NF-B pathway and cellular inflammation. In ad- dition, in vivo study showed that expression level of TERC as well as its downstream genes TYROBP, TPRG1L and USP16 were upregulated in patients with chronic inflam- mation disease. Gene Ontology and KEGG pathway analyses were per- formed for upregulated genes in TERC-U2OS cells using the online tool DAVID (18,19). TPRG1L, TYROBP and USP16) were validated to be tran- scriptionally regulated by TERC. Interestingly, these four genes, including three first defined, were related to activa- tion of NF-B pathway and cellular inflammation. In ad- dition, in vivo study showed that expression level of TERC as well as its downstream genes TYROBP, TPRG1L and USP16 were upregulated in patients with chronic inflam- mation disease. Quantitative RT-PCR Total RNA was isolated using TRIzol (Takara). Approxi- mately 1 g RNA per sample was used to generate cDNA by reverse transcription. Real-time PCR was carried out with q-PCR buffer containing SYBR Green I in Light- Cycler 480 (Roche). All PCR primer sequences were from PrimerBank (22). Mapping of TERC-ChIRP fragments to genome TERC CHIRP-seq dataset was downloaded from published article (13). The original dataset included 2198 fragments located in hg18. These fragments were re-localized in hg19. The nearest transcription start sites (TSSs) were identified. The distance between fragment and TSS was calculated and defined as “distance to TSS”. To further explore this hypothesis, we stimulated TERC- U2OS and control pBabe-U2OS cells with TNF- and de- tected the expression and secretion of inflammatory fac- tors. The results showed that mRNA levels of five cytokines (IL-6, IL-8, IL-32, TNF- and CSF2) were upregulated in TERC-U2OS compared to pBabe-U2OS (Figure 1C). Ac- cordingly, secreted cytokines (IL-6, IL-8 and CSF2) in cul- ture medium were also increased (Figure 1D). Conversely, when TERC was depleted by siRNAs in human astrocy- toma B2–17cells (siRNA with a scramble sequence was used as a control), both the expression and secretion level of cy- tokines significantly decreased (Figure 1E and F). Because B2–17 are telomerase positive cells, to exclude the engage- ment of telomerase in the inflammatory response, TERT was knocked down (Figure 1G) and cytokine secretion was determined. Our results showed that in contrast to knock- down of TERC, depletion of TERT only slightly decreased the secretion of CSF2 and had no effect on IL-6 and IL-8 (Figure 1F). To further confirm the role of TERC in the in- flammatory response, TERC was knocked down in human normal fibroblast BJ cells, which do not express TERT and therefore do not have telomerase activity. We observed that depletion of TERC significantly decreased secretion of IL-6, IL-8 and CSF2 in response to immune stimulation (Figure 1H). RNA sequencing and data analysis The RNA sample preparation, sequencing and data anal- ysis were performed as previously reported (17). The genes with log2-fold change ≥2 or ≤–2 as well as FDR<0.01 were considered up- or downregulated genes, respectively. The 8086 Nucleic Acids Research, 2019, Vol. 47, No. 15 8086 or STAT3 pathway, U2OS cells were treated with indicated inhibitors for 6 h. The culture medium was collected for ELISA analysis, and the cells were collected for Western blotting. TERC expression in clinical samples Processed gene expression profiles of clinical samples (GSE54350, GSE65561 and GES66988) were downloaded from GEO database (Gene Expression Omnibus). Expres- sion levels of TERC and its target genes were analyzed. TERC promotes cellular inflammatory response To explore the biologic functions of TERC beyond serving as a template for telomerase extension, we ectopically ex- pressed TERC in telomerase negative U2OS cells in which endogenous TERC was in the minutest amount and TERT was undetectable. Differentially expressed genes were then examined by RNA-seq. The result showed that 431 genes were up- or downregulated with log2-fold change ≥2 or ≤–2, respectively (Supplementary Table S1). Upregulated genes were then subjected to Gene Oncology (GO) anal- ysis. Strikingly, 8 of 15 enriched GO terms were related to immunology, including immune response, chemokine- mediated signaling pathway, macrophage chemotaxis, and inflammatory response (Figure 1A). Alternatively, when up- regulated genes underwent KEGG pathway analysis, 22 out of 28 KEGG terms were related to immunology such as im- mune system, immune related diseases or inflammation re- lated pathway (Figure 1B). These results strongly suggested that TERC is involved in cellular immune in a direct or in- direct manner. Luciferase reporter assays HEK293T cells were plated in 96-well plates ( 1×104 per well) and transfected with plasmids encoding NF-B luciferase reporter (firefly luciferase plasmid), pRL-TK- luc (renilla luciferase plasmid) and pBabe-TERC (Full- length TERC). For knockdown experiment, siTERC was transfected into HEK293T cells. Cells were harvested at 48 h after transfection and luciferase activity was measured with Dual-Luciferase Assay kit (Promega) according to the protocol provided by manufacturer. The firefly luciferase in- tensity of NF-B reporter was first normalized to renilla luciferase and then divided by corresponding control to ob- tain a relative activity of NF-B luciferase (Folds). Immunofluorescence (IF) (A) Enriched biological processes after TERC overexpression. Gene ontology was analyzed ERC-U2OS compared to pBabe-U2OS. GO terms in blue boxes were immune related biological processes. (B) Enriched signalin rexpression. KEGG pathway enrichment was analyzed with upregulated genes in TERC-U2OS compared to pBabe-U2OS. Di ays were boxed and labeled. (C) The mRNA levels of cytokines after TERC overexpression in U2OS cells. The stable cell lines pB OS were stimulated with TNF- for 1 h, and cells were collected for qPCR. (D) The secreted cytokines after TERC overexpressi cell lines pBabe-U2OS and TERC-U2OS were stimulated by TNF- for 6 h, and culture medium was collected for ELISA. (E) T nes after TERC knockdown in B2–17 cells. The cells were transfected with indicated siRNAs for 72 h and treated with LPS du on. Cells were then collected for qPCR. (F) The secreted cytokines in B2–17 cells after TERC or TERT knockdown. Cells were iRNAs for 72 h and treated with LPS during the last 6 h of transfection. Culture medium was then collected for ELISA. (G) of TERT. The B2–17 cells were transfected with TERT siRNAs for 72 h, and cells were collected for qPCR. (H) The secrete C knockdown. Cells were transfected with indicated siRNAs for 72 h and treated with TNF- during the last 6 h of transfecti llected for ELISA. Immunofluorescence (IF) All values are means ± SEM of more than three independent experiments (P < 0.05, P < 0.01,P < Enriched GO terms of up-regulated genes after TERC overexpression 0 5 10 15 GO:0050900~leukocyte migration GO:0070374~positive regulation of ERK1 and ERK2 cascade GO:0006955~immune response GO:0030198~extracellular matrix organization GO:0070493~thrombin receptor signaling pathway GO:0031663~lipopolysaccharide-mediated signaling pathway GO:0043114~regulation of vascular permeability GO:0050918~positive chemotaxis GO:0070098~chemokine-mediated signaling pathway GO:0010759~positive regulation of macrophage chemotaxis GO:0048246~macrophage chemotaxis GO:0045785~positive regulation of cell adhesion GO:0006954~inflammatory response GO:0010595~positive regulation of endothelial cell migration GO:0048566~embryonic digestive tract development 0.01 0.001 p value Enriched gene number A A Enriched GO terms of up-regulated genes after TERC overexpression A Enriched GO terms of up-regulated genes after TERC overexpression g Enriched KEGG pathway of up-regulated genes after TERC overexpression 0 5 10 15 hsa05164:Influenza A hsa05144:Malaria hsa05142:Chagas disease hsa05146:Amoebiasis hsa05168:Herpes simplex infection hsa05133:Pertussis hsa05143:African trypanosomiasis hsa05152:Tuberculosis hsa05145:Toxoplasmosis hsa05150:Staphylococcus aureus infection hsa05321:Inflammatory bowel disease (IBD) hsa05310:Asthma hsa05332:Graft-versus-host disease hsa05330:Allograft rejection hsa05323:Rheumatoid arthritis hsa04621:NOD-like receptor signaling pathway hsa04611:Platelet activation hsa04612:Antigen processing and presentation hsa04670:Leukocyte transendothelial migration hsa04668:TNF signaling pathway hsa04060:Cytokine-cytokine receptor interaction hsa04064:NF-kappa B signaling pathway hsa04514:Cell adhesion molecules (CAMs) hsa04151:PI3K-Akt signaling pathway hsa04940:Type I diabetes mellitus hsa04915:Estrogen signaling pathway hsa05200:Pathways in cancer hsa05416:Viral myocarditis >0.05 0.05 0.01 0.001 Others Immune system Immune diseases Infectious diseases Autoimmune disease Inflammation related pathway p value Enriched gene number B B F C IL -6 IL -8 IL -3 2 T N F -α C S F 2 0 1 2 3 4 5 5 1 0 1 5 2 0 p B a b e -U 2 O S T E R C -U 2 O S * * * * R e la tiv e fo ld o f m R N A G D ELISA IL-6 IL-8 CSF2 0 1 2 3 5 10 15 20 pBabe-U2OS TERC-U2OS * *** * n i e t o r p f o d l o f e vit al e R H hTR IL-6 IL-8 IL-32 TNF-α CSF2 0.0 0.5 1.0 1.5 NC TERC si1 TERC si2 * * * *** * * ns ** * * * B2-17 Relative fold of mRNA E G H hTR IL-6 IL-8 IL-32 TNF-α CSF2 0.0 0.5 1.0 1.5 NC TERC si1 TERC si2 * * * *** * * ns ** * * * B2-17 Relative fold of mRNA D ELISA IL-6 IL-8 CSF2 0 1 2 3 5 10 15 20 pBabe-U2OS TERC-U2OS * * * n i e t o r p f o d l o f e vit al e R E D E E C C F G H G ELISA NC TERC si1 TERC si2 TERT si NC TERC si1 TERC si2 TERT si NC TERC si1 TERC si2 TERT si 0.0 0.5 1.0 1.5 IL-6 IL-8 CSF2 * * * * * * ns ns B2-17 n i e t o r p f o d l o f e v it a l e R ELISA NC TERC si1 TERC si2 NC TERC si1 TERC si2 NC TERC si1 TERC si2 0.0 0.5 1.0 1.5 IL-6 CSF2 * * * * * IL-8 BJ n i e t o r p f o d l o f e vit al e R NC siTERT 0.0 0.5 1.0 1.5 B2-17 Relative fold of TERT H I TN C B2-17 F I T N C C G si2 TERT si CSF2 * ELISA NC TERC si1 TERC si2 NC TERC si1 TERC si2 NC TERC si1 TERC si2 0.0 0.5 1.0 1.5 IL-6 CSF2 * * * * * IL-8 BJ n i e t o r p f o d l o f e vit al e R NC siTERT 0.0 0.5 1.0 1.5 B2-17 Relative fold of TERT H T B2-17 Figure 1. Immunofluorescence (IF) The NF-B signaling pathway plays a fundamental role in inflammatory response, governing the release of inflamma- tory factors (23). We noticed that NF-B signaling path- way was enriched by KEGG pathway analysis in TERC overexpressing cells (Figure 1B). To explore whether TERC- induced stimulation of inflammatory response is mediated by NF-B pathway, inhibitors of p65 cofactors IB and STAT3 (Bay11-7082 and Stattic, respectively) were used to treat TERC-U2OS. Indeed, both Bay11-7082 and Stattic, but not the AKT inhibitor perifosine, suppressed the in- Cells were grown on coverslip, washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature, and then permeabilized in 0.5% Triton X-100 at room tempera- ture for 30 min. The cells were washed thrice with 1× PBST and blocked with 5% goat serum for 1 h at room temper- ature. The cells were incubated sequentially with anti-p65 antibody overnight at 4◦C and secondary antibody conju- gated with DyLight 488 for 1 h at room temperature. The coverslip was washed with PBST, mounted with DAPI, and visualized using a Zeiss microscope. Nucleic Acids Research, 2019, Vol. 47, No. Immunofluorescence (IF) 15 8087 Enriched GO terms of up-regulated genes after TERC overexpression 0 5 10 15 GO:0050900~leukocyte migration GO:0070374~positive regulation of ERK1 and ERK2 cascade GO:0006955~immune response GO:0030198~extracellular matrix organization GO:0070493~thrombin receptor signaling pathway GO:0031663~lipopolysaccharide-mediated signaling pathway GO:0043114~regulation of vascular permeability GO:0050918~positive chemotaxis GO:0070098~chemokine-mediated signaling pathway GO:0010759~positive regulation of macrophage chemotaxis GO:0048246~macrophage chemotaxis GO:0045785~positive regulation of cell adhesion GO:0006954~inflammatory response GO:0010595~positive regulation of endothelial cell migration GO:0048566~embryonic digestive tract development 0.01 0.001 p value Enriched gene number Enriched KEGG pathway of up-regulated genes after TERC overexpression 0 5 10 15 hsa05164:Influenza A hsa05144:Malaria hsa05142:Chagas disease hsa05146:Amoebiasis hsa05168:Herpes simplex infection hsa05133:Pertussis hsa05143:African trypanosomiasis hsa05152:Tuberculosis hsa05145:Toxoplasmosis hsa05150:Staphylococcus aureus infection hsa05321:Inflammatory bowel disease (IBD) hsa05310:Asthma hsa05332:Graft-versus-host disease hsa05330:Allograft rejection hsa05323:Rheumatoid arthritis hsa04621:NOD-like receptor signaling pathway hsa04611:Platelet activation hsa04612:Antigen processing and presentation hsa04670:Leukocyte transendothelial migration hsa04668:TNF signaling pathway hsa04060:Cytokine-cytokine receptor interaction hsa04064:NF-kappa B signaling pathway hsa04514:Cell adhesion molecules (CAMs) hsa04151:PI3K-Akt signaling pathway hsa04940:Type I diabetes mellitus hsa04915:Estrogen signaling pathway hsa05200:Pathways in cancer hsa05416:Viral myocarditis >0.05 0.05 0.01 0.001 Others Immune system Immune diseases Infectious diseases Autoimmune disease Inflammation related pathway p value Enriched gene number G ELISA NC TERC si1 TERC si2 TERT si NC TERC si1 TERC si2 TERT si NC TERC si1 TERC si2 TERT si 0.0 0.5 1.0 1.5 IL-6 IL-8 CSF2 * * * * * * ns ns B2-17 n i e t o r p f o d l o f e v it a l e R ELISA NC TERC si1 TERC si2 NC TERC si1 TERC si2 NC TERC si1 TERC si2 0.0 0.5 1.0 1.5 IL-6 CSF2 * * * * * IL-8 BJ n i e t o r p f o d l o f e vit al e R NC siTERT 0.0 0.5 1.0 1.5 B2-17 Relative fold of TERT A B H hTR IL-6 IL-8 IL-32 TNF-α CSF2 0.0 0.5 1.0 1.5 NC TERC si1 TERC si2 * * * * * * ns ** * * * B2-17 Relative fold of mRNA F C IL -6 IL -8 IL -3 2 T N F -α C S F 2 0 1 2 3 4 5 5 1 0 1 5 2 0 p B a b e -U 2 O S T E R C -U 2 O S * * * * * R e la tiv e fo ld o f m R N A D ELISA IL-6 IL-8 CSF2 0 1 2 3 5 10 15 20 pBabe-U2OS TERC-U2OS * *** * n i e t o r p f o d l o f e vit al e R E C promotes inflammatory response. Immunofluorescence (IF) (E) Total and phosphorylated p65 were upregulated by TERC. PBabe-U2OS and TERC-U2OS cells were treated with TNF- for indic times. Total and phosphorylated levels of p65 were determined by Western blotting. (F) Quantification of (E). Protein levels in (E) were quantified normalized by actin. (G) Inhibited nuclear translocation of p65 in TERC depleted cells. B2–17 cells were transfected with TERC siRNA or NC fo h and then treated with TNF- (10 ng/ml) for 20 min. Immunofluorescence (IF) using p65 antibody was performed to determine the amount of p nucleus and cytoplasm. (H) Quantification of (G). Nuclear p65 (Fluorescence intensity) was quantified as a percentage of overall p65 in cell. ∼100 were counted for each experiment. (I, J) NF-B luciferase reporter assay for analysis of activation of NF-B in TERC overexpressed (I) and depleted (J). HEK293T cells were transfected with TERC/pBabe (I) or siTERC/NC (J) together with NF-B-luc and pRL-TK. Luciferase activity was determ 48 h after transfection. All values are means ± SEM of more than three independent experiments (P < 0.05, P < 0.01, *P < 0.001). 8088 Nucleic Acids Research, 2019, Vol. 47, No. 15 A B C D A B B C D A E F F F E H G I J I H I J J G Figure 2. TERC activates the NF-B pathway. (A) Inhibition of NF-B or STAT3 activation by inhibitors in TERC-U2OS. TERC-U2OS cells were treated with inhibitors of AKT, IB and STAT3 (Perifosine, Bay11–7082 and Stattic, respectively) for 6h, and cells were collected for Western blotting of indicated proteins. (B-D) The TERC promotion of cytokine secretion was counteracted by inhibitors of p65 cofactors IB and STAT3. Stable cell lines pBabe- U2OS and TERC-U2OS were treated with TNF- plus indicated inhibitor for 6 h, and culture medium was collected for IL-6, IL-8 and CSF2 detection by ELISA. (E) Total and phosphorylated p65 were upregulated by TERC. PBabe-U2OS and TERC-U2OS cells were treated with TNF- for indicated times. Total and phosphorylated levels of p65 were determined by Western blotting. (F) Quantification of (E). Protein levels in (E) were quantified and normalized by actin. (G) Inhibited nuclear translocation of p65 in TERC depleted cells. B2–17 cells were transfected with TERC siRNA or NC for 72 h and then treated with TNF- (10 ng/ml) for 20 min. Immunofluorescence (IF) using p65 antibody was performed to determine the amount of p65 in nucleus and cytoplasm. Immunofluorescence (IF) TERC promotes inflammatory response. (A) Enriched biological processes after TERC overexpression. Gene ontology was analyzed for upregu- lated genes in TERC-U2OS compared to pBabe-U2OS. GO terms in blue boxes were immune related biological processes. (B) Enriched signaling pathways after TERC overexpression. KEGG pathway enrichment was analyzed with upregulated genes in TERC-U2OS compared to pBabe-U2OS. Different cate- gories of pathways were boxed and labeled. (C) The mRNA levels of cytokines after TERC overexpression in U2OS cells. The stable cell lines pBabe-U2OS and TERC-U2OS were stimulated with TNF- for 1 h, and cells were collected for qPCR. (D) The secreted cytokines after TERC overexpression in U2OS cells. The stable cell lines pBabe-U2OS and TERC-U2OS were stimulated by TNF- for 6 h, and culture medium was collected for ELISA. (E) The mRNA levels of cytokines after TERC knockdown in B2–17 cells. The cells were transfected with indicated siRNAs for 72 h and treated with LPS during the last 1 h of transfection. Cells were then collected for qPCR. (F) The secreted cytokines in B2–17 cells after TERC or TERT knockdown. Cells were transfected with indicated siRNAs for 72 h and treated with LPS during the last 6 h of transfection. Culture medium was then collected for ELISA. (G) The knock- down efficiency of TERT. The B2–17 cells were transfected with TERT siRNAs for 72 h, and cells were collected for qPCR. (H) The secreted cytokines in BJ after TERC knockdown. Cells were transfected with indicated siRNAs for 72 h and treated with TNF- during the last 6 h of transfection. Culture medium was collected for ELISA. All values are means ± SEM of more than three independent experiments (P < 0.05, P < 0.01,P < 0.001). 8088 Nucleic Acids Research, 2019, Vol. 47, No. 15 A B C D E G F H I J Figure 2. TERC activates the NF-B pathway. (A) Inhibition of NF-B or STAT3 activation by inhibitors in TERC-U2OS. TERC-U2OS cells were tre with inhibitors of AKT, IB and STAT3 (Perifosine, Bay11–7082 and Stattic, respectively) for 6h, and cells were collected for Western blotting of indic proteins. (B-D) The TERC promotion of cytokine secretion was counteracted by inhibitors of p65 cofactors IB and STAT3. Stable cell lines pB U2OS and TERC-U2OS were treated with TNF- plus indicated inhibitor for 6 h, and culture medium was collected for IL-6, IL-8 and CSF2 detec by ELISA. Immunofluorescence (IF) (H) Quantification of (G). Nuclear p65 (Fluorescence intensity) was quantified as a percentage of overall p65 in cell. ∼100 cells were counted for each experiment. (I, J) NF-B luciferase reporter assay for analysis of activation of NF-B in TERC overexpressed (I) and depleted cells (J). HEK293T cells were transfected with TERC/pBabe (I) or siTERC/NC (J) together with NF-B-luc and pRL-TK. Luciferase activity was determined 48 h after transfection. All values are means ± SEM of more than three independent experiments (P < 0.05, P < 0.01, P < 0.001). Nucleic Acids Research, 2019, Vol. 47, No. 15 8089 Nucleic Acids Research, 2019, Vol. 47, No. 15 8089 B2-17 TERC LIN37 SLC26A1 TPRG1L TYROBP USP16 UNC5A 0.0 0.5 1.0 1.5 2.0 NC siTERC * ns * * ns Relative fold of mRNA A ALPL LIN37 DUSP21 MSL1 SLC26A1 TPRG1L TYROBP RRAGA CIZ1 CNOT2 RPH3A CHD3 USP16 RAD17 CCR3 GBA2 CERCAM PYGO2 CEP63 LCN15 MTERFD3 ACTR8 SCRN1 EML3 ASTL SNAI1 ALG3 UNC5A APOM TMEM139 0 2 4 6 8 10 30 40 50 * * * * * pBabe-U2OS TERC-U2OS *** * *** * * * Relative fold of mRNA B TERC LIN37 SLC26A1 TPRG1L TYROBP USP16 UNC5A 0 1 2 3 NC siTERC HeLa * * *** * ** ns * Relative mRNA level E D TERC LIN37 SLC26A1 TPRG1L TYROBP USP16 UNC5A 0.0 0.5 1.0 1.5 NC siTERC 293T * * * * * Relative mRNA level F C TERC DNA TERC Figure 3. TERC targets gene’s promoters using its binding motif. (A) Schematic diagram of TERC targeting to gene’s promoters that contain the sequence of TERC binding motif. (B). Distribution of TERC targeted fragments on genome. The fragment was classified according to its distance to the nearest TSS. (C) The mRNA levels of 30 genes with TERC binding motif in their promoters in TERC-U2OS vs pBabe-U2OS cells. (D–F). Determination of mRNA levels of top 6 genes in (C) that have the highest fold change (up- and downregulation) in response to TERC expression. Gene expression was tested in indicated cells with or without TERC knockdown. Each panel represents different cells as indicated. All values are means ± SEM of more than three independent experiments (P < 0.05, P < 0.01, P < 0.001). Immunofluorescence (IF) crease of IL 6 IL 8 and CSF2 induced by TERC overex scription of target genes (24) The amount of p65 in nu A TERC DNA TERC B A A ALPL LIN37 DUSP21 MSL1 SLC26A1 TPRG1L TYROBP RRAGA CIZ1 CNOT2 RPH3A CHD3 USP16 RAD17 CCR3 GBA2 CERCAM PYGO2 CEP63 LCN15 MTERFD3 ACTR8 SCRN1 EML3 ASTL SNAI1 ALG3 UNC5A APOM TMEM139 0 2 4 6 8 10 30 40 50 * * * * * pBabe-U2OS TERC-U2OS *** * *** * * * Relative fold of mRNA B C TERC DNA TERC ALPL LIN37 DUSP21 MSL1 SLC26A1 TPRG1L TYROBP RRAGA CIZ1 CNOT2 RPH3A CHD3 USP16 RAD17 CCR3 GBA2 CERCAM PYGO2 CEP63 LCN15 MTERFD3 ACTR8 SCRN1 EML3 ASTL SNAI1 ALG3 UNC5A APOM TMEM139 0 2 4 6 8 10 30 40 50 * * * * * pBabe-U2OS TERC-U2OS * * *** * * * Relative fold of mRNA C TERC C B2-17 TERC LIN37 SLC26A1 TPRG1L TYROBP USP16 UNC5A 0.0 0.5 1.0 1.5 2.0 NC siTERC * * ns * * ns Relative fold of mRNA TERC LIN37 SLC26A1 TPRG1L TYROBP USP16 UNC5A 0 1 2 3 NC siTERC HeLa * * * * ** ns * Relative mRNA level E D TERC LIN37 SLC26A1 TPRG1L TYROBP USP16 UNC5A 0.0 0.5 1.0 1.5 NC siTERC 293T * * * * * Relative mRNA level F B2-17 TERC LIN37 SLC26A1 TPRG1L TYROBP USP16 UNC5A 0.0 0.5 1.0 1.5 2.0 NC siTERC * * ns * * ** ns Relative fold of mRNA D E D F Figure 3. TERC targets gene’s promoters using its binding motif. (A) Schematic diagram of TERC targeting to gene’s promoters that contain the sequence of TERC binding motif. (B). Distribution of TERC targeted fragments on genome. The fragment was classified according to its distance to the nearest TSS. (C) The mRNA levels of 30 genes with TERC binding motif in their promoters in TERC-U2OS vs pBabe-U2OS cells. (D–F). Determination of mRNA levels of top 6 genes in (C) that have the highest fold change (up- and downregulation) in response to TERC expression. Gene expression was tested in indicated cells with or without TERC knockdown. Each panel represents different cells as indicated. All values are means ± SEM of more than three independent experiments (P < 0.05, P < 0.01, *P < 0.001). Immunofluorescence (IF) crease of IL-6, IL-8 and CSF2 induced by TERC overex- pession (Figure 2A–D). scription of target genes (24). The amount of p65 in nu- cleus is indicative of activation extent of NF-B pathway. We observed that in response to TNF- treatment, TERC- depleted cells display much less p65 in nucleus than control cells, demonstrating that TERC positively regulates the ac- tivation of NF-B pathway (Figure 2G, H). Moreover, we also performed luciferase reporter assay, in which NF-B response element is placed in TATA promoter that drives the transcription of luciferase. The result showed that com- pared to control cells, overexpression of TERC increases lu- ciferase activity, whereas knockdown of TERC decreases its Furthermore, we observed that NF-B subunit p65 and phosphorylated p65 (p-p65) were higher in TERC-U2OS than in control pBabe-U2OS, suggesting the activation of the NF-B pathway (Figure 2E, 0min). When cells were fur- ther stimulated with TNF-, the level of p65 and p-p65 in TERC-U2OS were continuously higher than pBabe-U2OS throughout the activation procedure, suggesting that TERC stimulates activation of NF-B pathway (Figure 2E, F). Upon activation of NF-B pathway, p65 migrates from cy- toplasm to nucleus where it functions to initiate the tran- 8090 Nucleic Acids Research, 2019, Vol. 47, No. 15 8090 Nucleic Acids Research, 2019, Vol. 47, No. 15 Table 1. Promoters containing potential TERC binding site Gene Name Gene Promoter Seq. from EPD ALPL actcgggccccgcggccgcctttataaggcggcgggggtggtggcccggGCCGCGTTGCG LIN37 taataggaacaagctactgccgaaggggcccgcccacagaagggtggtgGCCACGGTCCA DUSP21 agatgggagtggtgagaggagacagaaagagggtggtggccgatagctgGTCCTCTTTCT MSL1 gcggccagcgagggcagatggaagagTATGAGGAAGAGCCCTCTCGGGGGTGGTGGCGGC SLC26A1 cagagtccagggcacagaccactgcctgcaggttggcgccaccacccccACTCTCCCCGC TPRG1L ccgcggggcggggccgggggcgcggccgggtggtggcggtggctgcggcgacggcggtcG TYROBP ccctgtctcctcctcccttctgccaccacccgcctcagacttcctccttCACTTGCCTGG RRAGA gagatcgccgccggaagtgggtggtggcggggacgcagcggctccctccCGGAAAGCGAG CIZ1 ggcaaaatggcgaaatccctctctacaaaaaatacaaacattagccagggtggtggcggg CNOT2 agggagggagggggtgtgtatgggggtggtggtggACCGGACGTAAAGCGTCGCTGTACT RPH3A aggagggagagggggtggtggaggagggagagagggtgggagaaggagtgatgaagatgg CHD3 ggggaggcgggcgggcggtgggtgggggggtggtgggggggccAGAGCCACAGGATGGCT USP16 GGGAGGTGGGGGTGGGGTGGTGGTGGCCTAGCCACTTCCCATAATGCCGCGTTCCGGAAG RAD17 CGAATATTTGAGCTTAGTATTCCCTGTTCACTGTGTGGGGTGGTGGTGGGTCGGCTAGGA CCR3 aggtggtggcctgcccctccccgcaggcactctgtcccagggagaaatcagaactcttta GBA2 CACGGCCACTTCTGCATCCAGGTGGGGATGCTGGCACTGAAGGTGGTGGCCCTTCTGGGA CERCAM GGAGCCGGGGAAGCCCGGGAGGTGGTGGCCGAGTGGGCGCCGCCCCTCTGGGTCTGCGGC PYGO2 ttgctccccctccccgcagcgctcagtggtggtggccgcgacgagttccGGTTCCGGTTG CEP63 GCCTCGCAGGCCACCACCATCCGCACCGTACGACAGGCCGTCCCTCAGCTGCGGCTTCCT LCN15 caggtggtggcctgggctataaagctggccccctggggcttggggactcAGCACCAGGGG MTERFD3 ggaagcaaatgcagctggtgcaggagagggaaatgggaattagggtggtGGCAGAGCCCA ACTR8 cccctggtggggggAGTGCGGAAGCGGTCGTTCTTTTCCGGGTGGTGGCGCGCCGGGACG SCRN1 tcccactcctctccacctccactgccaccaccctgcaccaagccaccaccatctccagcc EML3 CTCGGGGTGGTGGTACGGCGCCCTTCGCGCGCGCCCCGGGGTGCTTCCCCTTCCCCTCTC ASTL GTAACCTAATTGCAGAACCGGCACCACCACCCCCTCTTAAATAGCAGCTGctccacctcc SNAI1 CCACCACCCCCCCGGAGTACTTAAGGGAGTTGGCGGCGCTGCTGCATTCATTGCGCCGCG ALG3 aagcggaacctaagtgtcgaaggttcgggtttccgggggtggtgggcccACACAAGCGGC UNC5A GCCCACCACCCCAAGCCCCTCCCTGGGGGAGCCTCAGGCATCGCCCAGAGGGATTCCCGG APOM acacacccaccaccccgcggctccgcccccgacttccccacggaccgtcACTTCCGGTCT TMEM139 acctacccgctccggcccttcccaccaccccccaccccatctactttctACAGTCTGTGG Underline indicates potential binding sites Lower case indicates upstream of TSS Underline indicates potential binding sites Lower case indicates upstream of TSS activity (Figure 2I and J). Altogether, these data confirmed that TERC promotes the activation of NF-B pathway. by TERC using pBabe-U2OS and TERC-U2OS cells. 14 of 30 genes exhibited up- or downregulated transcription in response to TERC expression (Figure 3C). The top six up- or downregulated genes were LIN37, SLC26A1, TPRG1L, TYROBP, USP16 and UNC5A. Among them, four genes (LIN37, TPRG1L, TYROBP and USP16) showed consis- tent downregulation when TERC was knockdown in B2– 17, HeLa and 293T cells (Figure 3D–F). We thus focused on these four genes. TERC regulates gene transcription by targeting its promoter We next explored how TERC stimulates the NF-B path- way. As a typical lncRNA, we speculated that TERC may regulate gene transcription by targeting specific sites in the genome. Indeed, using chromatin isolation by RNA pu- rification (ChIRP), it was previously reported that TERC targets the genome with a high preference for the se- quence 5′-GGCCACCACCCC-3′ (termed the binding mo- tif) (13), which is exactly complementary to the sequence 5′-GGGGUGGUGGCC-3′ at site 25–36 of TERC. This strongly suggested that TERC binds to genomic DNA using its binding motif, which may form a triplex structure with target DNA (Figure 3A). TERC binds to gene’s promoters by forming RNA–DNA triplexes TERC binds to gene’s promoters by forming RNA–DNA triplexes To investigate whether TERC binds to promoters of LIN37, TPRG1L, TYROBP and USP16 in vivo, ChIRP-PCR was performed, in which biotin-labeled oligonucleotides were used to pull down TERC and associated chromatin (13,21). PCR was then performed using primers that cover the TERC binding site in the gene’s promoter (Table 1). The results showed that promoters of four genes were enriched by ChIRP, demonstrating association of these promoters with TERC (Figure 4A). To survey the locations of TERC target sites, we down- loaded 2198 TERC-ChIRP fragments from published study (13) and mapped them to the human genome. The distance to the nearest transcription start site (TSS) of each frag- ment was calculated. Most fragments (TERC targeted sites) were located within ±1000 bp of TSSs (Figure 3B), suggest- ing that TERC tends to target gene’s promoters. Then, we screened potential binding sequences using a TERC binding motif in Eukaryotic Promoter Database (EPD) (20), which contains all identified promoters and sequences adjacent to TSSs. As a result, as many as 30 sequences on gene pro- moters were identified that were listed in Table 1. We then verified whether the transcription of these genes was altered ( g ) To further validate the interaction between TERC and promoters, electrophoretic mobility shift assay (EMSA) was carried out in vitro by incubating TERC and synthe- sized 59 bp double-stranded (ds) DNA that is from in- dicated gene promoter and consists of binding motif se- quence of TERC. Indeed, shifted bands were observed for all four tested promoters, but not for scramble sequence (Figure 4B). In addition, with increased TERC, shift bands Nucleic Acids Research, 2019, Vol. 47, No. 15 8091 A E B D C ure 4. RNA–DNA triplex formation between TERC and gene’s promoters. (A) TERC binds to the promoter of indicated gene in vivo. TERC ChIRP was ormed using TERC probe. LacZ probe was used as control. Promoters of the indicated genes were detected by PCR. (B) TERC binds to the promoters ndicated genes in vitro. EMSA was performed to detect the triplex formation between synthesized gene promoters containing TERC binding motif and RC. RNA with random sequence was used as control. Products were analyzed on 2% agarose gels. (C) Triplex formation in a dose dependent manner. o picomoles of indicated gene promoters were incubated with increasing amounts of TERC. Products were analyzed on 2% agarose gels. TERC binds to gene’s promoters by forming RNA–DNA triplexes (D) Expression ls of indicated genes after RNH1 knockdown. U2OS cells were transfected with siRNH1 for 72 h, and corresponding mRNAs were detected by qPCR. Melting temperature decreased after triplex formation at neutral pH. Melting temperatures of gene’s promoters were detected in the presence or absence TERC or TERC1–49. All values are means ± SEM of more than three independent experiments (P < 0.05, P < 0.01, P < 0.001). B D C A B D B A D D E E Figure 4. RNA–DNA triplex formation between TERC and gene’s promoters. (A) TERC binds to the promoter of indicated gene in vivo. TERC ChIRP was performed using TERC probe. LacZ probe was used as control. Promoters of the indicated genes were detected by PCR. (B) TERC binds to the promoters of indicated genes in vitro. EMSA was performed to detect the triplex formation between synthesized gene promoters containing TERC binding motif and TERC. RNA with random sequence was used as control. Products were analyzed on 2% agarose gels. (C) Triplex formation in a dose dependent manner. Two picomoles of indicated gene promoters were incubated with increasing amounts of TERC. Products were analyzed on 2% agarose gels. (D) Expression levels of indicated genes after RNH1 knockdown. U2OS cells were transfected with siRNH1 for 72 h, and corresponding mRNAs were detected by qPCR. (E) Melting temperature decreased after triplex formation at neutral pH. Melting temperatures of gene’s promoters were detected in the presence or absence of TERC or TERC1–49. All values are means ± SEM of more than three independent experiments (P < 0.05, P < 0.01, P < 0.001). TERC-targeted genes activate the NF-B pathway (RNA–DNA) gradually increased and free promoter DNA decreased accordingly (Figure 4C). Because NF-B component such as p65 or STAT3 is not direct target of TERC, we hypothesized that TERC stim- ulates the NF-B pathway in an indirect manner, poten- tially through promoting the expression of targeted genes. We thus tested identified four genes for their ability to ac- tivate the NF-B pathway. Four genes (LIN37, TPRG1L, TYROBP and USP16) were individually knocked down in TERC-U2OS cells. We found that while knockdown of LIN37, TPRG1L and USP16 decreased the level of p65 and p-p65, the knockdown of LIN37, TPRG1L and TY- ROBP decreased the level of STAT3 and phosphorylated STAT3 (p-STAT3) (Figure 5A, B). Consistently, ELISA as- say showed that knockdown of LIN37, TPRG1L, TYROBP and USP16 counteracted enhanced secretion of IL-6 by TERC (Figure 5C). Altogether, these results demonstrated that TERC modulates the inflammatory response through regulating a group of genes such as LIN37, TPRG1L, TY- ROBP and USP16. While TYROBP has been previously re- TERC may form R-loop or triplex with targeted ds- promoters. To test the possibility that TERC may form R-loops by hybridizing with targeted DNA, we knocked down the RNH1 gene in TERC-U2OS cells. RNase H is an enzyme that digests RNA in Watson–Crick RNA–DNA hybrids (25). It is speculated that gene expression levels should be altered by either overexpression or knockdown of RNase H if they are regulated by R-loop (26). In con- trast to this, we observed no change in the expression levels of four genes when RNase H was knocked down (Figure 4D). In addition, we observed that melting temperatures of ds-promoters were reduced by approximately 1◦C after in- cubating with TERC or 49 nt TERC fragments that contain the binding motif (Figure 4E). This is consistent with the previous report that melting temperature decreases during conformation change from double-strand DNA into RNA– DNA triplexes (27). Altogether, these results suggested that TERC may target gene’s promoters by forming RNA–DNA triplexes. 8092 Nucleic Acids Research, 2019, Vol. 47, No. 15 Figure 5. TERC activates NF-B pathways through targeting immune-related genes. (A) The knockdown efficiency of indicated genes. The TERC-U2OS cells were transfected with indicated siRNAs for 72 h, and corresponding mRNAs were detected by qPCR. (B) P65 and STAT3 were downregulated by knockdown of TERC targeted genes. The TERC-U2OS cells were transfected with indicated siRNAs for 72 h. TERC-targeted genes activate the NF-B pathway Total and phosphorylated p65 and STAT3 expressions were determined by western blotting. (C) The TERC promotion of IL-6 secretion was counteracted by knockdown of TERC targeted genes. PBabe-U2OS and TERC-U2OS cells were transfected with indicated siRNAs for 72 h and treated with TNF- during the last 6 h of transfection. Culture medium was collected for IL-6 detection by ELISA. All values are means ± SEM of more than three independent experiments (P < 0.05, P < 0.01, P < 0.001). Figure 5. TERC activates NF-B pathways through targeting immune-related genes. (A) The knockdown efficiency of indicated genes. The TERC-U2OS cells were transfected with indicated siRNAs for 72 h, and corresponding mRNAs were detected by qPCR. (B) P65 and STAT3 were downregulated by knockdown of TERC targeted genes. The TERC-U2OS cells were transfected with indicated siRNAs for 72 h. Total and phosphorylated p65 and STAT3 expressions were determined by western blotting. (C) The TERC promotion of IL-6 secretion was counteracted by knockdown of TERC targeted genes. PBabe-U2OS and TERC-U2OS cells were transfected with indicated siRNAs for 72 h and treated with TNF- during the last 6 h of transfection. Culture medium was collected for IL-6 detection by ELISA. All values are means ± SEM of more than three independent experiments (P < 0.05, P < 0.01, P < 0.001). 6E and F). Therefore, the elevated inflammatory response is positively correlated with increased expression of TERC and TERC target genes in patients. ported to be involved in the activation of the inflammatory response (28–31), the other three genes are newly identified. DISCUSSION Because TERC regulates the expression of inflammation related genes in vitro, we then investigated its potential immune-regulation function in patients. First, we analyzed TERC levels in type II diabetes and multiple sclerosis, both displaying increased chronic inflammation (32,33). The ex- pression levels of TERC in CD14+ cells from type II di- abetes and multiple sclerosis patients were significantly higher than normal people (Figure 6A and B). We also ex- amined the expression level of LIN37, TPRG1L, TYROBP and USP16 in CD14+ cells from patients and normal peo- ple. For diabetic patients, TPRG1L and TYROBP were up- regulated, whereas TYROBP and USP16 were upregulated in multiple sclerosis patients (Figure 6C and D). Consis- tently, the expression of inflammatory cytokines such as IL-8 and TNF- in diabetic patients and IL-6, IL-8, CSF2 and TNF- in multiple sclerosis patients increased (Figure A high incidence of TERC mutation and an increased copy number of TERC genes has been reported to associate with the pathogenesis of the inherited disorder dyskeratosis con- genital (DC), aplastic anemia (AA) (34,35) and other ge- netic diseases (36–38). Interestingly, both DC and AA pa- tients also display immune abnormalities (39,40). More- over, many mutations in TERC do not affect its function as an RNA template and telomerase activity (41–43), imply- ing that TERC may have noncanonical functions beyond telomerase. In this study, we revealed that TERC stimulates cellular inflammatory response in a telomerase independent manner. The evidence supporting this includes: (i) RNA-seq data indicated that expression of immune-related genes are regulated by TERC; (ii) overexpression of TERC in telom- erase negative U2OS cells resulted in increased expression and secretion of inflammatory factors; (iii) knockdown of TERC, but not TERT, in telomerase positive cells decreased Nucleic Acids Research, 2019, Vol. 47, No. DISCUSSION 15 8093 G S E 5 4 3 5 0 & G S E 6 5 5 6 1 N o rm a l D ia b e tic 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 * C D 1 4 + C ells R e la tiv e fo ld o f T E R C G S E 5 4 3 5 0 & G S E 6 5 5 6 1 L IN 3 7 T P R G 1 L T Y R O B P U S P 1 6 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 H ea lth y V o lu n te er D ia b e tic ** * C D 1 4 + C ells R e la tiv e fo ld o f m R N A G S E 5 4 3 5 0 & G S E 6 5 5 6 1 IL 6 IL 8 C S F 2 T N F -α 0 1 2 3 4 ** * H ea lth y V o lu n te er D ia b e tic C D 1 4 + C ells R e la tiv e fo ld o f m R N A G S E 6 6 9 8 8 N o rm a l 0 .6 0 .8 1 .0 1 .2 1 .4 M u lt ip le S c le ro s is C D 1 4 + C ells R e la tiv e fo ld o f T E R C G S E 6 6 9 8 8 L IN 3 7 T P R G 1 L T Y R O B P U S P 1 6 0 .0 0 .5 1 .0 1 .5 H ea lth y V o lu n te er M u ltip le S cle ro sis * * C D 1 4 + C ells R e la tiv e fo ld o f m R N A G S E 6 6 9 8 8 IL 6 IL 8 C S F 2 T N F -α 0 .0 0 .5 1 .0 1 .5 2 4 6 8 H ea lth y V o lu n te er M u ltip le S cle ro sis * * * C D 1 4 + C ells R e la tiv e fo ld o f m R N A C D A B E F Figure 6. DISCUSSION G S E 5 4 3 5 0 & G S E 6 5 5 6 1 L IN 3 7 T P R G 1 L T Y R O B P U S P 1 6 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 H ea lth y V o lu n te er D ia b e tic ** * C D 1 4 + C ells R e la tiv e fo ld o f m R N A C G S E 5 4 3 5 0 & G S E 6 5 5 6 1 IL 6 IL 8 C S F 2 T N F -α 0 1 2 3 4 ** * H ea lth y V o lu n te er D ia b e tic R e la tiv e fo ld o f m R N A E C C E A A G S E 5 4 3 5 0 & G S E 6 5 5 6 1 N o rm a l D ia b e tic 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 * * C D 1 4 + C ells R e la tiv e fo ld o f T E R C A C D 1 4 + C ells C D 1 4 + C ells C D 1 4 + C ells C D 1 4 + C ells G S E 6 6 9 8 8 L IN 3 7 T P R G 1 L T Y R O B P U S P 1 6 0 .0 0 .5 1 .0 1 .5 H ea lth y V o lu n te er M u ltip le S cle ro sis *** * C D 1 4 + C ells R e la tiv e fo ld o f m R N A D G S E 6 6 9 8 8 IL 6 IL 8 C S F 2 T N F -α 0 .0 0 .5 1 .0 1 .5 2 4 6 8 H ea lth y V o lu n te er M u ltip le S cle ro sis * * * R e la tiv e fo ld o f m R N A F G S E 6 6 9 8 8 N o rm a l 0 .6 0 .8 1 .0 1 .2 1 .4 ** M u lt ip le S c le ro s is C D 1 4 + C ells R e la tiv e fo ld o f T E R C B D D F B C D 1 4 + C ells C D 1 4 + C ells T C D 1 4 + C ells C D 1 4 + C ells C D 1 4 + C ells Figure 6. DISCUSSION The expression levels of TERC and TERC targeted genes in inflammation related diseases. (A) TERC was upregulated in CD14+ cells of diabetic patients. Data were downloaded from the GEO database (GSE54350 & GSE65561), and TERC expression levels were analyzed (n ≥10, P < 0.05, P < 0.01). (B) TERC was upregulated in CD14+ cells of multiple sclerosis patients. Data were downloaded from the GEO database (GSE66988), and TERC expression levels were analyzed (n ≥10, P < 0.05, *P < 0.01). (C) Expression levels of TERC targeted genes in CD14+ cells of diabetic patients. Data analysis is the same as (A). (D) Expression levels of TERC targeted genes in CD14+ cells of multiple sclerosis patients. Data analysis is the same as (B). (E) Expression levels of cytokines in CD14+ cells of diabetic patients. Data analysis is the same as (A), except that ‘n’ of IL6 is 6 because IL6 is missing in GSE65561. (F) Expression levels of cytokines in CD14+ cells of multiple sclerosis patients. Data analysis is the same as (B). secretion of inflammatory factors; (iv) overexpression of TERC led to activation of the NF-B signaling pathway in the absence of allothogenic stimulation; (v) TERC upreg- ulated the expression of LIN37, TPRG1L, TYROBP and USP16 that is linked to the activation of NF-B signaling pathway, leading to increased inflammatory response and (vi) high TERC levels corresponded to high inflammation states in patients with type II diabetes or multiple sclerosis. LncRNAs may regulate gene expression in different man- ners (11). For example, eRNA, which is transcribed from enhancers, promotes chromatin accessibility by remodeling the chromatin (44), whereas lncRNA Khps1, HOTAIR and MEG3 regulate gene transcription by recruiting chromatin secretion of inflammatory factors; (iv) overexpression of TERC led to activation of the NF-B signaling pathway in the absence of allothogenic stimulation; (v) TERC upreg- ulated the expression of LIN37, TPRG1L, TYROBP and USP16 that is linked to the activation of NF-B signaling pathway, leading to increased inflammatory response and (vi) high TERC levels corresponded to high inflammation states in patients with type II diabetes or multiple sclerosis. DISCUSSION The expression levels of TERC and TERC targeted genes in inflammation related diseases. (A) TERC was upregulated in CD14+ cells of diabetic patients. Data were downloaded from the GEO database (GSE54350 & GSE65561), and TERC expression levels were analyzed (n ≥10, P < 0.05, *P < 0.01). (B) TERC was upregulated in CD14+ cells of multiple sclerosis patients. Data were downloaded from the GEO database (GSE66988), and TERC expression levels were analyzed (n ≥10, P < 0.05, **P < 0.01). (C) Expression levels of TERC targeted genes in CD14+ cells of diabetic patients. Data analysis is the same as (A). (D) Expression levels of TERC targeted genes in CD14+ cells of multiple sclerosis patients. Data analysis is the same as (B). (E) Expression levels of cytokines in CD14+ cells of diabetic patients. Data analysis is the same as (A), except that ‘n’ of IL6 is 6 because IL6 is missing in GSE65561. (F) Expression levels of cytokines in CD14+ cells of multiple sclerosis patients. Data analysis is the same as (B). DISCUSSION secretion of inflammatory factors; (iv) overexpression of TERC led to activation of the NF-B signaling pathway in the absence of allothogenic stimulation; (v) TERC upreg- ulated the expression of LIN37, TPRG1L, TYROBP and USP16 that is linked to the activation of NF-B signaling pathway, leading to increased inflammatory response and (vi) high TERC levels corresponded to high inflammation states in patients with type II diabetes or multiple sclerosis. LncRNAs may regulate gene expression in different man- ners (11). For example, eRNA, which is transcribed from enhancers, promotes chromatin accessibility by remodeling the chromatin (44), whereas lncRNA Khps1, HOTAIR and MEG3 regulate gene transcription by recruiting chromatin modifiers to target sites (13–16). A previous study identified the sequence ‘GGCCACCACCCC’ as a binding motif in TERC that may associate with genomic DNA (13). Here, we found that TERC associated sequences were largely located near the TSSs of genes (Figure 3B). Following this route, we identified 30 potential promoters TERC may bind to. Among them, 4 genes were experimentally verified to be transcriptionally regulated by TERC (LIN37, TPRG1L, TYROBP and USP16). Similar to many other lncRNAs (13–16), we demonstrated that TERC formed triplexes with promoter sequences of these genes and thereby promoted their transcription. It should be noted that identified four genes are representative of 14 genes, which are transcrip- p yp p LncRNAs may regulate gene expression in different man- ners (11). For example, eRNA, which is transcribed from enhancers, promotes chromatin accessibility by remodeling the chromatin (44), whereas lncRNA Khps1, HOTAIR and MEG3 regulate gene transcription by recruiting chromatin 94 Nucleic Acids Research, 2019, Vol. 47, No. 15 8094 luciferase plasmid, and for helpful suggestions on experi- mental design. tionally regulated by TERC (Figure 3). Considering that di- rect association is one of manners by which lncRNA regu- lates gene transcription, the total number of genes regulated by TERC might be much greater than 14. Further investi- gation is thus needed to identify whole genes regulated by TERC. Author Contributions: H. Liu and Y. Zhao designed the study, analyzed the data and wrote the paper. H. Liu and Y. Yang performed the majority of the experiments. J. Liu an- alyzed the clinical samples. Y. Ge offered ideas and helped to analyze the data. Y. Zhao supervised the project and re- vised the manuscript. All authors read and approved the manuscript. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. 11. Dykes,I.M. and Emanueli,C. (2017) Transcriptional and Post-transcriptional gene regulation by long Non-coding RNA. Genomics Proteomics Bioinformatics, 15, 177–186. CONCLUSIONS Here, we found that TERC regulates immune-related gene transcription by forming triplex with their promoters. In this way, four genes were identified that promote cellular in- flammatory response by enhancing the activation of NF-B signaling pathway. The axis of TERC/targeted-genes/NF- B/inflammatory response exists in patients with chronic inflammatory disease. 5. Xi,L. and Cech,T.R. (2014) Inventory of telomerase components in human cells reveals multiple subpopulations of hTR and hTERT. Nucleic Acids Res., 42, 8565–8577. 6. Blasco,M.A., Rizen,M., Greider,C.W. and Hanahan,D. (1996) Differential regulation of telomerase activity and telomerase RNA during multi-stage tumorigenesis. Nat. Genet., 12, 200–204. 7. Cairney,C.J., Hoare,S.F., Daidone,M.G., Zaffaroni,N. and Keith,W.N. (2008) High level of telomerase RNA gene expression is associated with chromatin modification, the ALT phenotype and poor prognosis in liposarcoma. Br. J. Cancer, 98, 1467–1474. FUNDING National Natural Science Foundation of China Grants [81771506, 31571410]; National Key R&D Program of China [2018YFA0107000]; Guangzhou Municipal People’s Livelihood Science and Technology Plan [201803010108]. Funding for open access charge: National Natural Science Foundation of China Grants [81771506, 31571410]; Na- tional Key R&D Program of China [2018YFA0107000]; Guangzhou Municipal People’s Livelihood Science and Technology Plan [201803010108]. It was previously discovered that TERC is implicated in angiogenesis, metastasis and proliferation of cancer cells by regulating the global gene expression (45,46). Similarly, we revealed that TERC promotes cellular inflammatory re- sponse by upregulating the expression of immune-related genes such as LIN37, TPRG1L, TYROBP and USP16 in human normal and cancer cells. Moreover, we observed that in patients with chronic inflammation such as Type II dia- betes and multiple sclerosis, the expression of TERC is up- regulated. Accordingly, the expression of TERC-targeted genes (TPRG1L, TYROBP and USP16) as well as secreted inflammatory factors (IL-6, IL-8, CSF2 and TNF-) are upregulated, which may contribute to increased inflamma- tory level in patients. In this context, TERC may be a poten- tial target for anti-inflammatory therapeutics for patients with chronic inflammation. gy [ ] Conflict of interest statement. None declared. gy [ ] Conflict of interest statement. None declared. DATA AVAILABILITY 8. Gazzaniga,F.S. and Blackburn,E.H. (2014) An antiapoptotic role for telomerase RNA in human immune cells independent of telomere integrity or telomerase enzymatic activity. Blood, 124, 3675–3684. The raw RNAseq data of pBabe-U2OS and TERC-U2OS (hTR-U2OS in the website) have been submitted to GEO database. The GEO accession number is GSE125024. The link of the dataset is: https://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi?acc=GSE125024. 9. Kedde,M., le Sage,C., Duursma,A., Zlotorynski,E., van Leeuwen,B., Nijkamp,W., Beijersbergen,R. and Agami,R. (2006) Telomerase-independent regulation of ATR by human telomerase RNA. J. Biol. Chem., 281, 40503–40514. 10. Ting,N.S., Pohorelic,B., Yu,Y., Lees-Miller,S.P. and Beattie,T.L. (2009) The human telomerase RNA component, hTR, activates the DNA-dependent protein kinase to phosphorylate heterogeneous nuclear ribonucleoprotein A1. Nucleic Acids Res., 37, 6105–6115. REFERENCES 1. 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https://openalex.org/W3011416235	https://iris.unife.it/bitstream/11392/2486610/1/Transpulmonary%20thermodilution%20detects%20rapid%20and%20reversible%20increases%20in%20lung%20water%20induced.pdf	English	null	Transpulmonary thermodilution detects rapid and reversible increases in lung water induced by positive end-expiratory pressure in acute respiratory distress syndrome	Annals of intensive care	2,020	cc-by	7,319	Correspondence: francesco.gavelli@uniupo.it 1 Service de médecine intensive ‑ réanimation, Hôpitaux universitaires Paris-Saclay, Hôpital de Bicêtre, APHP, 78, rue du Général Leclerc, 94270 Le Kremlin‑Bicêtre, France Full list of author information is available at the end of the article Abstract This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. Gavelli et al. Ann. Intensive Care (2020) 10:28 https://doi.org/10.1186/s13613-020-0644-2 Gavelli et al. Ann. Intensive Care (2020) 10:28 https://doi.org/10.1186/s13613-020-0644-2 RESEARCH Transpulmonary thermodilution detects rapid and reversible increases in lung water induced by positive end‑expiratory pressure in acute respiratory distress syndrome Francesco Gavelli1,2,3 , Jean‑Louis Teboul1,2, Danila Azzolina3, Alexandra Beurton1,2, Temistocle Taccheri1,2, Imane Adda1,2, Christopher Lai1,2, Gian Carlo Avanzi3 and Xavier Monnet1,2 Open Access Open Access Abstract Purpose: It has been suggested that, by recruiting lung regions and enlarging the distribution volume of the cold indicator, increasing the positive end-expiratory pressure (PEEP) may lead to an artefactual overestimation of extravas‑ cular lung water (EVLW) by transpulmonary thermodilution (TPTD). Methods: In 60 ARDS patients, we measured EVLW (PiCCO2 device) at a PEEP level set to reach a plateau pressure of 30 cmH2O (HighPEEPstart) and 15 and 45 min after decreasing PEEP to 5 cmH2O (LowPEEP15′ and LowPEEP45′, respec‑ tively). Then, we increased PEEP back to the baseline level (HighPEEPend). Between HighPEEPstart and LowPEEP15′, we estimated the degree of lung derecruitment either by measuring changes in the compliance of the respiratory system (Crs) in the whole population, or by measuring the lung derecruited volume in 30 patients. We defined patients with a large derecruitment from the other ones as patients in whom the Crs changes and the measured derecruited volume were larger than the median of these variables observed in the whole population. Results: Reducing PEEP from HighPEEPstart (14 ± 2 cmH2O) to LowPEEP15′ significantly decreased EVLW from 20 ± 4 to 18 ± 4 mL/kg, central venous pressure (CVP) from 15 ± 4 to 12 ± 4 mmHg, the arterial oxygen tension over inspired oxygen fraction (PaO2/FiO2) ratio from 184 ± 76 to 150 ± 69 mmHg and lung volume by 144 [68–420] mL. The EVLW decrease was similar in “large derecruiters” and the other patients. When PEEP was re-increased to HighPEEPend, CVP, PaO2/FiO2 and EVLW significantly re-increased. At linear mixed effect model, EVLW changes were significantly deter‑ mined only by changes in PEEP and CVP (p < 0.001 and p = 0.03, respectively, n = 60). When the same analysis was performed by estimating recruitment according to lung volume changes (n = 30), CVP remained significantly associ‑ ated to the changes in EVLW (p < 0.001). Conclusions: In ARDS patients, changing the PEEP level induced parallel, small and reversible changes in EVLW. These changes were not due to an artefact of the TPTD technique and were likely due to the PEEP-induced changes in CVP, which is the backward pressure of the lung lymphatic drainage. Trial registration ID RCB: 2015-A01654-45. Registered 23 October 2015 © The Author(s) 2020. Transpulmonary thermodilution detects rapid and reversible increases in lung water induced by positive end‑expiratory pressure in acute respiratory distress syndrome Francesco Gavelli1,2,3* , Jean‑Louis Teboul1,2, Danila Azzolina3, Alexandra Beurton1,2, Temistocle Taccheri1,2, Imane Adda1,2, Christopher Lai1,2, Gian Carlo Avanzi3 and Xavier Monnet1,2 © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. Haemodynamic measurements In addition to arterial pressure and CI, we measured the central venous pressure (CVP) at the base of the C wave, at end-expiration. The value of three successive respira- tory cycles was averaged. The pressure transducer was attached to the arm, at a height corresponding to the level of the right atrium. g Thus, the goal of our study, conducted in ARDS patients, was to investigate whether the estimation of EVLW by TPTD is artefactually influenced by the lung derecruitment potentially secondary to the decrease in the PEEP level. Estimation of alveolar derecruitment induced by PEEP decreasesi In a subgroup of 30 patients ventilated with an Infinity V500 ventilator (Dräger, Lübeck, Germany), we directly estimated the volume of derecruited lung during the PEEP decrease. For this purpose, after transiently reduc- ing the respiratory rate to 10 breaths/min to reduce the risk of air trapping, a prolonged expiration was per- formed while abruptly reducing PEEP from its base- line value to 5 cmH2O for one breath. The difference in end-expiratory Vt between the breath while PEEP was decreased and the one before was defined as the total change in lung volume [34]. At the same time, we Gavelli et al. Ann. Intensive Care (2020) 10:28 Page 2 of 8 Gavelli et al. Ann. Intensive Care Keywords: Pulmonary oedema, Pulmonary lymphatic drainage, Central venous pressure, Lung recruitment, Mechanical ventilation Keywords: Pulmonary oedema, Pulmonary lymphatic drainage, Central venous pressure, Lung recruitment, Mechanical ventilation TPTD measurements TPTD The large majority of these studies were conducted in animals [3–5, 8–27], with various models of ARDS and methods of EVLW estimation. Today, the routine measurement of EVLW at the bedside is allowed by transpulmonary thermodilution (TPTD). One animal [8] and three human studies [6, 7, 28] have investigated the effects of PEEP changes on TPTD-estimated EVLW, three suggesting that EVLW increases with PEEP [6–8] and another one that it remains unchanged [28]. How- ever, these studies did not investigate the potential arte- fact that may induce an increase in EVLW along with the PEEP level. TPTD measurements were performed by injecting 15-mL boluses of saline (< 8 °C) through a jugular vein catheter. In order to allow the detection of small changes in EVLW, the average of the results obtained by five suc- cessive thermodilution measurements was used. With this number of replicates, the least significant change of EVLW is 8% [32]. With TPTD, we also measured the pulmonary vascular permeability index (PVPI) [1, 33] (also averaged from five successive thermodilution measurements) and cardiac index (CI). Indeed, the PEEP-induced lung recruitment may relieve the hypoxic vasoconstriction of the recruited regions, which eventually become accessible to the cold indicator while they were not at a lower PEEP level. This may lead to an artefactual overestimation of the PEEP- induced EVLW augmentation. Background able to give consent, the same information was delivered to them, with possibility for them to deny the participa- tion. All patients and/or relatives accepted to participate. Inclusion criteria were age ≥ 18 years, presence of ARDS according to the Berlin definition [29] and moni- toring with a TPTD device (PiCCO2 device, Pulsion Medical Systems, Feldkirchen, Germany). Exclusion criteria were contraindications to PEEP increase (pneu- mothorax, uncontrolled shock state) and extracorporeal membrane oxygenation, which impedes the measure- ment of EVLW by TPTD. Patients could be under contin- uous venovenous haemofiltration since it does not affect the TPTD estimation of EVLW [30, 31]. Extravascular lung water (EVLW) is the amount of fluid present in the lungs, outside the pulmonary blood vessels [1]. In acute respiratory distress syndrome (ARDS), lung injury leads to increases in the pulmonary capillary per- meability and in EVLW, which reflect the severity of the disease [2]. Many studies have investigated the changes in EVLW induced by a positive end-expiratory pressure (PEEP), which is the cornerstone of ARDS treatment (Additional file 1: Table S1). However, they have provided very dis- cordant results, some showing that EVLW augmented when increasing levels of PEEP were applied [3–8], some that it decreased [9–17] and some others that it did not change [18–28].h Methods This prospective, one-centre study was approved by the Institutional Review Board of our institution (Comité pour la Protection des Personnes, Ile-de-France VII, IDCRB 2015-A01654-45). At the time of inclusion, patients’ relatives were informed of the study protocol and possibility was given to them to refuse participation. As soon as clinical condition improved and patients were Gavelli et al. Ann. Intensive Care (2020) 10:28 Gavelli et al. Ann. Intensive Care (2020) 10:28 Gavelli et al. Ann. Intensive Care (2020) 10:28 Page 3 of 8 estimated the minimal predicted change in lung volume determined by the PEEP change, as previously described [35]. Briefly, the respiratory system compliance at low PEEP was multiplied by the pressure difference between the two PEEP levels. Then, this value was subtracted from the total change in lung volume and the result was considered as an estimation of derecruited lung volume induced by PEEP reduction [34, 35]. In addition, in the whole population, we estimated the degree of derecruit- ment during the PEEP decrease by observing the simul- taneous changes in compliance of the respiratory system (Crs) [36]. For this purpose, Crs was calculated as the ratio of tidal volume (Vt) over the driving pressure (pla- teau pressure—PEEP). Data are expressed as mean ± standard deviation for normally distributed variables or median [interquartile range] for skewed data. A Shapiro–Wilk test was con- sidered to determine if a variable was well-modelled by a normal distribution. The analysis of patients with a large derecruitment compared to the other ones was planned a priori. A linear mixed factor ANOVA for repeated meas- urements was used to evaluate both within-subject effect (PEEP/time effect) and between-subject effects (recruiting effect). Both High-PEEPstart and Low- PEEP45′ have been considered as reference categories for comparisons. Multiple comparisons of means have been performed using Tukey contrasts. The covariate effect on EVLW outcome was then estimated using a linear mixed model for repeated measurements (random intercept model) adjusting the estimates for PEEP, position (prone/supine) and recruiting effect according either to the Crs changes and the recruited lung volume. Sample size calculation and statistical analysis were performed with MedCalc 18.2.1 software (Mariakerke, Belgium) and R 3.5.2 sta- tistical software with lme4 package. We defined patients with a large derecruitment from the other ones as patients in whom Crs changes and the measured derecruited volume were larger than the median of their value observed in the whole population. Study design b l At baseline, patients were ventilated in the assist-control mode with a Vt at 6 mL/kg (predicted body weight). PEEP was set to reach a plateau pressure of 28–30 cmH2O (High-PEEP) [37]. Sedation was provided by propofol and remifentanil. Results Patients At this time (High-PEEPstart), a first set of measure- ments was performed including heart rate, arterial pres- sure, CVP, EVLW and blood gas analysis. PEEP was then decreased, while the derecruited volume was estimated in the 30 patients in whom it was possible. After 15 min (Low-PEEP15′) and 45 min (Low-PEEP45′), we meas- ured the same variables as at baseline. A time interval of 45 min appeared to us as reasonably long enough for allowing potential fluid transfer through the pulmonary capillary barrier. Thereafter, PEEP was increased back to its baseline level. After 15 min, the variables measured at baseline were measured again (High-PEEPend). Sixty consecutive patients were included. On average, ARDS developed for 3 [1–5] days at the time of inclu- sion. Septic shock was present in 54 (90%) patients (Table 1). Pneumonia was the cause of ARDS in all patients. The number of chest X-ray quadrants involved was two in 21 (35%) cases, three in 35 (58%) cases and four in 4 (7%) patients. At baseline, blood lactate was 2.5 [1.6–3.4] mmol/L, creatinine 98 [66–106] μmol/L and 15 (25%) patients had renal replacement therapy in place (conventional haemodialysis in three, continu- ous venovenous hemofiltration in 12 patients, without weight loss). Eleven (18%) patients were in prone posi- tion at the time of inclusion, whereas 28 (47%) other ones had required prone positioning before the inclu- sion (Table 1). Seventeen (28%) patients were paralysed at the time of inclusion and the Richmond Agitation- Sedation Scale was − 4 [− 5 to − 3]. Sedative drugs, Vt, respiratory rate, and the fraction of inspired oxygen (FiO2) remained unchanged during the study. Volume expansion, fluid removal, recruitment manoeuvres, administration of inhaled nitric oxide or nebulization were not performed during this time. Statistical analysis Considering an α risk of 5% and a β risk of 20%, to evi- dence a PEEP-induced change in EVLW by 2 ± 4 mL/kg, we estimated that 54 patients should be included into the study, a number that was rounded to 60. The PEEP- induced change in EVLW was estimated by considering that the least significant change of the measurement is 8% if five values of TPTD are averaged [32] and by expecting a baseline EVLW of 20 ± 6 mL/kg [38]. Effects of PEEP changes on haemodynamic variables Effects of PEEP changes on haemodynamic variables The decrease in PEEP from High-PEEPstart decreased CVP by 21 ± 13% (p < 0.01) (Table 2). When PEEP was increased from Low-PEEP45′ to High-PEEPend, opposite and symmetrical changes were observed (Table 2; see Additional file 2: Table S2 for post hoc comparisons). Gavelli et al. Ann. Intensive Care (2020) 10:28 Page 4 of 8 Table 1 Patient characteristics ARDS: acute respiratory distress syndrome; E/e′: ratio of the amplitude of the E wave over the amplitude of the e′ wave of the mitral flow with echocardiography; ICU: intensive care unit; PBW: predicted body weight Patient characteristics (n = 60) Age (years) 69 ± 10 Male gender (n, %) 34 (57%) Body mass index (kg/m2) 25 ± 4 Simplified Acute Physiologic Score II on inclusion 51 ± 18 ARDS severity (n, %) Mild 21 (35%) Moderate 31 (52%) Severe 8 (13%) Aetiology of ARDS (n, %) Community acquired pneumonia 46 (77%) Aspiration pneumonia with neurologic disorders 5 (8%) Ventilator associated pneumonia 9 (15%) ICU length of stay (days) 12 [11–38] Total duration of mechanical ventilation (days) 11 [10–37] Mortality at day-28 (n, %) 26 (43%) Norepinephrine Number of patients (%) 54 (90%) Dose of norepinephrine (µg/kg/min) 0.53 [0.27–1.00] Left ventricular ejection fraction (%) 44 ± 11 E/e′ ratio 9 ± 2 Ventilator settings Tidal volume (mL/kg of PBW) 5.5 [5.0–6.0] Respiratory rate (breaths/min) 27 ± 5 Fraction of inspired oxygen 0.64 ± 0.20 Patients requiring prone position (n, %) 39 (65%) Effects of PEEP changes on EVLW Decreasing PEEP from High-PEEPstart induced a sig- nificant decrease in EVLW by 8 ± 7% (p < 0.01) (Table 2, Fig. 1). This decrease in EVLW was observed in all the patients but two (Fig. 1). It persisted at Low-PEEP15′ and Low-PEEP45′. When PEEP was increased from Low- PEEP45′ to High-PEEPend, opposite and symmetrical changes in EVLW were observed (Table 2).f Table 1 Patient characteristics Effects of PEEP changes on EVLW Patient characteristics (n = 60) When we evaluated the covariate effect on EVLW at the linear mixed model for repeated measures, adjusted for PEEP, prone/supine position and recruitment accord- ing to Crs changes, only the changes in PEEP and CVP were significantly associated to the changes in EVLW (p < 0.001 and p = 0.03, respectively) (Table 3). Were the PEEP‑induced changes in EVLW due to artefacts of the TPTD technique? ARDS: acute respiratory distress syndrome; E/e′: ratio of the amplitude of the E wave over the amplitude of the e′ wave of the mitral flow with echocardiography; ICU: intensive care unit; PBW: predicted body weight At the bedside, the only technique that allows the meas- urement of EVLW is TPTD. Although it can detect interstitial oedema, lung ultrasound does not allow the quantification of the EVLW total volume, and CT scan cannot be used routinely. The estimation of EVLW by TPTD in humans has been demonstrated to correlate with the one provided by gravimetry [39], which is the reference technique. Even small and rapid changes in EVLW can be measured [40]. The value of EVLW has been regularly demonstrated to be correlated with mor- tality in critically ill patients [41, 42], especially in septic shock [43, 44] and ARDS [38, 45]. Effects of PEEP changes on respiratory variables From High-PEEPstart to Low-PEEP15′, PEEP decreased by 9 ± 2 cmH2O and this was accompanied by a decrease in the plateau pressure by 8 ± 3 cmH2O. When decreasing PEEP from High-PEEPstart, the change in Crs was 0.0 [− 3.8 to 3.8] mL/cmH2O (n = 60) (Table 2). Since the median value of Crs changes was 0.0 mL/ cmH2O, we defined derecruiters as patients in whom decreasing PEEP induced a decrease in Crs. When decreasing PEEP from High-PEEPstart, the estimated derecruited lung volume was 144 [68–420] mL in the patients in whom it was measured (n = 30). Nevertheless, the ability of TPTD to assess the changes in EVLW induced by PEEP has been only scarcely inves- tigated, despite its important role in ARDS management [46]. Moreover, the few available studies did not specifi- cally investigate the artefact that may affect the TPTD estimation of PEEP-induced changes in EVLW [6–8, 28]. As a matter of fact, by relieving the hypoxic vaso- constriction in recruited areas, PEEP may allow the cold indicator to reach these regions, increasing the volume of EVLW that is accessible to measurement. In our study, the changes in EVLW were the same among patients with high or low derecruitment, when derecruitment No differences were found in terms of EVLW changes between derecruiters and the other patients, defined according to either the Crs change (n = 60) or the dere- cruited volume (n = 30). Discussionh This study shows that decreasing PEEP in ARDS patients induces a small, reversible and rapid decrease in EVLW measured by TPTD. The recruited lung volume was not independently associated with this change in EVLW, while it was the case for the change in CVP. Effects of PEEP changes on haemodynamic variables When we performed the same analysis by estimating recruitment according to lung volume changes (n = 30), CVP but not the recruited lung volume remained significantly associ- ated to the changes in EVLW (p < 0.001). Were the PEEP‑induced changes in EVLW due to artefacts of the TPTD technique? All the significant changes in respiratory variables reversed with a similar amplitude when PEEP was increased from Low-PEEP45′ to High- PEEPend (Table 2). Gavelli et al. Ann. Intensive Care (2020) 10:28 Page 5 of 8 Table 2 Haemodynamic and respiratory variables FiO2 inspired oxygen fraction, PEEP positive end-expiratory pressure, Pplateau plateau pressure, PaO2 arterial oxygen partial pressure, SaO2 arterial oxygen saturation, SpO2 pulse oxygen saturation p < 0.05 vs. High-PEEPstart, p < 0.05 vs. Low-PEEP45′. See Additional file 2: Table S2 for post hoc comparisons Variables High-PEEPstart Low-PEEP15′ Low-PEEP45′ High-PEEPend Heart rate (min−1) 89 ± 18 90 ± 19 91 ± 19 91 ± 19 Systolic arterial pressure (mmHg) 130 ± 25 132 ± 19 129 ± 19 124 ± 17 Diastolic arterial pressure (mmHg) 65 ± 20 63 ± 10 62 ± 11 62 ± 10 Mean arterial pressure (mmHg) 86 ± 16 86 ± 13 85 ± 14 83 ± 11 Central venous pressure (mmHg) 15 ± 4 12 ± 4 12 ± 4* 15 ± 4** Cardiac index (L/min/m2) 2.77 ± 0.79 3.08 ± 0.85* 3.12 ± 0.90* 2.81 ± 0.89** Cardiac function index (min−1) 4.0 ± 1.2 4.3 ± 1.3* 4.3 ± 1.3* 4.1 ± 1.3** Global end-diastolic volume indexed (mL/m2) 750 ± 116 787 ± 168* 791 ± 150* 748 ± 127** Extravascular lung water (mL/kg) 20 ± 4 18 ± 4* 18 ± 4* 20 ± 5** Pulmonary vascular permeability index 3.6 ± 1.0 3.5 ± 1.0* 3.5 ± 1.0* 3.6 ± 0.9 Pulse pressure variation (%) 7 [4–14] 6 [4–12] 8 [4–12] 7 [5–11] Stroke volume variation (%) 8 [5–13] 7 [4–13] 7 [5–13] 8 [6–13] PEEP (cmH2O) 14 ± 2 5 ± 0* 5 ± 0* 14 ± 2** Pplateau (cmH2O) 28 ± 2 20 ± 3* 20 ± 3* 28 ± 2** Respiratory system compliance (mL/cmH2O) 27 [22–32] 26 [24–28] 25 [23–28] 27 [23–31] SpO2 (%) 98 ± 2 96 ± 3* 95 ± 4* 98 ± 2** SaO2 (%) 97 ± 3 94 ± 5* 93 ± 6+* 96 ± 4** PaO2/FiO2 ratio 184 ± 76 150 ± 69* 147 ± 68* 178 ± 76** Table 2 Haemodynamic and respiratory variables FiO2 inspired oxygen fraction, PEEP positive end-expiratory pressure, Pplateau plateau pressure, PaO2 arterial oxygen partial pressure, SaO2 arterial oxygen saturation, SpO2 pulse oxygen saturation p < 0.05 vs. High-PEEPstart, p < 0.05 vs. Low-PEEP45′. Were the PEEP‑induced changes in EVLW due to artefacts of the TPTD technique? See Additional file 2: Table S2 for post hoc comparisons Table 3 Linear mixed model estimation on extravascular lung water changes adjusted by presence or absence of prone position and by changes in positive end- expiratory pressure, central venous pressure, and compliance of the respiratory system Fig. 1 Individual values of extravascular lung water (EVLW) at different study times. PEEP positive end-expiratory pressure. p < 0.05 vs. High-PEEP, *p < 0.05 vs. 45′ after decreasing PEEP at 5 cmH2O CVP central venous pressure, DF degrees of freedom, PEEP positive end- expiratory pressure, Crs respiratory system compliance Value Standard error DF t value p value Intercept 15.58 1.15 178 13.53 CVP 0.15 0.07 178 2.16 0.03 PEEP 0.13 0.03 178 4.79 < 0.01 Prone position − 0.78 1.56 57 − 0.5 0.62 Crs 0.61 1.21 57 0.5 0.62 was assessed by the PEEP-induced change in lung vol- ume, the method that is today the best one for estimat- ing recruitment/derecruitment at the bedside [36]. It was also the same in the whole population, when we defined derecruitment as a decrease in Crs. Moreover, neither the estimated derecruited lung volume nor the Crs changes were independently associated with EVLW changes at linear mixed model analysis. Another argument against the explanation of EVLW changes by artefacts due to lung recruitment is that the changes in EVLW were observed rapidly both after Fig. 1 Individual values of extravascular lung water (EVLW) at different study times. PEEP positive end-expiratory pressure. p < 0.05 vs. High-PEEP, *p < 0.05 vs. 45′ after decreasing PEEP at 5 cmH2O Fig. 1 Individual values of extravascular lung water (EVLW) at different study times. PEEP positive end-expiratory pressure. p < 0.05 vs. High-PEEP, **p < 0.05 vs. 45′ after decreasing PEEP at 5 cmH2O Gavelli et al. Ann. Intensive Care (2020) 10:28 Page 6 of 8 Page 6 of 8 reducing and increasing the PEEP level. Incrementing and decrementing PEEP have different impact on the time required to reach equilibrium in the respiratory sys- tem [47]. The fact that specular changes were observed after opposite PEEP changes strongly suggests that a haemodynamic mechanism may be a more plausible explanation for the observed results. in humans comes from animal studies, and it is much of an assumption that lymph flow is the same per kilo- gram of bodyweight in humans as in dogs [51]. Mechanisms of the PEEP‑induced changes in EVLW Since the PEEP-induced changes in EVLW we observed were not due to artefacts in the TPTD estimation, one should consider that EVLW was really decreased when the PEEP level was reduced, and that this small and rapid change was reversible. Although of small amplitude, the EVLW changes were actually significant. Moreover, Fig. 1 shows well how EVLW changes were very consistent among patients. Also, we took the precaution to measure EVLW by averaging not three but five TPTD measure- ments, which enabled us to reliably detect small changes in EVLW [32]. In particular, it might be possible that part of the changes in EVLW we observed were related to changes in lung permeability, although this seems to be unlikely in such a short time. The decrease in PEEP was associ- ated with a significant but slight decrease in PVPI, which reflects alveolo-capillary permeability. Nevertheless, this change was very small, and was not significantly reversed when PEEP was re-increased. Our results are in accordance with the previous stud- ies which, amongst very discrepant ones, suggested that PEEP induces small increases in EVLW [3–8]. In theory, three mechanisms might explain why EVLW varies in the same direction as PEEP (Additional file 3: Figure S1). First, decreasing PEEP decreases CVP, which is the back- ward pressure of the drainage through the thoracic duct. This may happen by direct transmission of the intratho- racic pressure to the right atrial pressure, or as the result of the decrease in the right ventricular afterload. Although the changes in EVLW were of lower amplitude than those of CVP, the results of the linear mixed effect model make this pathophysiological hypothesis accept- able. Of note, even though it may increase CVP in ARDS patients [48, 49], prone position in our population was not an element influencing the relationship between CVP and EVLW. Practical implicationsi First, our findings show that TPTD is not flawed by the level of PEEP, as it has been previously suspected [53]. Second, our observation that increasing PEEP increases EVLW does not challenge the benefit of PEEP in ARDS. The increase in EVLW we report was small and might be easily counterbalanced by the potential benefits of PEEP such as increase in end-expiratory lung volume induced by recruitment, decrease in pulmonary shunt in recruit- ers and redistribution of alveolar fluid to extra-alveolar spaces [36]. Nevertheless, when using TPTD at the bed- side [54], clinicians should be aware that changing PEEP might slightly change EVLW and that it is not due to a worsening of the disease or to the deleterious effects of some fluid administration. Were the PEEP‑induced changes in EVLW due to artefacts of the TPTD technique? Then, this is compatible with the amount of changes in EVLW we observed. The PEEP decrease led to a reduction of EVLW by 1.6 ± 1.6 mL/kg, which was equivalent to roughly 100 mL of lung water accumulated in 15 min, and vice versa when PEEP was re-increased. Nevertheless, since we did not directly measure the lymphatic flow and since the link between EVLW and CVP observed in our results was imperfect, we cannot exclude the contribution of other mechanisms. Conclusions In ARDS patients, changing the PEEP level induced parallel, small and reversible changes in EVLW. These changes were not due to an artefact of the TPTD tech- nique and are likely due to the PEEP-induced changes in CVP. Received: 7 June 2019 Accepted: 21 February 2020 Author details 1 Service de médecine intensive ‑ réanimation, Hôpitaux universitaires Paris-Saclay, Hôpital de Bicêtre, APHP, 78, rue du Général Leclerc, 94270 Le Kremlin‑Bicêtre, France. 2 Inserm UMR S_999, Univ Paris-Saclay, 78, rue du Général Leclerc, 94270 Le Kremlin‑Bicêtre, France. 3 Dipartimento di Medicina Traslazionale, Università del Piemonte Orientale, Via Solaroli 17, 28100 Novara, Italy. Competing interests J.-L. Teboul and X. Monnet are members of the Medical Advisory Board of Pulsion Medical Systems. J.-L. Teboul and X. Monnet gave lectures for Masimo. The other authors have no conflict of interest to declare. No financial support. Availability of data and materials we did in the whole population, has many limitations [36]. Fourth, the number of saline boluses required for averaging EVLW measurements may have provoked fluid-induced changes in EVLW. However, the fact that EVLW decreased at the first study step indicates that this limitation probably had a very small impact. Fifth, because this was a human study, we could not directly measure the lymphatic flow, a procedure that could have strengthened our conclusions. Finally, we did not insert either a pulmonary artery catheter or an oesophageal balloon and thus could not estimate the hydrostatic lung filtration pressure and the trans- mural pressure. We thus cannot exclude that changing PEEP also changed the degree of pulmonary oedema formation. we did in the whole population, has many limitations [36]. Fourth, the number of saline boluses required for averaging EVLW measurements may have provoked fluid-induced changes in EVLW. However, the fact that EVLW decreased at the first study step indicates that this limitation probably had a very small impact. Fifth, because this was a human study, we could not directly measure the lymphatic flow, a procedure that could have strengthened our conclusions. Finally, we did not insert either a pulmonary artery catheter or an oesophageal balloon and thus could not estimate the hydrostatic lung filtration pressure and the trans- mural pressure. We thus cannot exclude that changing PEEP also changed the degree of pulmonary oedema formation. Individual, de-identified participant data are available from the corresponding author on reasonable request. References k 1. Jozwiak M, Teboul J-L, Monnet X. Extravascular lung water in critical care: recent advances and clinical applications. Ann Intensive Care. 2015;5:38. Supplementary information accompanies this paper at https://doi. org/10.1186/s13613-020-0644-2. Supplementary information acc /10 1186/ 13613 020 0644 2 Supplementary information ac org/10.1186/s13613-020-0644-2. Supplementary information accompanies this paper at https://doi. org/10.1186/s13613-020-0644-2. Supplementary information ac org/10.1186/s13613-020-0644-2. 2. Kushimoto S, Endo T, Yamanouchi S, Sakamoto T, Ishikura H, Kitazawa Y, et al. Relationship between extravascular lung water and severity categories of acute respiratory distress syndrome by the Berlin definition. Crit Care. 2013;17:R132. Additional file 1: Table S1. Previous literature regarding positive end- expiratory pressure effects on lung water in acute respiratory distress syndrome. 3. Toung T, Saharia P, Permutt S, Zuidema GD, Cameron JL. Aspiration pneu‑ monia: beneficial and harmful effects of positive end-expiratory pressure. Surgery. 1977;82:279–83. Additional file 2: Table S2. Mean difference of haemodynamic and res‑ piratory variables for post hoc comparisons using Tukey HSD approach. g y 4. Carlile PV, Lowery DD, Gray BA. Effect of PEEP and type of injury on thermal-dye estimation of pulmonary edema. J Appl Physiol. 1985;1986(60):22–31. Additional file 3: Figure S1. Possible haemodynamic effects of positive end-expiratory pressure (PEEP) decrease on extravascular lung water (EVLW) levels, not taking into consideration possible artefactual effects related to the transpulmonary thermodilution (TPTD) method. 5. Nieman GF, Bredenberg CE, Paskanik AM. Positive end-expiratory pres‑ sure accelerates lung water accumulation in high surface tension edema. Surgery. 1990;107:156–62. 6. Szakmany T, Heigl P, Molnar Z. Correlation between extravascular lung water and oxygenation in ALI/ARDS patients in septic shock: pos‑ sible role in the development of atelectasis? Anaesth Intensive Care. 2004;32:196–201. Ethics approval and consent to participate Information and consent obtained for each patient.Name of the ethics committee that approved the study and the Committee’s reference number: Comité pour la Protection des Personnes, Ile-de-France VII. Trial registration ID RCB: 2015-A01654-45. Registered 23 October 2015. The patients were included prospectively. Authors’ contributions JLT and XM conceived and designed the study. AB, CL, FG, IA and TT recruited the patients and collected the data. DA, FG, JLT and XM analysed and inter‑ preted the data. GCA supervised the data interpretation. FG and XM drafted the report and all authors contributed to review it. All authors read and approved the final manuscript. 10. Russell JA, Hoeffel J, Murray JF. Effect of different levels of positive end- expiratory pressure on lung water content. J Appl Physiol. 1982;53:9–15. 11. Myers JC, Reilley TE, Vento JM, McDonald JS, Carey LC, Cloutier CT. Does compliance reflect oxygen delivery in porcine septic respiratory failure treated with positive end-expiratory pressure? Crit Care Med. 1987;15:38–40. Abbreviations ARDS A ARDS: Acute respiratory distress syndrome; CVP: Central venous pressure; CI: Cardiac index; Crs: Respiratory system compliance; EVLW: Extravascular lung water; FiO2: Inspired oxygen fraction; PaO2: Arterial oxygen partial pressure; PaO2/FiO2: Arterial oxygen tension over inspired oxygen fraction ratio; PEEP: Positive end-expiratory pressure; Pplateau: Plateau pressure; PVPI: Pulmonary vascular permeability index; SaO2: Arterial oxygen saturation; TPTD: Transpul‑ monary thermodilution; Vt: Tidal volume. 7. Krebs J, Pelosi P, Tsagogiorgas C, Alb M, Luecke T. Effects of positive end-expiratory pressure on respiratory function and hemodynamics in patients with acute respiratory failure with and without intra-abdominal hypertension: a pilot study. Crit Care. 2009;13:R160. 7. Krebs J, Pelosi P, Tsagogiorgas C, Alb M, Luecke T. Effects of positive end-expiratory pressure on respiratory function and hemodynamics in patients with acute respiratory failure with and without intra-abdominal hypertension: a pilot study. Crit Care. 2009;13:R160. 8. Wu X, Zheng R, Zhuang Z. Effect of transpulmonary pressure-guided positive end-expiratory pressure titration on lung injury in pigs with acute respiratory distress syndrome. J Clin Monit Comput. 2020;34:151–9 Limitations The second mechanism which may explain why the PEEP decrease diminished EVLW is a decrease in the formation of lung water (Additional file 3: Figure S1). Indeed, the intrathoracic pressure is transmitted to the left atrium, such that when PEEP is decreased, the intra- mural pulmonary capillary pressure is decreased as well. It is well known that, on the opposite, augmenting PEEP increases the intramural pulmonary artery occlusion pressure [50]. We could not assess this mechanism, since we estimated neither the pulmonary capillary pressure nor the pulmonary artery occlusion pressure in our study.hl The second mechanism which may explain why the PEEP decrease diminished EVLW is a decrease in the formation of lung water (Additional file 3: Figure S1). Indeed, the intrathoracic pressure is transmitted to the left atrium, such that when PEEP is decreased, the intra- mural pulmonary capillary pressure is decreased as well. It is well known that, on the opposite, augmenting PEEP increases the intramural pulmonary artery occlusion pressure [50]. We could not assess this mechanism, since we estimated neither the pulmonary capillary pressure nor the pulmonary artery occlusion pressure in our study. The normal pulmonary lymphatic flow is estimated to be 8–9 mL/h in humans [51]. Nevertheless, it has been reported that the pulmonary lymphatic flow could increase to tenfold, or even more, during ARDS [52]. Moreover, the estimation of pulmonary lymphatic flow First, we only observed the short-term effects of PEEP. We judged it was ethically unacceptable to maintain these patients with ARDS at a low PEEP level for a long time. Moreover, it would have been impossible to avoid confounding events (changes in ventilatory set- ting and fluid administration or removal) over longer periods. Second, we estimated the derecruited volume during the PEEP decrease and not the recruited volume during the PEEP re-increase. Indeed, we speculated that derecruitment may occur faster than recruitment and be easier to detect [34]. Third, we directly meas- ured the PEEP-induced changes in lung volume in 30 patients only, though it is the best method to estimate lung recruitment or derecruitment at the bedside. Estimating derecruitment through changes in Crs, as The normal pulmonary lymphatic flow is estimated to be 8–9 mL/h in humans [51]. 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https://openalex.org/W2106941578	https://casesjournal.biomedcentral.com/track/pdf/10.1186/1757-1626-3-30	English	null	Non-traumatic fractures following seizures: two case reports	Cases journal	2,010	cc-by	2,596	* Correspondence: ines.slim@yahoo.fr Department of Endocrinology and Diabetology, Farhat Hached University Hospital, Ibn Jazzar Street, 4002 Soussa, Tunisia © 2010 Ach et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Open Access Open Access Abstract Introduction: Seizures with or without trauma may cause fractures that occur commonly in epileptic seizures. Fracture risk is less reported in non-epileptic seizures. Some metabolic conditions leading to a decrease in bone mineral density may cause fractures secondary to non-epileptic seizure. Case presentation: We describe two cases of non-traumatic acetabular and vertebrae fractures following seizures without history of epilepsy. They occurred in two male patients, 18 and 48 years old suffering respectively from hypercorticism and poorly controlled diabetes mellitus. Seizures, occurring inside hospital, were secondary to hypertensive encephalopathy crisis with hypokaliemia in the first case and severe hypoglycaemia in the second one. Fracture was promoted by a decrease in mineral bone density caused respectively by hypercorticism and diabetic chronic renal failure. Conclusion: These observations emphasize that fracture prevention among patients with decreased mineral bone density should include the avoidance of metabolic causes of seizure. Non-traumatic fractures following seizures: two case reports Koussay Ach, Ines Slim, Sihem Trimech Ajmi, Molka Chadli Chaieb, Amel Maaroufi Beizig, Larbi Chaieb Introduction generalized tonic-clonic seizures that was not followed by trauma and that regressed after parenteral hypertonic glucose serum perfusion. Seizures increase fracture risk [1], occurring in the whole of the skeleton even without trauma [2]. This risk is mainly reported in epileptic seizures because of repeated occurrence of attacks and osteopenia due to the use of anti-epileptic drugs [1]. Literature data have shown that fractures are more related to the seizure itself rather than to the iatrogenic factor [1]. Moreover, some metabolic conditions may lead to a decrease in bone mineral density and hence may be considered as risk factors of fracture when seizures occur. We report here two cases of this unusual situation to resort its eventual prevention. Laboratory assessment showed a severe hypoglycemia (capillary glycemia at 1.8 mmol/l) and nephrotic syn- drome with renal failure (creatininemia at 241 μmol/l). After seizure, the patient presented a disability of the left lower limb. X-ray examination showed a fracture in the left iliac bone localized in the acetabular floor (Fig- ure 1) while brain CT-scan was normal. The patient was referred to orthopedic center where he was treated con- servatively. Six months later, the patient was able to walk. Insulin therapy was adjusted aiming to avoid hypoglycemia. Ach et al. Cases Journal 2010, 3:30 http://www.casesjournal.com/content/3/1/30 Ach et al. Cases Journal 2010, 3:30 http://www.casesjournal.com/content/3/1/30 Case report 1 An Arabic 18-year-old man from North Africa (Tunisia) was referred to our department for suspicion of Cushing syndrome. On physical examination, the patient was obese (BMI at 32 kg/m2), he had purplish abdominal striae and hypertension (systolic pressure between 15 and 17 mmHg). Neurological examination was normal. An Arabic 48-year-old man from North Africa (Tunisia) with a ten year history of diabetes mellitus is treated by insulin twice a day for seven years. His diabetes is poorly controlled mainly due to lack of observance. Additionally, he suffered from depression with suicide ideas. Shortly after an overdose of insulin, he developed Laboratory examination showed hypokalemia at 2.1 mmol/l confirmed in several samples separately per- formed while glycemia and calcemia were at normal range. Urinary free cortisol excretion was high (462 Ach et al. Cases Journal 2010, 3:30 http://www.casesjournal.com/content/3/1/30 Page 2 of 4 Figure 1 Radiography of hip after seizures showing left acetabular fracture (Case 1). Figure 1 Radiography of hip after seizures showing left acetabular fracture (Case 1). Figure 1 Radiography of hip after seizures showing left acetabular fracture (Case 1). reported fractures during seizures. Fracture risk is nota- bly increased mainly among epileptic patients. It occurs in 11% of patients mainly within the first two years of diagnosis. This risk is directly related to seizures in 43% of cases in one of the largest series [3]. μmol/24 h) and was not suppressed after both low and high doses of dexamethasone confirming the diagnosis of Cushing syndrome. X-ray examination (Figure 2) and bone densitometry revealed decrease of bone mineral density with a Z-score of -2. Brain MRI imaging showed a double pituitary adenoma without any other hemi- spheric lesion. The diagnosis of corticotropic adenoma was retained. Fractures concern the whole of skeleton particularly the skull, members, vertebras and the hip and may be multiples, bilateral or associated to other lesions such as dislocations of the shoulder [4,5]. They induce an increase of management cost of epileptic disease [6]. Acetabular fracture observed in the first patient has also been reported and may be serious leading to fatal pelvic haemorrhage [7,8]. During an acute hypertensive peak at 220 mm Hg, the patient developed tonic-clonic seizures without any trauma. His glycemia was normal (5.6 mmol/l). Seizures regressed after intravenous load of Loxen and Diaze- pam* however the patient suffered from mild low back pain. Case report 1 Radiographies identified L2, L3, L4 and L5 burst fractures (Figure 3). Non-epileptic seizures reported in our cases are unu- sual. In our first case, seizures are related to severe hypoglycaemia, which is common cause of seizures. They were reported in 4.7% of all the hospitalisations for hypoglycaemias especially in old and depressive patients [9]. Concerning the second patient, seizures are caused by encephalopathy hypertensive crisis in addition to hypokaliemia due to hypercorticism. These results are in line with database that emphasizes the role of hyper- tensive encephalopathy [10], and ionic disorders in sei- zures such as hypokaliemia and hypocalcaemia [5]. Cushing disease was surgically treated with regression of hypertension and weight. Bone mineral density was checked two years later and showed an improved Z- score. Discussion We reported here two cases of hip and vertebrae frac- tures following non-epileptic generalized tonic-clonic seizures without any trauma or fall. Several data have Ach et al. Cases Journal 2010, 3:30 http://www.casesjournal.com/content/3/1/30 Page 3 of 4 Figure 2 Radiography of lumbar vertebras before seizures showing bone demineralization (patient n 2). Figure 3 Radiography of lumbar vertebras after seizures showing fractures (patient n 2). Figure 3 Radiography of lumbar vertebras after seizures showing fractures (patient n 2). Figure 2 Radiography of lumbar vertebras before seizures showing bone demineralization (patient n 2). Fractures occurring without any trauma suggest pre- vious bone fragility due to other illness [5]. Bone fragi- lity has been confirmed in our second patient by bone mineral densitometry assessment before seizures. Figure 3 Radiography of lumbar vertebras after seizures showing fractures (patient n 2). as in our patient [13] or when diagnosis is delayed [16] leading to definitive bone sequels. Our cases concern male young patients who are not at risk of primary osteoporosis. Among classic reported causes of male secondary osteoporosis [11], we can incriminate secondary hyperparathyroidism caused by diabetic renal failure in the first patient [12] and hyper- corticism in the second one [13]. Nevertheless, diabetes mellitus is an unusual cause of osteoporosis and usually we find associated fracture risk factors [17]. Diabetes could play an indirect role through increasing fall risk-related fractures due to feet injuries [18]. Of interest, some prospective data showed that dia- betes mellitus is a statistically risk factor of osteoporosis especially in insulin treated diabetes, long history of dia- betes or in elderly [19]. Hypercorticism is one of the main diseases that cause osteoporosis leading to fractures. Vertebral bodies and ribs are the typical sites of fracture both in Cushing’s syndrome and in patients using glucocorticoid drugs long-term [14]. The frequency of vertebral fractures has ranged from 16 to 20% [14]. Decreased bone mineraliza- tion is particularly pronounced in children and in young females with Cushing’s syndrome [14]. Interestingly, ear- lier studies had shown that young and adolescent patients undergoing glucocorticoid therapy may lose bone mass more rapidly than older patients [15]. Frac- ture risk is higher at the onset of disease and may reveal it. Discussion It may also occur during clinical investigations such The role of metabolic control in osteopenia is still not well established despite that some data suggest that insulin, growth factors or osmotic factors such as extra- cellular glucose levels may play a significant role in the osteoblast metabolism [20]. References Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge 12. Akcali O, Kosay C, Gunal I, Alici E: Bilateral trochanteric fractures of the femur in a patient with chronic renal failure. Int Orthop 2000, 24:179-180. 12. Akcali O, Kosay C, Gunal I, Alici E: Bilateral trochanteric fractures of the femur in a patient with chronic renal failure. Int Orthop 2000, 24:179-180. 13. Mancini T, Doga M, Mazziotti G, Giustina A: Cushing’s syndrome and bone. Pituitary 2004, 7:243-246. 13. Mancini T, Doga M, Mazziotti G, Giustina A: Cushing’s syndrome and bone. Pituitary 2004, 7:243-246. 14. Khanine V, Fournier JJ, Requeda E, Luton JP, Simon F, Crouzet J: Osteoporotic fractures at presentation of Cushing’s disease: two case reports and a literature review. Joint Bone Spine 2000, 67:341-345. 14. Khanine V, Fournier JJ, Requeda E, Luton JP, Simon F, Crouzet J: Osteoporotic fractures at presentation of Cushing’s disease: two case reports and a literature review. Joint Bone Spine 2000, 67:341-345. 15. Consent Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review from the journal’s Editor-in-Chief. doi:10.1186/1757-1626-3-30 Cite this article as: Ach et al.: Non-traumatic fractures following seizures: two case reports. Cases Journal 2010 3:30. Conclusion These cases demonstrate the possibility of non-trau- matic fracture following seizure. Diabetes mellitus and Page 4 of 4 Page 4 of 4 Ach et al. Cases Journal 2010, 3:30 http://www.casesjournal.com/content/3/1/30 Ach et al. Cases Journal 2010, 3:30 http://www.casesjournal.com/content/3/1/30 17. Forsen L, Meyer HE, Midthjell K, Edna TH: Diabetes mellitus and the incidence of hip fracture: results from the Nord-Trondelag Health Survey. Diabetologia 1999, 42:920-925. 17. Forsen L, Meyer HE, Midthjell K, Edna TH: Diabetes mellitus and the incidence of hip fracture: results from the Nord-Trondelag Health Survey. Diabetologia 1999, 42:920-925. hypercorticism may cause, even in males, osteoporosis and subsequently are considered as risk factors of sei- zures and fractures. Importantly, these conditions may be well-managed to prevent osteoporosis. We insist not only on avoiding falls and trauma but also on preventing metabolic causes of seizures such as hypoglycaemia, ionic disturbances and hypertensive encephalopathy. y 18. Wallace C, Reiber GE, LeMaster J, Smith DG, Sullivan K, Hayes S, Vath C: Incidence of falls, risk factors for falls, and fall-related fractures in individuals with diabetes and a prior foot ulcer. Diabetes Care 2002, 25:1983-1986. 19. Nicodemus KK, Folsom AR: Iowa Women’s Health Study. Type 1 and type 2 diabetes and incident hip fractures in postmenopausal women. Diabetes Care 2001, 24:1192-1197. 20. Leidig-Bruckner G, Ziegler R: Diabetes mellitus a risk for osteoporosis?. Exp Clin Endocrinol Diabetes 2001, 109(Suppl 2):S493-S514. Competing interests Received: 30 November 2009 Accepted: 18 January 2010 Published: 18 January 2010 Received: 30 November 2009 Authors’ contributions f ll d h KA, IS, STA followed the patients reported here. KA, IS wrote the paper, MCC, AMB, LC reviewed the paper. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. References 1. Vestergaard P, Tigaran S, Rejnmark L, Tigaran C, Dam M, Mosekilde L: Fracture risk is increased in epilepsy. Acta Neurol Scand 1999, 99:269-275 2. Takahashi T, Tominaga T, Shamoto H, Shimizu H, Yoshimoto T: Seizure- induced thoracic spine compression fracture: case report. Surg Neurol 2002, 58:214-216. 3. Persson HB, Alberts KA, Farahmand BY, Tomson T: Risk of extremity fractures in adult outpatients with epilepsy. Epilepsia 2002, 43:768-772. 4. Heckmann JG, Stangl R, Erbguth F, Rutherford H, Neundorfer B: Non traumatic seizure-associated bilateral fractures of the head of the humerus. Intensive Care Med 1999, 25:548-549. g g traumatic seizure-associated bilateral fractures of the head of the humerus. Intensive Care Med 1999, 25:548-549. 5. Gosens T, Poels PJ, Rondhuis JJ: Posterior dislocation fractures of the shoulder in seizure disorders - two case reports and a review of literature. Seizure 2000, 9:446-448. 6. Davidson DL, Mac Donald S: The costs of trauma caused by seizures: can they be reduced?. Seizure 2002, 11:344-347. 7. Sikkink CJ, Tol van der A: Unilateral transverse acetabular fracture with medial displacement of the femoral head after an epileptic seizure. J Trauma 2000, 48:777-778. 8. Hughes CA, O’Briain D: Sudden death from pelvic haemorrhage after bilateral central fracture dislocations of the hip due to an epileptic seizure. Am J Forensic Med Pathol 2000, 21:380-384. 9. Stepka M, Rogala H, Czyzyk A: Hypoglycaemia: a major problem in the management of diabetes in the elderly. Aging (Milano) 1993, 5:117-121. 9. Stepka M, Rogala H, Czyzyk A: Hypoglycaemia: a major problem in the management of diabetes in the elderly. Aging (Milano) 1993, 5:117-121. 9. Stepka M, Rogala H, Czyzyk A: Hypoglycaemia: a major problem in the management of diabetes in the elderly. Aging (Milano) 1993, 5:117-121. 10. Zgurzynski P, Manno M: Coccygeal fracture, constipation, convulsion, and confusion: a case report of malignant hypertension in a child. Pediatr Emerg Care 1999, 15:425-428. 10. Zgurzynski P, Manno M: Coccygeal fracture, constipation, convulsion, and confusion: a case report of malignant hypertension in a child. Pediatr Emerg Care 1999, 15:425-428. 10. Zgurzynski P, Manno M: Coccygeal fracture, constipation, convulsion, and confusion: a case report of malignant hypertension in a child. Pediatr Emerg Care 1999, 15:425-428. g 11. Seeman E: Osteoporosis in men. Baillieres Clin Rheumatol 1997, 11:613-629. g 11. Seeman E: Osteoporosis in men. Baillieres Clin Rheumatol 1997, 11:613-629. References Ruegsegger P, Medici T, Anlider M: Corticosteroid-induced bone loss: a longituidal study of alternate day therapy in patients with bronchial asthma using quantitative computed tomography. Eur J Clin Pharmacol 1983, 25:615-620. 16. Yoshihara A, Okubo Y, Tanabe A, Sata A, Nishimaki M, Kawamata T, Kubo O, Hori T, Takano K: A Juvenile Case of Cushing’s Disease Incidentally Discovered with Multiple Bone Fractures. Intern Med 2007, 46:583-587.
https://openalex.org/W2613542736	https://europepmc.org/articles/pmc5429186?pdf=render	English	null	Fabrication of silicon films from patterned protruded seeds	AIP advances	2,017	cc-by	4,584	Fabrication of silicon ﬁlms from patterned protruded seeds Huang Zeng,1,2,a Wei Zhang,2,a Jizhou Li,1,2 Cong Wang,2 Hui Yang,2 Received 1 March 2017; accepted 3 May 2017; published online 12 May 2017) (Received 1 March 2017; accepted 3 May 2017; published online 12 May 2017) Thin, ﬂexible silicon crystals are starting up applications such as light-weighted ﬂex- ible solar cells, SOI, ﬂexible IC chips, 3D ICs imagers and 3D CMOS imagers on the demand of high performance with low cost. Kerﬂess wafering technology by direct conversion of source gases into mono-crystalline wafers on reusable substrates is highly cost-effective and feedstock-effective route to cheap wafers with the thick- ness down to several microns. Here we show a prototype for direct conversion of silicon source gases to wafers by using the substrate with protruded seeds. A reliable and controllable method of wafer-scaled preparation of protruded seed patterns has been developed by ﬁlling liquid wax into a rod array as the mask for the selective removal of oxide layer on the rod head. Selectively epitaxial growth is performed on the protruded seeds, and the voidless ﬁlm is formed by the merging of neighboring seeds through growing. And structured hollows are formed between the grown ﬁlm and the substrate, which would offer the transferability of the grown ﬁlm and the reusability of the protruded seeds. © 2017 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). [http://dx.doi.org/10.1063/1.4983575] AIP ADVANCES 7, 055307 (2017) aThese authors contributed equally. bAuthors to whom correspondence should be addressed. Electronic mails: yigangchen@shu.edu.cn; liudf@sari.ac.cn. aThese authors contributed equally. bAuthors to whom correspondence should be addressed. Electronic mails: yigangchen@shu.edu.cn; liudf@sari.ac.cn. aThese authors contributed equally. bAuthors to whom correspondence should be addressed. Electronic mails: yigangchen@shu.edu.cn; liudf@sari.ac.cn. 2158-3226/2017/7(5)/055307/9 7, 055307-1 © Author(s) 2017 I. INTRODUCTION Silicon is the dominant semiconductor material in the modern electronic and photovoltaic indus- tries. It is mostly used in the form of wafers sliced by costly wire-sawing from ingots.1–5 However, due to kerf loss, the slicing of ingot into wafers wastes great amount of expensive, high pure feed- stock. Additionally, ultrathin wafers are hardly manufactured by wire-sawing because of concern about mechanical stability when sawn.5 In recent years, a direct conversion of feedstock gas to wafers technology based on epitaxial growth on porous silicon has been well developed.6–15 In this technology, a porous silicon bilayer was built by anodization of traditional Si mono-crystal wafers in HF solution with platinum as cathode; the pores on the surface were sealed by hydrogen annealing, forming a thin quasi-mono-crystal template for subsequent epitaxial growth of silicon; the unsealed porous layer was used as a sacriﬁcial layer when the grown wafer was lifted off; and the thickness of grown wafer was controlled just by growth duration, available from several microns to hundreds of microns. And multiple use of the mother substrate can be achieved by rebuilding the porous bilayer after the grown wafer detached. Therefore, this gas-to-wafer technology provided a greatly raw-material-effective and cost-effective route to wafers by skipping the traditional Siemens’ poly-silicon synthesis, ingoting, and wire-sawing steps, and facilitated to realize ﬂexible devices with the ultrathin wafers. Nowadays, thin ﬂexible silicon crystals are spurring applications such as high efﬁcient light-weight and ﬂexible solar cells,8–11,16,17 SOI,12 ﬂexible IC chips,13 monolithic 3D ICs and CMOS imager,14,15 and ﬂexible transistors.18 © Author(s) 2017 2158-3226/2017/7(5)/055307/9 7, 055307-1 055307-2 Zeng et al. AIP Advances 7, 055307 (2017) However, it is rather challenging for the epitaxial technology based on porous silicon layers to avoid metallic contaminants from the electrolyte,8 and the reuse of mother substrates is limited by the re-creating step of the porous double layer in electrolyte solution.19,20 In this work, we demonstrate a prototype using patterned protruded seeds to epitaxially grow silicon ﬁlms,21 intending to form uniformly hollows between the grown ﬁlm and the mother sub- strate by the seed supporting structures, in this way, one approach might be achieved that the grown ﬁlm / wafer could be lifted off without any sacriﬁcial layer and the seeded mother substrate could be reused without any rebuilding steps in liquid. B. Fabrication of protruded seed patterns 4′′, n-type, (100) oriented, 500 µm thick commercial Cz-Si wafers were used as the mother sub- strates. Traditional UV-photolithography was used to form photo-resist mask pattern for inductively coupled dry etching silicon to fabricate rod array; the rods had the diameter of 4µm and the length of 10-15µm, and the array had the period of 10µm. µ y p µ Among the procedure of the prototype, selective removal of the oxide layer on the rod head is vital for the successful fabrication of patterned protruded seeds. Figure 2 shows the schematic procedure of selective removal of oxide layer on the rod head by using wax as the mask. Firstly, after the thermal oxidization, the substrate is immersed into a molten wax liquid which pushes away the air between the rods and completely ﬁlls the space among the rods. Secondly, the substrate is taken out from the wax liquid and kept inclined to run away the surplus liquid on the top of the rods, at the same time, the ﬁlled liquid will be retained because of its adhesiveness and the blocking effect of the rods. Thirdly, the substrate is placed horizontally and the ﬁlled wax liquid will get self-ﬂattened. Finally, the heating system is shut off and the ﬁlled wax liquid gets cooled to room temperature and becomes solid, and the wax will contract a little because of the phase transition from liquid to solid, thus showing up the rod heads for selective removal of the oxide layer by wet etch. By the way, due to the adhesiveness of the wax liquid, there is always a thin wax layer sticky on the rod heads when the wax liquid becomes solid and shrinks. Hence, before the wet etch, the substrate should be put into a slightly soluble organic solvent of wax to get rid of its thin layer on the rod heads. The selective wet etch was here performed by buffered oxide etcher (BOE), and the etching duration was used to control the etched depth down to the rods, i.e., the length of the seeds. A. The strategy of the prototype The strategy of the prototype is schematically shown in Figure 1, a periodically patterned silicon rod array is prepared by the traditional photolithography and inductive coupled plasma (ICP) dry etching method using Czochralski mono-crystal silicon (Cz-Si) wafers as mother substrates; thermal oxidization is performed to form SiO2 layer on the surface of rods and the mother substrate, and this oxide layer will be used as a mask for later selectively epitaxial ﬁlm growth; the oxide layer is selectively removed on the rod head, exposing the inner silicon core to form protruded seed; subsequently, selectively epitaxial growth is performed using the exposed silicon cores as seeds in a chemical vapor deposition system; As the growth evolved, the seeds grow larger and merge into a continuous ﬁlm leaving uniformly structured hollows between the mother substrate and the grown ﬁlm. The formation of the structured hollows is the most important goal of our prototype, which, on one hand, makes the grown ﬁlm in weak mechanical connection with the mother substrate only by the rods, thus will facilitate to mechanically lift off the grown ﬁlm; on the other hand, can provide a passage-way for gases, liquids to run into under the grown ﬁlm, allowing to perform such processing FIG. 1. Schematic strategy of the prototype. a) Cz-Si wafer, i.e., the mother substrate. b) Photo-resist patterning the mother substrate by traditional photolithography. c) Fabrication of rod array by ICP dry etch. d) Preparation of oxide layer by thermal oxidization. e) Selective removal of oxide layer on the rod heads, forming patterned protruded seeds. f) Selective epitaxial growth of silicon ﬁlm on the seeds. FIG. 1. Schematic strategy of the prototype. a) Cz-Si wafer, i.e., the mother substrate. b) Photo-resist patterning the mother substrate by traditional photolithography. c) Fabrication of rod array by ICP dry etch. d) Preparation of oxide layer by thermal oxidization. e) Selective removal of oxide layer on the rod heads, forming patterned protruded seeds. f) Selective epitaxial growth of silicon ﬁlm on the seeds. 055307-3 Zeng et al. AIP Advances 7, 055307 (2017) as etching and coating under the ﬁlm. Furthermore, another goal of our prototype is that the uniformly distributed rods can give a stable mechanical support when devices are processed while the ﬁlm still stands on the mother substrate, especially when the ﬁlm is ultrathin. B. Fabrication of protruded seed patterns Speciﬁcally, the fabrication of protruded seeds took the following procedure: i) kept the substrate with patterned rods at 1000◦C under pure oxygen atmosphere, and got 300-500nm thick oxide layer on the rods and the substrate; ii) immersed the substrate into a wax liquid in an oven at 60◦C, and kept it there for 10min to let air come off from the space between the rods, so got the rod array completely ﬁlled with liquid wax; iii) took the substrate out of the wax liquid and positioned it in the oven by the incline of 60 degree for at least 30min, thus let the surplus wax automatically run away from the top of the rod array; iv) ﬂattened the ﬁlled wax liquid between rods by horizontally placement in the oven for 1 hour; v) shut down the oven and the substrate cooled down to room temperature, and the ﬁlled wax liquid became solid; vi) got rid of the wax thin layer on the rod heads by acetone bath for 5min; vii) selectively removed the oxide layer on the rod heads by BOE solution etching for 4min under the masking of the ﬁlled wax solid; viii) completely removed the ﬁlled wax solid by the dissolution of tetrahydrofuran. D. Morphology characterization The morphologies of the samples were characterized by the scanning electronic microscope (Hitachi S-3400N) and the optical microscope (OM, Keyence, VK-9710). C. Epitaxial growth of silicon ﬁlms When the substrate with patterned protruded seeds was ready, the epitaxial growth of silicon ﬁlm was performed at 1050◦C under atmosphere pressure in a chemical vapor deposition system with cold walls. SiHCl3 gas (Arkonic Gases & Chemicals Inc., 99.999%) was used as the silicon source material with H2 (Airgas, 99.9999%) as the carrier gas. The HCl gas (APK gas, 99.999%.) FIG. 2. Schematic of selective removal of the oxide layer on the rod head to form patterned protruded seed. a) Immerse the substrate into a molten wax liquid. b) Take out the substrate and run away the surplus liquid by inclined placement. c) Flatten the liquid among the rods by upright placement. d) Cool down the ﬁlled wax liquid, and it becomes solid and contracted, thus showing up the rod heads for selective removal of the oxide layer by wet etch. FIG. 2. Schematic of selective removal of the oxide layer on the rod head to form patterned protruded seed. a) Immerse the substrate into a molten wax liquid. b) Take out the substrate and run away the surplus liquid by inclined placement. c) Flatten the liquid among the rods by upright placement. d) Cool down the ﬁlled wax liquid, and it becomes solid and contracted, thus showing up the rod heads for selective removal of the oxide layer by wet etch. 055307-4 Zeng et al. AIP Advances 7, 055307 (2017) AIP Advances 7, 055307 (2017) was used to inhibit silicon nucleation on the SiO2 mask layer, i.e., to enhance the selective growth on the seeds.21 After the seeds grew up and merged into a continuous ﬁlm, the HCl gas was shut off, and the growth of ﬁlm went on till the desired thickness was obtained. The typical growth duration was 20min. III. RESULTS AND DISCUSSION Figure 3 shows the optical-microscopic morphologic images of the substrates when the space between the rods was ﬁlled by wax solid. The duration of inclined placement when the wax was still liquid is different, 30min, 1hour, 5 days, and 20 days, respectively for Figure 3(a) to Figure 3 (d). Obviously, in all the four cases, when the wax liquid was cooled, it didn’t bring a ﬂat solid surface between the rods, and there were also some random pits in the ﬁlled surface, but there was always solid wax sticky around the rods; and the rods shot out of the solid wax, meanwhile, the amount of solid wax held between the rods decreased with the duration of inclined placement. Additionally, the ﬁlled amount of wax didn’t show much difference when the inclined placement duration was 30min and 1hour, same when 5 days and 20 days. It is not hard to understand that because of the blocking effect of the rods, viscous wax liquid will run very slowly across the rods, thus the difference was hardly discernible between Figure 3 (a) and Figure 3 (b); at the same time, because of the adhesiveness of the wax liquid, no more wax liquid can run away from around the rods when the duration of inclined placement is more than days, so the ﬁlling appeared fairly same in Figure 3 (c) and Figure 3 (d). As for the unevenness and pits of the ﬁlled wax solid, we thought the reason was that the used wax was an alkane mixture with short carbon chains, these alkane molecules didn’t tightly tangle and bind FIG. 3. Optical-microscopic images of the substrates with the space between the rods ﬁlled by wax solid with different duration of inclined placement when the wax was still liquid. (a) 30min; (b) 1 hour; (c) 5 days; (d) 20 days. FIG. 3. Optical-microscopic images of the substrates with the space between the rods ﬁlled by wax solid with differe duration of inclined placement when the wax was still liquid. (a) 30min; (b) 1 hour; (c) 5 days; (d) 20 days. 055307-5 Zeng et al. AIP Advances 7, 055307 (2017) AIP Advances 7, 055307 (2017) AIP Advances 7, 055307 (2017) each other like polymer molecules, and they preferred to agglomerate around the rods when the wax liquid cooled down, then the pits turned up if some spots without enough liquid. III. RESULTS AND DISCUSSION And we also found that the bulky ﬁlm of wax on a glass slide showed a rugged surface. The obtained protruded seeds are displayed in Figure 4. Across the whole 4′′ wafer substrate, the seeds are patterned with a uniform size exhibiting the excellence of wax as the ﬁlled mask for fabrication of wafer-sized protruded seeds. In particularly, the exposed silicon core has same length for each rod, this indicates that the oxide layer was just selectively removed on the rod head and controlled by its part out of the wax solid. When KOH solution was used to etch the exposed silicon head, it was found inverted pyramidal pits on each rod head, indicating the oxide layer was completely removed on the rod head (shown in Figure 4f). Evidently, although the ﬁlled wax solid didn’t show a ﬂat surface and was distributed many random pits, these phenomena didn’t prevent the wax from functioning as a good selective wet-etch mask for the rods. In consideration of the hydrophobic properties of solid wax surface, we thought that when the etchant solution ran past and covered the substrate, the air in the pits was trapped and also worked as a mask for the substrate below it; and the etching just happened to the outer part of the rod on the wax solid; moreover, the length of the outer part of the rod was inherently depended on the viscosity of the wax liquid, this was same to each rod on a single substrate, therefore, the length of exposed silicon core for each rod was same. According to our experiments, there are some virtues for wax used as a ﬁlled mask: i) Owing to the ﬂuidity of molten wax liquid, its ﬁlling is uniform no matter what size of the substrate is. ii) Due to the ﬂuidity, the surplus wax liquid is easy to dislodge by adequately inclined placement, keeping the ﬁlling right happen in the empties between the rods, and the uniformity of the liquid ﬁlling is recoverable by horizontal placement of the substrate. iii) The solid of wax is soft at room temperature, and the softness will relieve the stress of phase transition from liquid to solid, thus keep the wax sticky to the rods when the wax liquid becomes solid, and make the ﬁlled wax be a good etch resist for the rods. III. RESULTS AND DISCUSSION AIP Advances 7, 055307 (2017) AIP Advances 7, 055307 (2017) FIG. 6. Images revealing the growth of silicon ﬁlms using the prepared patterned protruded seeds. (a) Top view of the growing seeds. (b) Side view of the growing seeds. (c) Top view of the just immerged neighboring seeds. (d) Cross sectional view of the grown continuous ﬁlm with the substrate. (e) Magniﬁed cross sectional view of the grown ﬁlm with the substrate. (f) Top view of the grown ﬁlm. FIG. 6. Images revealing the growth of silicon ﬁlms using the prepared patterned protruded seeds. (a) Top view of the growing seeds. (b) Side view of the growing seeds. (c) Top view of the just immerged neighboring seeds. (d) Cross sectional view of the grown continuous ﬁlm with the substrate. (e) Magniﬁed cross sectional view of the grown ﬁlm with the substrate. (f) Top view of the grown ﬁlm. had the same periodicity as the original rod pattern. At the same time, no particles were observed on the surfaces of rods and the mother substrate (Figure 6b and Figure 6e), indicating that there was no growth started on the oxide mask layer. Since the nucleation and growth rate is much faster on silicon surface than on SiO2 surface under SiHCl3+HCl+H2 reactants system, if nuclei existed on the oxide layer, they would grow into big observable particles dotted the rods and substrate.22 On the other hand, the cleanness of the rods and substrate also manifested that the trapping effect of the rod jungle for reactant species didn’t signiﬁcantly improve the hetero nucleation on the oxide layer. The original empties between the rods was preserved intact, leaving channels for further investigation in future on the lift-off of the grown ﬁlms, reuse of the seeds, and surface engineering on both sides of the grown ﬁlms when still on the mother substrate. The observed preferred growth of silicon on the protruded seeds, the obtained voidless ﬁlm from discretely distributed seeds, and the as-formed structured hollows between rods veriﬁed our prototype with the potential to fabricate of transferrable ﬁlms/wafers by reusable substrates. The crystal quality of the grown ﬁlms have been characterized by X-ray diffraction (XRD) spectra, as shown in Figure 7, indicating that the grown ﬁlm from the patterned protruded seeds FIG. 7. III. RESULTS AND DISCUSSION iv) There is no course of solvent evaporating and solution condens- ing, which keeps the ﬁlled solid tightly seamed with the rods (when using the photo-resist as the ﬁlling matter, there were gaps between the rod and ﬁlled photo-resist, shown in the supplementary documents). Figure 5 presents the manipulation of the length of the seeds by wet etch time. In order to completely remove the oxide layer on the outer part of the rod on the wax solid, the wet-etching was often performed for 4min (Figure 5a). As the etching time was extended, the etching would go down along the rods through the wax solid, then showing longer seeds (Figure 5b and 5c). The controllability of seed length further proved the superior masking effect of the ﬁlled wax. FIG. 4. Images of the protruded seeds. (a) Photo-picture of a 4′′ Cz-Si wafer all covered with protruded seeds. (b) Scanning electronic microscopy (SEM) image of the patterned protruded seeds in a large area. (c) Zoomed-in SEM image on several seeds. (d) Magniﬁed SEM image on a single seed. (e) Magniﬁed SEM image on a broken seed. (f) SEM image of seeds etched by KOH solution. FIG. 4. Images of the protruded seeds. (a) Photo-picture of a 4′′ Cz-Si wafer all covered with protruded seeds. (b) Scanning electronic microscopy (SEM) image of the patterned protruded seeds in a large area. (c) Zoomed-in SEM image on several seeds. (d) Magniﬁed SEM image on a single seed. (e) Magniﬁed SEM image on a broken seed. (f) SEM image of seeds etched by KOH solution. 055307-6 Zeng et al. AIP Advances 7, 055307 (2017) 5. SEM images of the protruded seeds with controlled length by different wet-etching time in BOE solution. (a) 4min. min. (c) 20min. FIG. 5. SEM images of the protruded seeds with controlled length by different wet-etching time in BOE solution. (a) 4mi (b) 8min. (c) 20min. The selectively epitaxial growth of silicon ﬁlm was shown in Figure 6. At the ﬁrst stage, the seeds were grown into polyhedral crystal particles, showing the nature of crystallographic anisotropic growth. As the growth evolved, the seeds grew larger and the neighboring seeds were merged (Figure 6c), and eventually became an integral ﬁlm without any apertures (Figure 6d, Figure 6e, and Figure 6f), but there were square-like lightly texturized surface structures on the grown ﬁlm which 055307-7 Zeng et al. SUPPLEMENTARY MATERIAL See supplementary material for experiments about acetone soaking to get rid of wax thin laye on the rod head, photo-resist used as a ﬁlling matter between rods by spin-coating. IV. CONCLUSION In summary, we demonstrated the prototype using discrete protruded seeds to grow voidless and continuous silicon ﬁlm, and the method using wax as the ﬁlled mask to selectively remove the oxide layer on the heads of the patterned rods has been developed for fabrication of protruded seeds. The method of fabricating ﬁlled mask for patterned rod array using liquid wax is greatly applicable to wafer-sized preparation of protruded seeds now that the ﬂuidity of the wax liquid can facilitate uniformly ﬁlling all over any sized area. Moreover, the length of seeds along the rods was controllable by wet-etching time. The selective epitaxial growth of silicon ﬁlms was performed on the ready patterned protruded seeds, and voidless ﬁlms were obtained while the original empties between the rods were intact. It has to be pointed out that the primary target of our prototype is to build uniformly structured hollows between the grown ﬁlm and the mother substrate to realize the functions such as transferability of the grown ﬁlm, accessibility of gases or solutions to the below surface of the ﬁlm when it still staying on the mother substrate, and reusability of the seeds. According to the experiment results, the structured hollows were read- ily obtained by our prototype. Although much effort should be further paid to the investigation on lifting off the grown ﬁlm and reuse of the seeds, and even the prototype should be further upgraded, the principle from our prototype did offer a potential solution to kerﬂess wafers and ﬂexible crystals. III. RESULTS AND DISCUSSION XRD spectra of the grown ﬁlm from the patterned protruded seeds, of the epitaxial ﬁlm directly on the Si (100) substrate, and of the Si (100) substrate. FIG. 7. XRD spectra of the grown ﬁlm from the patterned protruded seeds, of the epitaxial ﬁlm directly on the Si (10 substrate, and of the Si (100) substrate. 055307-8 Zeng et al. AIP Advances 7, 055307 (2017) AIP Advances 7, 055307 (2017) has the almost same crystalline quality as the epitaxial one directly on the Si(100) substrate and the substrate itself. This conﬁrmed the fact that complete and continuous ﬁlm with the same crystal quality as the mother substrate could be grown from the discretely distributed protruded seeds. ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China under contract No 11374313 and 11504392. 1 H. Wu, Precision Eng. 43, 1 (2016). 2 , g , ( ) 2 M. Hecini, N. Drouiche, and O. Bouchelaghem, J. Cryst. Growth 453, 143 (2016). 2 M. Hecini, N. Drouiche, and O. Bouchelaghem, J. Cryst. Growth 453, 143 (2016). 3 3 H. J. Moller, Adv. Eng. Mater. 6(7), 501 (2004). 4 4 A. Goodrich, P. Hacke, Q. Wang, B. Sopori, R. Margolis, T. L. James, and M. Woodhouse, Solar Energ. Mater. & Solar Cells 114, 110 (2013). 4 A. Goodrich, P. Hacke, Q. Wang, B. Sopori, R. Margolis, T. L. James, and M. Woodhouse, Solar Energ. Mater. & Sol Cells 114, 110 (2013). 5 X. G. Yu, P. Wang, X. Q. Li, and D. R. Yang, Solar Energ. Mater. & Solar Cells 98, 337 (2012). 6 5 X. G. Yu, P. Wang, X. Q. Li, and D. R. Yang, Solar Energ. Mater. & Solar Cells 98, 337 (2012). 6 J. H. Peterman, D. Zielke, J. Schmidt, F. Haasw, E. G. Rojas, and R. Brendel, Prog. Photovolt.: Res. Appl. 20, 1 (2012). 6 J. H. Peterman, D. Zielke, J. Schmidt, F. Haasw, E. G. Rojas, and R. Brendel, Prog. Photovolt.: Re 7 7 C. Berge, M. Zhu, W. Brendle, M. B. Schubert, and J. H. Werner, Solar Energ. Mater. & Solar Cells 90, 3102 (2006). 8 D. M. Powell, V. P. Markevich, J. Hofstetter, M. A. Jensen, A. E. Morishige, S. Castellanos, B. Lai, A. R. Peaker, and T. Buonassisi, J. Appl. Phys. 119, 065101 (2016). 9 M. M. Moslehi, P. Kapur, J. Kramer, V. Rana, S. Seutter, A. Deshpande, T. Stalcup, S. Kommera, J. Ashjaee, A. Calcaterra, D. Grupp, D. Dutton, and R. Brown, “World record 20.6% efﬁciency 156mm×156mm full- square solar cells using low-cost kerﬂess ultrathin epitaxial silicon and porous silicon lift-off technology for industry leading high-performance smart PV modules,” PV Asia Paciﬁc Conference (APVIA/PVAP), 24 October, 2012. 9 M. M. Moslehi, P. Kapur, J. Kramer, V. Rana, S. Seutter, A. Deshpande, T. Stalcup, S. Kommera, J. Ashjaee, A. Calcaterra, D. Grupp, D. Dutton, and R. Brown, “World record 20.6% efﬁciency 156mm×156mm full- square solar cells using low-cost kerﬂess ultrathin epitaxial silicon and porous silicon lift-off technology for industry leading high-performance smart PV modules,” PV Asia Paciﬁc Conference (APVIA/PVAP), 24 October, 2012. 10 11 E. Kobayashi, Y. Watabe, R. 19 V. Steckenreiter, J. Hensen, A. Knorr, S. Kajari-Schroder, and R. Brendel, IEEE J. Photovolt. 6(3), 783 (2016). g g ( 18 D. Y. Khang, H. Q. Jiang, Y. Huang, and J. A. Rogers, Science 311, 208 (2006). 20 A. Jakokafarassar, K. Wan Nieuwenhuysen, I. Sharlandziev, V. Depauw, H. S. Radhakrishnan, T. Bearda, M. Debucquoy, I. Gordon, J. Szlufcik, Y. Abdulraheem, J. Poormans, and L. Magagnin, “Multiple reuse of the silicon substrate in a porous silicon based layer transfer process,” 32nd European Photovoltaic Solar Energy Conference and Exhibition. Proceedings, 2016, 313. ACKNOWLEDGMENTS Y. Hao, and T. S. Ravi, Appl. Phys. Lett. 106, 223504 (2015). 12 yashi, Y. Watabe, R. Y. Hao, and T. S. Ravi, Appl. Phys. Lett. 1 1 E. Kobayashi, Y. Watabe, R. Y. Hao, and T. S. Ravi, Appl. Ph 12 T. Yonehara, “ELTRAN®(SOI-Epi waferTM) technology,” Chapter 4, pp 53, INSPEC, IEE, (2002). 13 13 http://www.ims-chips.de/home.php?id=a3b8clen&adm=. 14 http://besang.com/. 15 http://www.monolithic3d.com. 16 R. J. Knuesel and H. O. Jacobs, Adv. Mater. 23, 2727 (2011). 17 18 D. Y. Khang, H. Q. Jiang, Y. Huang, and J. A. Rogers, Science 311, 208 (2006). 19 21 D. F. Liu, W. Zhang, X. Y. Chen, H. Yang, C. Wang, and L. F. Lu, U.S. patent 9,502,240 B2 (22 November, 2016); Chin Patent ZL 2013 1 0167373.3 (28 December, 2016). 22 22 L. Jastrzebski, J. Crystal Growth 63, 493 (1983). 055307-9 Zeng et al. AIP Advances 7, 055307 (2017) AIP Advances 7, 055307 (2017) 22 L. Jastrzebski, J. Crystal Growth 63, 493 (1983).
https://openalex.org/W2143372889	https://zenodo.org/records/2472500/files/article.pdf	English	null	Note on the falls of the Tsang-po	Scottish geographical magazine	1,914	public-domain	1,839	Scottish Geographical Magazine To cite this article: Captain F.M. Bailey (1914) Note on the falls of the Tsang-po, Scottish Geographical Magazine, 30:2, 90-92, DOI: 10.1080/14702541408555159 o cite this article: Captain F.M. Bailey (1914) Note on the falls of the Tsang-po Geographical Magazine, 30:2, 90-92, DOI: 10.1080/14702541408555159 To link to this article: http://dx.doi.org/10.1080/14702541408555159 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. 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Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions 90 SCOTTISH GEOGRAPHICAL MAGAZINE. days they met only some strips of ice, the southern points of which they rounded. On September 5 Bennett Island was reached, when on the 1'aimir the coil of the condenser broke, and the water in the boilers became salt. The wind blew heavily from the south, and obliged the vessels to stop on the north shores of Bennett Island. A party was landed and took Baron Toll's collections, and a cross was erected on Cape Emmelina to the memory of Baron Toll and his companions. y p On September 19 the wind became lighter, and the expedition surveyed the island and started to the south. At first it was proposed to take the great circle route to Wrangell Island, but the heavy ice met with on the way obliged the vessels to go by zigzags about three hundred miles along the meridian of 160 ° . Sometimes the ships met new ice, and it was very difficult to choose the route during the darkness of the nights. g On September 12 the expedition came to open water and im- mediately trawled for zoological purposes. 1 The accompanying sketch-map llas been constructed from the best available sources to show the supposed position of the falls~ prior to Captain Bailey's journey. It has not been found possible to include all the places mentioned in the text~ and the map does not cover tile whole of the area visite,l.--ED, ,% G. M. PLEASE SCROLL DOWN FOR ARTICLE y g p p The next day the expedition met ice up to the meridian of Tchaunski Bay, but the water was open from 70 ° N. in 180 ° longitude. On September 16 the ships reached Kolutehinski Bay and surveyed it. The ships started on September 20, but a heavy gale compelled them to anchor ; the route was then continued to Cape Dejnef ; on the 22nd it was passed, and forty miles from the Bay of Providence a heavy storm caught the ships, which rolled about 55 ° , and the speed against the wind was not more than half a knot. In the night the tiller-rope broke on the Taimir, and only on the next evening the ships were able to approach the shores of the island of St. Lawrence, where they stopped. pp y pp On September 26 the expedition was at St. Michael's Fort, and on October 14 at Petropavlovsk in Kamtchatka, from which point the above news was sent by wireless. By Captain F. M. BAILEY, WE followed the Tsang-po valley from Rinchenpung up to the village of Lagong, but no altitude in the river-bed was obtained higher up-stream than the confluence of the Chimdro Chu with the Tsang-po, a point some forty miles by road below Lagong; the altitude of the river by hypsometer at this point was 3070 feet. At Lagong we left the Tsang- po, and, crossing a pass, the Su La, entered the valley of the Nagong Chu, here called the Po Chu. This river was followed from Showa down to Trulung (Kinthup's "Poh-toi-]ung "). The altitude of the river at NOTE ON THE FALLS OF THE TSANG-PO. 91 Trulung was 6600 f~c~ by hypsumctcr. We wished to followthis river down to its junction with the Tsang-po at Gompo No, but were prevented, as the rope bridge had been carried away; the height of the Tsang-po at Gompc Ne must be about 6000 feet, or some 600 feet below Trulung. From Trulung we followed Kinthup's road as far as Kongbu Lunang, from which place we took a more direct route to Phe (Phea) than that followed by Kinthup. We then went down to Gyala, opposite which is a waterfall on a small tributary, in which a god, Sinji Chogye, is carved on the rock behind the cascade. The god is only visible in SKETCH MAP SHOWING WHERE FALLS WERE SUPPOSED TO EXIST Enjtll~h Mile# D " " ~ 6 ,'5 ,,~ non# 2 Ton~-juk-Jonj~ co Trul~n~g Calls were t to ~xlst one # / / 8inchenpunj? fl t / / ~,,~ /f I I rll winter when the stream is small. From Gyala we went down to Pemako Chung, a small lamasery which was visited by Kinthup. Near this are the falls which he described. The river nearly the whole way from Gyala to this point is a foaming rapid, though in one or two places it flows quietly. At Kinthup's falls the rapid develops into a fall of about 30 feet; here rainbows were seen. We succeeded in pushing about twelve miles below Pemako Chung. Our lowest hypsometer observation, taken about two miles above the lowest point mapped, gave an altitude of 7200 feet. The gap in the river which is unmapped we estimate at forty-five miles, i.e. from the lowest point reached below Pemako Chung to Lagong. By Captain F. M. BAILEY, The gap between the two boiling point observations (below Pemako Chung, and at the confluence of the Chimdro Chu and the SKETCH MAP SHOWING WHERE FALLS WERE SUPPOSED TO EXIST Enjtll~h Mile# D " " ~ 6 ,'5 ,,~ non# 2 Ton~-juk-Jonj~ co Trul~n~g Calls were t to ~xlst one # / / 8inchenpunj? fl t / / ~,,~ /f I I rll SKETCH MAP SHOWING WHERE FALLS WERE SUPPOSED TO EXIST Enjtll~h Mile# D " " ~ 6 ,'5 ,,~ non# 2 Ton~-juk-Jonj~ co Trul~n~g Calls were t to ~xlst one # / / 8inchenpunj? fl t / / ~,,~ /f I I rll winter when the stream is small. From Gyala we went down to Pemako Chung, a small lamasery which was visited by Kinthup. Near this are the falls which he described. The river nearly the whole way from Gyala to this point is a foaming rapid, though in one or two places it flows quietly. At Kinthup's falls the rapid develops into a fall of about 30 feet; here rainbows were seen. We succeeded in pushing about twelve miles below Pemako Chung. Our lowest hypsometer observation, taken about two miles above the lowest point mapped, gave an altitude of 7200 feet. The gap in the river which is unmapped we estimate at forty-five miles, i.e. from the lowest point reached below Pemako Chung to Lagong. The gap between the two boiling point observations (below Pemako Chung, and at the confluence of the Chimdro Chu and the SCOTTISH GEOGRAPHICAL MAGAZINE. 9~ Tsang-po) is, however, about ninety miles. In the forty-five miles that are unmapped we have the estimated height of 6000 feet of the Pc Chu- Tsang-po confluence at Gompo Ne, which cannot be much in excess. The road down from Gompo Ne to Lagong is used a great deal in winter, as when the Su La is closed by snow, it is the only road from the Po Chu valley to the lower Tsang-po valley. We met a great many people who had seen this part of the river, all of whom agreed that there was nothing in the way of falls on it, though at the confluence of the rivers at Gompo Ne there are remarkable rapids and whirlpools. By Captain F. M. BAILEY, As regards the portion between Pemako Chung and Gompo Ne it was more difficult to collect information, as there is no road, but the distance can only be about fifteen miles, and we met people who had hunted in the jungles in the neighbourhood who all said that there was no big waterfall on that section of fhe river, though the rapids must be extraordinarily steep. ownloaded by [University of Cambridge] at 09:50 03 January 2015 g p The heights mentioned are liable to revision. A NEW ANTARCTIC EXPEDITION. (]l~th McqQ IT was amlounced in the daily press at the end of December last that Sir Ernest Shackleton, who has been desirous for some time of returning to the Polar Regions, has been able, through the generosity of a friend, to arrange an expedition with the object of crossing the Antarctic Continent from the Weddell Sea to the Ross Sea. The plans of the expedition, as detailed below, should be compared with those already drawn up by Dr. W. S. Bruce (cf. this Ma(/a':'i~e, xxiv. p. 200, and xxvi. p. 192), and the accompanying map, reproduced by perinission from the Tiv~es, with that in vol. xxvi. p. 193, which shows Dr. Bruce's proposed route. It is understood that the estimated minimum cost of the expedition, ~50,000, has been already provided for, but £60,000 or £70,000 would be required to equip it with full efficiency. The Royal Geographical Society of London has subscribed £1000 to the funds. Sir Ernest Shackleton has supplied us with the following official programme of the expedition :-- "THE hIPERIAL TRANs-ANTARCTIC Ex~,~Drrlo~,. " O/,]cc[~" of the E.,2Jeditiot~.--To cross the South Polar Continent from sea to sea--from the Weddell Sea to the Ross Sea. The crossing of the South Polar Continent will be the biggest Polar journey ever yet attempted. It is a longer journey than to the Pole and back, and the first 800 miles will be on quite a new route, and with good fortune it is probable that the whole journey may be on entirely new ground. The minimum distance from sea to sea is about 1500 geographical miles--that is roughly 1700 statute miles. "From a geographical point of view the complete continental nature c~ o~ r~ r~ q~ o~ rJ2 c~ o~ r~ r~ q~ o~ rJ2 Downloaded by [University of Cambridge] at 09:50 03 January 2015 c~ o~ r~ r~ q~ o~ rJ2
https://openalex.org/W2234292234	https://nottingham-repository.worktribe.com/file/772539/1/Atkins%20S%20et%20al%20Simulated%20consultations%20-%20a%20sociolinguistic%20perspective.pdf	English	null	Simulated consultations: a sociolinguistic perspective	BMC medical education	2,016	cc-by	9,437	* Correspondence: Sarah.Atkins@nottingham.ac.uk 1Centre for Research in Applied Linguistics, Trent Building, University of Nottingham, Nottingham NG7 2RD, UK Full list of author information is available at the end of the article Sarah Atkins1, Celia Roberts2, Kamila Hawthorne3 and Trisha Greenhalgh4 Sarah Atkins1, Celia Roberts2, Kamila Hawthorne3 and Trisha Greenhalgh4 © 2016 Atkins et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Atkins et al. BMC Medical Education (2016) 16:16 DOI 10.1186/s12909-016-0535-2 Atkins et al. BMC Medical Education (2016) 16:16 DOI 10.1186/s12909-016-0535-2 Open Access Abstract Background: Assessment of consulting skills using simulated patients is widespread in medical education. Most research into such assessment is sited in a statistical paradigm that focuses on psychometric properties or replicability of such tests. Equally important, but less researched, is the question of how far consultations with simulated patients reflect real clinical encounters – for which sociolinguistics, defined as the study of language in its socio-cultural context, provides a helpful analytic lens. Discussion: In this debate article, we draw on a detailed empirical study of assessed role-plays, involving sociolinguistic analysis of talk in OSCE interactions. We consider critically the evidence for the simulated consultation (a) as a proxy for the real; (b) as performance; (c) as a context for assessing talk; and (d) as potentially disadvantaging candidates trained overseas. Talk is always a performance in context, especially in professional situations (such as the consultation) and institutional ones (the assessment of professional skills and competence). Candidates who can handle the social and linguistic complexities of the artificial context of assessed role-plays score highly – yet what is being assessed is not real professional communication, but the ability to voice a credible appearance of such communication. Summary: Fidelity may not be the primary objective of simulation for medical training, where it enables the practising of skills. However the linguistic problems and differences that arise from interacting in artificial settings are of considerable importance in assessment, where we must be sure that the exam construct adequately embodies the skills expected for real-life practice. The reproducibility of assessed simulations should not be confused with their validity. Sociolinguistic analysis of simulations in various professional contexts has identified evidence for the gap between real interactions and assessed role-plays. The contextual conditions of the simulated consultation both expect and reward a particular interactional style. Whilst simulation undoubtedly has a place in formative learning for professional communication, the simulated consultation may distort assessment of professional communication These sociolinguistic findings contribute to the on-going critique of simulations in high-stakes assessments and indicate that further research, which steps outside psychometric approaches, is necessary. Keywords: Simulated consultations, OSCE, Communication skills, Interpersonal skills, Assessment, Diversity, Sociolinguistics © 2016 Atkins et al. * Correspondence: Sarah.Atkins@nottingham.ac.uk 1Centre for Research in Applied Linguistics, Trent Building, University of Nottingham, Nottingham NG7 2RD, UK Full list of author information is available at the end of the article © 2016 Atkins et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background However, given that such exams can demonstrate significant differences in pass rates between demographic groups, such as, the Membership of the Royal College of Physicians ‘Practical Assessment of Clinical Examination Skills’ (PACES) exams and Membership of the Royal College of General Practitioners’ Clinical Skills Assessment (CSA) [9–11], the construct validity of simula- tions is an important research question. Recreating a lin- guistically authentic medical interaction may not be the primary objective of simulation when used for medical training, where it enables practice and focussing in on par- ticular skills, particularly technical skills. Recent research evidence has also shown the important uses of simulation in communication skills training for medical teams, not least because of the facility it provides practitioners to rec- ord and reflect on how an interaction has unfolded [12]. However, the linguistic differences and difficulties of simu- lation are of considerable importance in assessment. In many high-stakes summative exams, the simulated consult- ation is not used to enable the medical practitioner to re- flect on and develop their own communication skills, but rather for an external party to measure a candidate’s com- petence and assign a grade. When using the simulated con- sultation for assessment, particularly where interpersonal and communication skills are being marked, we must be sure that the exam construct and the linguistic require- ments placed on candidates adequately embody the skills expected for real-life practice. Sociolinguistic analysis can unpick the subtly different interactional discrepancies that a simulation produces. It looks at direct evidence from the interactions pro- duced in situ, rather than relying on asking participants to reflect back on an interaction. Sociolinguistics often employs techniques of ‘discourse analysis’ to identify important features and fine-grained characteristics of talk, such as grammatical structure, turn-taking be- tween speakers, intonation and the integration of non- verbal communication [22]. These can systematically evidence the characteristic linguistic features that occur in a particular setting - and how the talk is created by, as well as constitutive of, the social relationships in that context. Sociolinguistics is therefore interested in the choices that speakers make when they use language and what those choices and variations might mean for the evaluation of speakers (page 16) [23]. The authenticity of the interaction is particularly per- tinent when assessing communication skills. The clinical consultation is not a technical procedure, but an emo- tionally charged interpersonal interaction of high social significance and linguistic complexity [13]. Background language and society inter-relate. It looks at how people use language in their everyday lives and how language- in-context creates the complex social world. The tools of sociolinguistic research are real recordings of spoken language, to examine evidence of how different contexts and social backgrounds affect the talk we produce and how it is evaluated by others. This focus on evidence from actual interactions is an important one here. Prior research on simulated consultations has largely addressed psychometric properties of particular tests and scenarios such as internal consistency (e.g. using Cronbach’s alpha), generalisability, inter-rater reliability, predictability (e.g. of subsequent examination success), discriminatory power (ability to distinguish consistently between ‘good’ and ‘poor’ examinees), as well as protocols and procedures for quality control [3, 14–18]. A crucial question remains as to how far such scenarios reflect real consulting abilities. Both simulated patients and those being assessed, when asked after the event, tend to rate their experience as ‘real- istic’ [19, 20]. Various studies have also found that un- announced simulated patients, trained to present with a particular scenario in a kind of ‘mystery shopper’ ap- proach, went undetected by medical practitioners [6, 21]. Yet there remain crucial differences between real and sim- ulated consultations, particularly as they are used in as- sessment, that cannot easily be evidenced by the mystery shopper method or participants’ retrospective accounts. This paper addresses issues arising from the use of simu- lated patients in assessments of clinical consulting, in particular the linguistic difficulties of interacting in such settings and how far they reflect a practitioner’s real con- sulting abilities. Simulated patients are lay people or pro- fessional actors trained to portray a patient with a particular condition in a standardised way. As well as their use in practice and training for medical practi- tioners, they play an important role in formal assess- ment, such as the objective structured clinical examination (OSCE) for undergraduates [1, 2] and licen- sure examinations for postgraduates [3–5]. One advan- tage of this assessment format is that it helps ensure everyone has a standardised, equitable and repeatable experience [6–8]. Abstract Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Atkins et al. BMC Medical Education (2016) 16:16 Page 2 of 9 Atkins et al. BMC Medical Education (2016) 16:16 Page 2 of 9 Background When con- sidering the appropriateness of simulation in assessing this complexity, we need a different kind of research - sited in a humanistic rather than psychometric para- digm. Sociolinguistic research provides a useful means of interrogating and debating these issues. Sociolinguis- tics is a field which systematically studies the way Integrated with this theoretical approach in sociolinguis- tics, is a fundamental interest in how language produces power relations in society. Professional discourses in par- ticular, which largely consist of goal-oriented encounters, often demonstrate a degree of power-imbalance [24]. We can evidence these power relationships in the ‘microphys- ics’ and fine-grained detail of our everyday practices [25], including our professional talk. There are asymmetrical relationships in terms of who is expected to speak at cer- tain points, who should show politeness and which Atkins et al. BMC Medical Education (2016) 16:16 Page 3 of 9 Page 3 of 9 speakers are meant to demonstrate their domain-specific, professional knowledge [26]. Power relations in the setting of an exam role-play differ from real-life clinical encoun- ter, since the role-player has a very different position to that of a patient and there is also a more powerful third participant in the examiner, observing the interaction. Given the different relationships of participants, inter- actional differences in simulated consultations compared to real-life are perhaps to be expected. The environment in assessed simulations is (intentionally) decontextualized and scenarios involving invasive physical examinations [5] and a range of patient groups such as those with multi- morbidity, limited English or a very different communica- tive style [27] are often (though not invariably) excluded. Yet candidates are asked to behave as if these scenarios were real [28, 29]. degree of standardisation of any scenario (hence, its replic- ability) and its reflection of the real (its authenticity), since 100 % standardisation would require the simulated patient to reproduce a script robotically. In reality, while the sim- ulated patient plays a character and helps depict a context- ual hinterland in answer to candidates’ questions, he or she must draw on their own interactional resources to manage the interaction itself [28]. To understand how simulations are experienced differ- ently from real consultations, we must ask, “what ‘main- tenance work’ needs to be done by both parties to maintain the semblance of reality?”. Background To do this, we draw on the work of sociologist Erving Goffman, whose sem- inal essay ‘Frame Analysis’ addressed the question, “under what circumstances do we think things are real?” [39]. Goffman argued that the sense of feeling an activity is real depends upon our sense of self as we relate to others. Each interaction creates and reinforces a shared reality to keep the relationship going [40]. We attend to others, become involved in the to and fro of talk, how- ever momentarily, since we have what Goffman calls a moral requirement to display ourselves in ways that others expect of us. This sociolinguistic approach to language and professional communication was recently used in a 3-year study of the Royal College of General Practitioners’ Clinical Skills Assessment (CSA) [27, 30], which we draw upon in this paper. As well as this we draw on analytic work around final-year undergraduate medical OSCEs by Roberts et al. [31]. De La Croix and Skelton on under- graduate OSCEs [32–34], Seale et al.’s linguistic study of OSCE examinations [28], Niements on the use of role- play in training [35], and O’Grady and Candlin on the Royal Australian College of General Practitioners' licens- ing exam [36]. Some linguistic evidence on the use of simulation for assessing professional communication out- side the medical field is also drawn upon, particularly Sto- koe’s [37, 38] account of the linguistic patterns in simulated police interviews. Evaluating this evidence col- lectively helps us flesh out this debate paper with a fuller picture of the complexities of using simulation. Although simulated consultations and OSCE exams do vary in their setup, some of the essential commonalities, like the semi- scripted standardised part of role-players, the timed cases and marking descriptors, render our sociolinguistic dis- cussion relevant to this wide genre of assessment. The paper addresses the following four themes, identified from the literature review and our own research, in considering the simulated consultation: (a) as a proxy for the real; (b) as performance; (c) as a context for assessing talk and (d) as potentially disadvantaging candidates trained overseas. p His concept of ‘frame’ describes this socially defined reality. In any given stage of an encounter, speakers and listeners establish or negotiate what is going on: we are in the frame of a passing conversation, a preliminary chat about the weather before the consultation proper begins, an examination and so on. Background The frame constitutes what is happening and also works as a filtering process through which general principles of conduct apply. For example, when a doctor tells a patient that chances of recovery are high, both sides can understand that they are in a ‘reassurance’ frame within this shared moment of reality. Different frames can be invoked, and indeed evidenced, through changes in linguistic behaviour by the participants. For example, in a case from our CSA research [27], a simulated patient presents with menor- rhagia. The candidate indicates that he wants to do a "quick abdominal examination", in the frame of a rou- tine element of the diagnosis. But when he responds to the simulated patient’s query by saying “we look for any abnormal growth”, the simulated patient becomes alarmed. The candidate then shifts the frame from infor- mation giving to one of reassurance and self-correction. Discussion At any time in an encounter, Goffman argues (page 156–200) [39], we can experience multiple frames. For example, in an OSCE-style exam, the frame of showing empathy to a role-playing patient is nested in a frame of displaying competence to an examiner, which in turn is nested in the institutional frame of the overall assessment process. For this reason, the values associated with em- pathy are not seriously committed to or felt as real be- cause they are anchored in a more fundamental frame, The simulated consultation as a proxy for the real Social interaction cannot ultimately be standardised. While there are some relatively stable, overarching fea- tures of a consultation, such as the phases to be per- formed and the general history to be conveyed by the patient, at the minute level of turn-by-turn talk stand- ardisation becomes difficult and small differences in de- livery are inevitable. There is a tension, then, between the Atkins et al. BMC Medical Education (2016) 16:16 Page 4 of 9 Page 4 of 9 related to simulated performance in the exam. While role- player and trainee/candidate can put on a surface per- formance that is realistic, the assessor must decide whether the candidate is demonstrating ‘real caring’. This makes any simulated consultation a hybrid activity in which real qualities (subjectively experienced) are assessed through the unreal, requiring a considerable amount of interactional work to sustain the talk and illusion of a real consultation [28]. roles and identities at play in simulations and these can be evidenced in the communication. In answering the question on ‘authenticity’, Seale et al. ultimately do sug- gest that experience of participants in a role-play is fun- damentally different from that of a real-life interaction and that the candidate must do much more inter- actional ‘work’ to keep the illusion up (page 181). Of course, real consultations also require some level of performance, but to properly understand the differences we must unpack what ‘acting’ and ‘performing’ mean in these interactional situations. To do so, we can draw on Goffman’s depiction of life as drama – i.e. we present our- selves on the world as a stage, ‘performing’ in different ways to different ‘audiences’ in different settings (everyday, professional, institutional and so on) [43]. We perform all the time in the everyday, managing impressions of our- selves in what Goffman called ‘facework’ [44]. Discussion Goffman calls an activity that does not fit within the frame of the moment a ‘frame-break’. For example, candidates in simulated consultations often do not know whether they are expected to carry out a physical examination ‘for real’. They may commence a physical examination frame, only to be interrupted by the examiner either verbally or by handing them a card with key physical findings. Candidates must then rap- idly shift frame to the preliminaries of diagnosis. We found such shifts were typically marked by disfluencies and/or hesitations, even with highly successful candi- dates, as the candidate worked to maintain the simu- lated case and ignore any interaction with the examiner (page 53) [27]. To justify a simulated con- sultation as a proxy for the real obscures its limitations and complexities, many of which only become appar- ent when analysing their interactional detail. It is in this linguistic detail of simulations that we can really identify the different communicative competences that come to the fore in simulated consultations, which may not be the competences required for real-life practice. Goffman distinguished the banal and intimate per- formances of the everyday that occur ‘backstage’ from professional behaviour, which is largely ‘front-stage’ [43] – a term he used to refer to activities like the waiter at table, the doctor in the surgery or the teacher in class. Here there are constraints on behaviour in terms of manner, quality of attention and emotions, and the performance has an ‘audience’ that evaluate the competence displayed [45, 46]. Importantly, under- standing professional behaviour as a performance does not undercut its values. For example, to care for a pa- tient may involve masking frustration or fatigue in order to care better. When institutions require this professional behaviour to be monitored and assessed, however, it becomes an institutional performance. Evaluation of professional performance becomes insti- tutionalised as observers rate and record performance and implement rewards and sanctions. There is a heightened awareness of the need, on the part of the professional, to perform expressively, a “heightened mimicry” [28] and, on the part of the assessor, “a license … to regard the act of expression and the performer with special intensity” (page 11) [46]. However, it is im- portant to make the distinction between a heightened performance for institutional purposes (e.g. Discussion someone pointedly looking in the mirror when taking a driving test) and a simulated performance (someone pretending to look in the mirror). The simulated consultation as performance p One of the concerns voiced about OSCE examina- tions is that they test acting skills as much as they do professional communication [30, 41]. Niements de- scribes how role-played interactions "cannot reproduce the orientations of real interactions...[W]hat is authentic to those users when they “live” a specific situation can- not be authentic to trainers/trainees when they play it" [42] (p. 317). A number of studies have addressed the types of ‘acted’ behaviour such settings consequently produce, what de la Croix and Skelton have called "the language game of role-play" [32]. Seale et al. explore how different ‘frames’, real and fictitious, are invoked through talk in simulations [28]. They find subtle mo- ments in which attention is drawn to the fictitious na- ture of role-play, citing an example of humorous comments made about an entirely invented paediatric patient, that both the candidate and the role-player are pretending is present (page 183). In their analysis, using the fictitious nature of role-play to create humour is a means for the candidate to achieve rapport with the actor, not rapport with a ‘patient’. So there are multiple In simulation, the environment is mutually con- structed as an unreal activity. In her analysis of emotions in theatre acting, Konijin discusses the way actors must monitor how far the emotions they are acting out accord with the inner model of what the play should convey [47]. The actor’s task is not to convey sincere emotions but to play out words and actions that convince the audience of the authenticity of their character within the terms of the drama. At the same time they monitor their Page 5 of 9 Atkins et al. BMC Medical Education (2016) 16:16 simultaneous amplification and reduction is most appar- ent in the interpersonal domains of assessment, as we discuss below. own experience of acting and so experience a ‘dual con- sciousness’. In a simulation, likewise, the trainee or candi- date has to work hard to create a synthetic reality – one that convinces the audience/observer, but not one that is real to candidates in terms of consequences for patients: an institutionalised display rather than a professional in- vestment, all the while monitoring their conduct vis-à-vis the examiner. In sum, simulation is a multi-layered per- formance for both role-player and candidate requiring some of the skills of an actor. The design and timing of cases The design of cases for simulated consultations moves the focus from the how of patient care to the why of the par- ticular selected case. Both students and candidates are primed to fear the trip-wire that comes with the case: “learners sometimes think there are hidden aspects…they are being asked to discover, akin to peeling away the skins of an onion until the flesh is found” (page 67) [52]. The simulated consultation as a context for assessing talk The design of OSCE-style exams bring five other com- plexities, relating to the quality of talk to the candidate’s task, adding burdens and reducing the ‘real’. We con- sider: (i) the talk-heavy nature of the consultations; (ii) the design and timing of cases; (iii) the shift of power to the role-player; (iv) standardised scenarios but individual emotional responses and (v) who fails such assessments – and why? We establish these themes from the authors’ study of the CSA [27] and from an overview of the lin- guistic research on simulations [28, 32–37], but draw on these findings to debate the particular implications for assessment. g In a high-stakes examination, this ‘Sherlock Holmes’ factor can mar or make success [27]. It turns the candi- date into a timekeeper, dealing with concerns superfi- cially so that the putative puzzle of the case can be resolved. They may stop in mid-sentence when the whis- tle blows or pack in questions or information as the last minute ticks by. The strictly timed structure for simu- lated consultations produces very different openings and closings from that identified in real consultations. For example, in real clinical encounters, doctors raising new topics at the likely end of the encounter is rare but, con- versely, closings are often extended conversational exchanges which build the doctor-patient relationship more generally [53]. The talk-heavy nature of the consultations In simulated consultations, it is primarily talk-in-interaction that is assessed. To succeed in simulated scenarios, candi- dates must work harder or ‘over perform’, holding a higher proportion of the conversational floor (between 67–77 %) than in everyday consultations [34]. Research by Seale et al. identifies the complex, additional linguistic work required from candidates in simulations [28] and research on the Royal Australian College of General Practitioners’ licensing examination identified how role-played scenarios require a complex, hybrid discourse from the GP candidate [36]. Col- lectively, these findings suggest that simulated consultations require actions and skills to be verbalised by the candidate to a much greater degree than in everyday clinical work. Who fails such assessments – and why? We have noted the heavy focus on talk in simulated con- sultations. Communication or interpersonal skills are often explicitly assessed with their own marking criteria in medical OSCEs, but can also become implicitly judged across all other domains, since professional ac- tions like data-gathering and clinical management must also be performed through effective communication [27]. The metric of reliability also tends to reinforce the unspoken assumption that there is an implicit ‘best way’ of scoring highly in the interpersonal domain. Candidates in simulated consultations routinely produce formulaic phrases such as "Can you tell me a bit more about…" "I understand how you feel" or "I’m sorry to hear about that", with a greater frequency and often in different sequential positions, than is found in real life practice [27]. Some of these mimic the phrases recommended in communication skills textbooks and their extensive use in simulations may be inevitable in an environment in which talk is being ob- served and assessed. This is a finding corroborated by Roberts et al. [31] in a study of undergraduate medical OSCEs, where the use of elicitation phrases such as "How do you feel about that?" could be interpreted as sounding overly trained if used in the wrong location (page 8–9). In an essay on the experience of being a role-player evalu- ating candidates in US medical exams, Jamison points out that to gain marks, empathy and compassion must be ‘voiced’ and that (perhaps as a consequence) candi- dates seemed either aggressively formulaic in their in- sistence, "that must be really hard", or saturated with humility "Would you mind if I – listened to your heart?" (page 4–5) [56]. There have been similar findings on simulations in professional settings outside medicine, such as Stokoe’s research on police interview role-plays, in which communication directives from training man- uals are overtly used in the openings, in a way which they are not in real-life police interviews, potentially for the benefit of a marker [38]. Standardised scenarios but individual emotional responses The role-player’s power is made more complex by the shift from the institutional persona of the actor/patient to the instinctive resources of the private person. In other words, the role-player works with a hybrid of act- ing behaviour and their own, individual interactional re- sources. While careful training is used to standardise ‘patients’, the role-player is usually not working to a tightly scripted part. Who fails such assessments – and why? He or she is given guidance to react to the candidate in a natural way, to fulfil interactional criteria (Table 1). If the candidate’s performance is un- clear or irritating to the role-player, then the role-player can respond in accord with their inner emotions (the ir- ritation feels real, even though the setting is simulated). g g All parties in fact, must draw on their own inter- actional resources to make sense of the encounter. Even where a middle class actor acts a convincingly troubled and inarticulate teenager, they cannot gainsay their own interpretive processes (e.g. they can mumble or remain silent but they cannot not understand). Examiners not only have to judge this hybrid of simulation and instinct- ive resources, they also have to manage their own mix of instinctive reactions to how others interact, their own professional expectations and the formal categories of the examination. As one said, “A lot of the time, I am comparing them to me and what I’m used to” [27]. This mix of habits of talk, interpretation and evaluation (on the one hand) and standardised judgements (on the other) are most problematic in the domain of interper- sonal skills, where subjective interpretation is necessary to interpret what counts as ‘rapport’ or ‘sensitivity’. This It seems to be a consequence of the assessed, simulated setting then, that participants use these formulaic, trained professional phrases and interactional moves with a much higher frequency than real-life. In exams such as the CSA, high scoring candidates also produce 32 % more of these exam-modelled utterances than weaker candidates. Yet these phrases appear much less frequently in real consul- tations [27]. Interestingly, weaker CSA candidates who also produce these types of phrases, albeit slightly less fre- quently, were assessed as formulaic in examiner feedback: Table 1 Examples of instructions to role-player from the CSA: Behaviour/Demeanour/Body language ‘Case 1’ Reticent, trying to appear unconcerned. A bit resentful if the doctor appears to be telling you off. ‘Case 2’ Try to build a good rapport with doctor and don’t keep information back. You are familiar with GPs and hospitals, so you are comfortable with the doctor. Table 1 Examples of instructions to role-player from the CSA: Behaviour/Demeanour/Body language Try to build a good rapport with doctor and don’t keep information back. You are familiar with GPs and hospitals, so you are comfortable with the doctor. The shift of power to the role-player account of the conversational inauthenticities of role-play for police interviews [37], particularly the more elaborate and sometimes humorous way in which conversational ac- tions are performed in these false settings, where the stakes for participants are entirely different from those where a real defendant is being interviewed. There is lin- guistic evidence, then, for how participants must orient themselves in acted, simulated settings, monitoring their performance and conducting extra linguistic work to maintain the illusion of a real interaction. account of the conversational inauthenticities of role-play for police interviews [37], particularly the more elaborate and sometimes humorous way in which conversational ac- tions are performed in these false settings, where the stakes for participants are entirely different from those where a real defendant is being interviewed. There is lin- guistic evidence, then, for how participants must orient themselves in acted, simulated settings, monitoring their performance and conducting extra linguistic work to maintain the illusion of a real interaction. Who fails such assessments – and why? The shift of power to the role-player The shift of power to the role player Sociolinguistic research has identified how asymmetrical interactions, where one speaker has more power than another, show small-scale differences in talk. Medical consultations are necessarily asymmetrical. The move- ment in recent years towards patient-centredness and shared decision making has not fundamentally altered this, since asymmetry stems at least partly from the doctor’s knowledge [54]. But in simulated consultations, candidates must manage the fact that “the power rela- tion is inverted, because knowledge and judgment rest with the simulated patient rather than with the physician student” (page 266) [55]. De la Croix and Skelton iden- tify a higher number of interruptions from role-players across 100 third-year OSCE exams, suggesting a position of greater interactional power compared to findings on the linguistic behaviour of real-life patients [32–34]. Not only do the simulated patients know the case and how it should play out [34], but in examiner feedback sessions for research on simulations [27], examiners noted that the simulated patients positioned themselves in an actorly manner. They put demands on candidates that patients usually would not and showed familiarity with the exigencies of the case through their language [27]. Talk in simulated assessments is also relatively decon- textualized, without the shaping role of the computer [27, 48] or any of the other props and interruptions of real consultations. Decontextualised environments incur more talk [49] and, in an environment such as this, lead to talk becoming intensely focussed on. In addition, there is no continuity of care, so shared, unspoken knowledge between doctor and patient can play no part. This potentially diminishes the types of relationships and interactions that can be experienced by doctor and patient in the simulated consultation. Relationship build- ing over time and the deep values inherent in building professional capability [50, 51] are overshadowed by an externally timed case where surface skills must be made explicit (e.g. enacted or voiced) for assessment. This Evidence from outside medical education has also shown the shift in power relations between speakers when interacting in simulations. Stokoe conducts a nuanced Atkins et al. BMC Medical Education (2016) 16:16 Page 6 of 9 became particularly evident in feedback sessions with examiners, as the following section explores. Who fails such assessments – and why? It seems just very formulaic and a lot of it seems learned. ‘I understand why you would be worried’, Atkins et al. BMC Medical Education (2016) 16:16 Page 7 of 9 Page 7 of 9 Atkins et al. BMC Medical Education (2016) 16:16 As indicated above, simulations lead to more talk, more formulaic phrases and more work to ensure that such talk sounds sincere. This focus on talk and how it sounds in contexts of intense assessment puts particular pressure on those whose style of communicating is dif- ferent from the majority of examiners and also, perhaps, the patient role-players. Small differences in such subtle features as intonation, word stress and other small markers of speech can be amplified and read off as showing negative characteristics, such as formulaic re- sponses or not engaging, attracting lower marks in the interpersonal (page 32–73) [27]. Additionally, since it can be difficult to make standardised, simulated cases re- flect the same variation as real-life consulting, perform- ance will not reflect the ability to interact effectively and flexibly with a diverse patient population. In many such exams, while UK graduates are not assessed on consult- ing in linguistically challenging situations, International Medical Graduates, many of whom consult regularly in another expert language within the British multi-cultural context, have no opportunity to display this skill as they might use it in their everyday practice. Such competence in linguistically and culturally challenging situations is increasingly important for medical practitioners treating diverse patient populations, both in the UK and globally. It is perhaps the biggest challenge for assessing medical practitioners’ interpersonal competence in our modern- day context of globalised, mobile and diverse societies. ‘What kind of thought went through your mind when you made this appointment’ which kind of is an attempt to do the right thing but to me it just felt very crass… [27] ‘What kind of thought went through your mind when you made this appointment’ which kind of is an attempt to do the right thing but to me it just felt very crass… [27] Detailed analysis of stronger candidates’ talk showed that they knew how to play the game: they customised formulaic phrases so they sounded more real and sin- cere, adding in little hesitations, colloquialisms and changes in intonation (page 59–61) [27]. Summary A review of sociolinguistic approaches to simulations demonstrates that simulated assessment, even when it is ‘realistic’, shows some crucial differences to the communi- cative competences found in real-life practice. Talk is al- ways a performance in context and in simulations, the role-playing patient, the candidate and the examiner all have to work hard to maintain the illusion. Candidates who can handle the social and linguistic complexity of this somewhat artificial, standardised situation score highly – yet what is being assessed is not real communication but the ability to voice a credible appearance of such commu- nication. It follows that if communication skills are assessed purely through simulated patients, this may not reflect the real consulting abilities of candidates. We must question whether simulations replace the values–led de- velopment of medical students with ‘playing the game’ of simulation [50, 51, 59]. The ability of doctors to form en- during therapeutic relationships with patients may not be adequately reflected in the “colonisation [of medicine] by the technologies of the unreal” [60]. Who fails such assessments – and why? In such cir- cumstances, the ‘empathy telling’, already a simulation of feeling and perception, has to be further worked on to invoke a convincing suspension of disbelief: a double simulation or to extend Konijin’s concept of ‘dual con- sciousness’ [47], a ‘triple consciousness’, consisting of the candidate’s own sense of themselves as professionals, the consciousness that they must simulate a professional en- counter – and in addition within the institutional frame – must work on the formulaic phrases of the simulation so that they sound more sincere to examiners. It is in these small details of talk, here in the small variations in delivery of exam-modelled phrases, that we can see how power and social relationships are constituted in the micropractices of interaction and its evaluation [25]. In terms of construct validity, does the simulated con- sultation measure what it purports: the interpersonal capabilities expected of a doctor? The answer is a com- plex one. Though the simulation may be good at testing skills such as giving explanations and structuring the consultation, there are a number of linguistic features which do not mimic real-life practice. For example, it examines competence in using additional communica- tive resources to make exam-induced ‘voiced’ phrases sound sincere and to manage the triple consciousness required to perform to examiners. In terms of assess- ment theory, there is a "construct-irrelevant variance" [57] in which certain know-how is assessed which is not a requirement of real consultations. Received: 18 September 2015 Accepted: 6 January 2016 Received: 18 September 2015 Accepted: 6 January 2016 Received: 18 September 2015 Accepted: 6 January 2016 Acknowledgements Th h f The authors are grateful to the research funders who facilitated the work with the Royal College of General Practitioners, which is referred to in this paper. This included a Knowledge Transfer Partnership award (KTP008346, 2011–2013), from the Technology Strategy Board and the Academy of Medical Royal Colleges in the United Kingdom. SA was additionally funded by an Economic and Social Research Council ‘Future Research Leaders’ grant at the University of Nottingham (ES/K00865X/1, 2013–2016). The authors are grateful to the research funders who facilitated the work with the Royal College of General Practitioners, which is referred to in this paper. This included a Knowledge Transfer Partnership award (KTP008346, 2011–2013), from the Technology Strategy Board and the Academy of Medical Royal Colleges in the United Kingdom. SA was additionally funded by an Economic and Social Research Council ‘Future Research Leaders’ grant at the University of Nottingham (ES/K00865X/1, 2013–2016). We are also grateful to the Royal College of General Practitioners for the access and close advice they gave the authors throughout the original research, on which this debate article is built, and to all the exam candidates who gave their consent to be part of the study. We are also grateful to the Royal College of General Practitioners for the access and close advice they gave the authors throughout the original research, on which this debate article is built, and to all the exam candidates who gave their consent to be part of the study. Simulated consultations as potentially disadvantaging candidates trained overseas While all assessments may require ‘exam skills’ to some degree, when one group of candidates fares much worse than another, as occurs in many of these assessments [27], the fairness of these exam-constructed requirements needs to be carefully considered. There is wide recogni- tion that for many candidates trained outside their home country for the assessment, simulations are often a new phenomenon and that, like any type of assessment, lack of familiarity affects performance [58]. The simple solution offered is that this group need more practice with simula- tions. However, detailed sociolinguistic analysis suggests that simulations may cause difficulties for this group of candidates in other ways as well. The discipline of sociolinguistics offers an evidenced approach to these questions around professional com- munication. In this paper, we have introduced three core sociolinguistic concepts relevant to the assessment of Atkins et al. BMC Medical Education (2016) 16:16 Page 8 of 9 Page 8 of 9 Page 8 of 9 communication in medicine: that the particular variety of talk in simulated consultations separates it out from the talk in real consultations; that the notion of ‘frame’ is used to understand how we relate to and make our talk real to each other and that this reality breaks down in institutionally assessed communication; and that micro-features of talk feed constantly into our evaluation of others and, in high-stakes assessments, can have large consequences on the trajectory of an interaction. While a single awkward moment is unlikely to lead to failure, in settings of intense evaluation, perceived infelicities such as an unfilled pause or formulaic phrase become amplified. The cumulative effect of such micro-features may lead to a candidate being judged as "not developing rapport" or as showing inadequate responsiveness to "verbal and non-verbal cues" and an overall negative im- pression of interpersonal abilities. as an adviser on the original project with the Royal College of General Practitioners. TG has conducted extensive research in the field of medical education and has drawn on this in substantially rewriting the initial and subsequent versions of this article. All authors contributed to conceptualizing and writing the paper, sourcing material. All authors have seen and approved the final manuscript. Competing interests KH i f KH was an examiner for the Royal College of General Practitioners’ (RCGP) Clinical Skills Assessment during the data-collection and analytic phases of the research cited in this article. The authors have no other competing interests to declare. KH was an examiner for the Royal College of General Practitioners’ (RCGP) Clinical Skills Assessment during the data-collection and analytic phases of the research cited in this article. The authors have no other competing interests to declare. 11. McManus IC, Wakeford R. PLAB and UK graduates' performance on MRCP(UK) and MRCGP examinations: data linkage study. Br Med J. 2014;348:g2621. 12. Korkiakangas T, Weldon S-M, Bezemer J, Kneebone R. Video-Supported Simulation for Interactions in the Operating Theatre (ViSIOT). Clin Simul Nurs. 2015;11(4):203–7. Abbreviations CSA li i l kill CSA: clinical skills assessment; MRCGP: Examinations for ‘Membership of the Royal College of General Practitioners’; OSCE: objective structured clinical examination; RCGP: Royal College of General Practitioners. 9. Dewhurst NG, McManus C, Mollon J, Dacre JE, Vale AJ. Performance in the MRCP(UK) Examination 2003–4: analysis of pass rates of UK graduates in relation to self-declared ethnicity and gender. BMC Med. 2007;5:8. 10. McManus IC, Elder AT, Dacre J. Investigating possible ethnicity and sex bias in clinical examiners: an analysis of data from the MRCP(UK) PACES and nPACES examinations. BMC Med Educ. 2013;13:103. References 1. Khan KZ, Gaunt K, Ramachandran S, Pushkar P. The Objective Structured Clinical Examination (OSCE): AMEE Guide No. 81. Part II: Organisation & Administration. Med Teach. 2013;35(9):e1447–63. 1. Khan KZ, Gaunt K, Ramachandran S, Pushkar P. The Objective Structured Clinical Examination (OSCE): AMEE Guide No. 81. Part II: Organisation & Administration. Med Teach. 2013;35(9):e1447–63. 2. Khan KZ, Ramachandran S, Gaunt K, Pushkar P. The Objective Structured Clinical Examination (OSCE): AMEE Guide No. 81. Part I: An historical and theoretical perspective. Med Teach. 2013;35(9):e1437–46. 2. Khan KZ, Ramachandran S, Gaunt K, Pushkar P. The Objective Structured Clinical Examination (OSCE): AMEE Guide No. 81. Part I: An historical and theoretical perspective. Med Teach. 2013;35(9):e1437–46. 3. Swanson DB, van der Vleuten CP. Assessment of clinical skills with standardized patients: state of the art revisited. Teaching and Learning in Medicine. 2013;25(sup1):S17–25. 3. Swanson DB, van der Vleuten CP. Assessment of clinical skills with standardized patients: state of the art revisited. Teaching and Learning in Medicine. 2013;25(sup1):S17–25. 4. First LR, Chaudhry HJ, Melnick DE. Quality, cost, and value of clinical skills assessment. N Engl J Med. 2013;368(10):963–4. 4. First LR, Chaudhry HJ, Melnick DE. Quality, cost, and value of clinical skills assessment. N Engl J Med. 2013;368(10):963–4. 5. Boulet JR, Smee SM, Dillon GF, Gimpel JR. The use of standardized patient assessments for certification and licensure decisions. Simul Healthc. 2009; 4(1):35–42. 5. Boulet JR, Smee SM, Dillon GF, Gimpel JR. The use of standardized patient assessments for certification and licensure decisions. Simul Healthc. 2009; 4(1):35–42. 6. 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Healthcare interaction as an expert communicative system. New Advent Lang Interac. 2010;196:167. 11. McManus IC, Wakeford R. PLAB and UK graduates' performance on MRCP(UK) and MRCGP examinations: data linkage study. Br Med J. 2014;348:g2621. 12. Korkiakangas T, Weldon S-M, Bezemer J, Kneebone R. Video-Supported Simulation for Interactions in the Operating Theatre (ViSIOT). Clin Simul Nurs. 2015;11(4):203–7. Author details 1 1Centre for Research in Applied Linguistics, Trent Building, University of Nottingham, Nottingham NG7 2RD, UK. 2Department of Education & Professional Studies, King’s College London, Franklin-Wilkins Building, Waterloo Road, London SE1 9NH, UK. 3Duke of Kent Building, Faculty of Health and Medical Sciences, University of Surrey, Surrey GU2 7XH, UK. 4Nuffield Department of Primary Care Health Sciences, University of Oxford Although a number of studies have identified that sim- ulated interactions show important differences from real-life professional communication [27, 28, 33–37], we are not arguing that simulation has no place in teaching or assessment. Much of medical practice consists of skills that are more or less technical in nature and which can be both taught and assessed effectively using simu- lated patients (the rationale behind the ‘skills lab’) [61]. Formative simulated consultations have great value in the safety they afford learners to make and learn from mistakes, as well as to ‘slow down’ the consultation to study what has happened. Summative simulated assess- ments, however, must carefully consider the difficulties of assessing interpersonal skills in this setting. Hence, we do not seek to bury the OSCE, but in introducing the sociolinguistic perspective, we do seek to debate its level of validity for assessing communicative and interactional aspects of clinical performance. Furthermore, we believe the evidence identified in a number sociolinguistic studies of simulated interaction [27, 30, 58] requires us to con- sider carefully what we mean by ‘fairness’ in assessment and how we might better assess communication skills in settings of cultural and linguistic diversity. 4Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford OX2 6GG, UK. 10. McManus IC, Elder AT, Dacre J. Investigating possible ethnicity and sex bias in clinical examiners: an analysis of data from the MRCP(UK) PACES and nPACES examinations. BMC Med Educ. 2013;13:103. 6. Cleland JA, Abe K, Rethans J-J. The use of simulated patients in medical education: AMEE Guide No 42 1. Med Teach. 2009;31(6):477–86. CSA: clinical skills assessment; MRCGP: Examinations for ‘Membership of the Royal College of General Practitioners’; OSCE: objective structured clinical examination; RCGP: Royal College of General Practitioners. 8. Gormley G, Sterling M, Menary A, McKeown G. Keeping it real! Enhancing realism in standardised patient OSCE stations. Clin Teach. 2012;9(6):382–6. 7. Holmboe ES, Ward DS, Reznick RK, Katsufrakis PJ, Leslie KM, Patel VL, et al. Faculty development in assessment: the missing link in competency-based medical education. Acad Med. 2011;86(4):460–7. 14. Brannick MT, Erol-Korkmaz HT, Prewett M. A systematic review of the reliability of objective structured clinical examination scores. 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Cambridge: Cambridge University Press; 2006. 54. Peräkylä A. Communicating and responding to diagnosis. Authors’ contributions S ll h SA was originally the Research Associate who conducted the analytic work on the Clinical Skills Assessment described in this article and drafted the first version of this debate article. CR was originally the Principle Investigator on the research project with the Royal College of General Practitioners and substantially contributed to the first draft and subsequent versions of this article. KH contributed a significant amount of analytic work and discussion 14. Brannick MT, Erol-Korkmaz HT, Prewett M. A systematic review of the reliability of objective structured clinical examination scores. Med Educ. 2011;45(12):1181–9. 14. Brannick MT, Erol-Korkmaz HT, Prewett M. A systematic review of the reliability of objective structured clinical examination scores. Med Educ. 2011;45(12):1181–9. Page 9 of 9 Atkins et al. 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Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit y p and we will help you at every step: 35. Niements N. From Role-Playing to Role-Taking: Interpreter’s Role(s) in Healthcare. 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https://openalex.org/W4205511605	http://jurnal.umb.ac.id/index.php/JAKTA/article/download/1960/2902	Indonesian	null	ANALISIS FAKTOR-FAKTOR YANG BERPENGARUH TERHADAP PERATAAN LABA PADA PERUSAHAAN MANUFAKTUR SUB SEKTOR MAKANAN DAN MINUMAN YANG TERDAFTAR DI BURSA EFEK INDONESIA	Jurnal Akuntansi, Keuangan dan Teknologi Informasi Akuntansi	2,022	cc-by-sa	7,151	ISSN: 2723-1399 e-ISSN: 2723-1488 ISSN: 2723-1399 e-ISSN: 2723-1488 ISSN: 2723-1399 e-ISSN: 2723-1488 Keywords: Kata kunci: Profitabilitas, Ukuran Perusahaan, Risiko Keuangan, Perataan Laba. ANALISIS FAKTOR-FAKTOR YANG BERPENGARUH TERHADAP PERATAAN LABA PADA PERUSAHAAN MANUFAKTUR SUB SEKTOR MAKANAN DAN MINUMAN YANG TERDAFTAR DI BURSA EFEK INDONESIA Irna Astriana 1, Nurmala1 1Program Studi Akuntansi, Fakultas Ekonomi 1Universitas PGRI Palembang Email : nurmala@univpgri-palembang.ac.id http://jurnal.umb.ac.id/index.php/JAKTA/index INFORMASI ARTIKEL This study aims to determine the effect of profitability, firm size, and financial risk on income smoothing in food and beverage sub-sector manufacturing companies listed on the Indonesia Stock Exchange for the 2015-2019 period. Indonesia Stock Exchange with a total sample of 13 companies. The sampling technique was done by purposive sampling technique. The data used in this study is the company's annual financial report data for 2015-2019. Data analysis and discussion was carried out using quantitative descriptive methods by testing the truth of the hypothesis. The results partially show that the profitability of Return On Assets (ROA) obtained a significant value of 0.041 ˂ 0.05, this means that Ha is rejected and Ho is accepted, then there is a significant effect between Return On Assets on income smoothing of firm size, a significant value is 0.031 ˂ 0.05, this means Ha is rejected and Ho is accepted, then there is a significant effect between firm size on income smoothing and financial risk as measured by the Leverage Ratio, a significant value is obtained at 0.390 ˃ 0.05, this means Ha is accepted and Ho is rejected, so there is no significant influence between financial risk on income smoothing. While the results of the study simultaneously state that the Fsig value is 0.140 ˃ 0.05, this means that Ha is accepted and Ho is rejected, so profitability, firm size, and financial risk simultaneously have no significant effect on income smoothing. Riwayat Artikel: Diterima : 25 Agustus 2022 Direvisi : 10 September 2022 Disetujui : 29 Desember 2022 Keywords: Profitability, Company Size, Financial Risk, Income Smoothing. Keywords: Profitability, Company Size, Financial Risk, Income Smoothing. PENDAHULUAN Situasi perekonomian negara yang tidak menentu dan ketatnya persaingan di dunia usaha mendorong manajemen untuk bekerja lebih efektif dan efisien agar perusahaan mampu bertahan dan menjaga eksistensinya sekaligus meningkatkan kinerja manajemen untuk mendapatkan hasil yang optimal bagi perusahaan. Bagi investor, kinerja manajemen menjadi faktor pendorong dalam menilai suatu perusahaan dan membuat keputusan. Dalam perkembangan dunia usaha yang meningkat pesat, kemajuan teknologi yang semakin canggih, persaingan yang semakin ketat antar perusahaan, serta situasi perekonomian negara yang tidak menentu saat ini mendorong manajemen perusahaan berlomba-lomba menjadi unggul dari perusahaan pesaingnya, salah satu dari banyaknya media yang dapat digunakan oleh pihak manajemen untuk menampilkan kinerja dan performa perusahaannya adalah informasi akuntansi dan salah satunya ialah informasi dalam laporan keuangan. Informasi akuntansi yang berhubungan dengan kinerja perusahaan merupakan kebutuhan yang paling mendasar dalam proses pengambilan keputusan bagi investor di pasar modal. Menurut (Muhammad Rifky Santoso, 2014), laporan keuangan sebagai bentuk pertanggungjawaban dari pengeluaran biaya dan penerimaan semua pendapatan dari kegiatan operasional dalam mendapatkan keuntungan yang menjadi tujuan perusahaan. Menurut Hans Kartikahadi 2016 laporan keuangan bertujuan menyediakan informasi tentang posisi keuangan, kinerja, serta perubahan posisi keuangan suatu perusahaan yang bermanfaat bagi sejumlah besar pemakai dalam pengambilan keputusan ekonomi. Untuk memenuhi tujuan tersebut, laporan keuangan menyediakan informasi tentang suatu entitas yang terdiri dari: aset, liabilitas, ekuitas, pendapatan dan beban serta kontribusi dan distribusi kepada pemilik dalam kapasitasnya sebagai pemilik, serta arus kas. Informasi tersebut beserta informasi lain yang terdapat dalam catatan atas laporan keuangan membantu pengguna laporan keuangan dalam prediksi arus kas masa depan dan kinerja entitas. Laporan keuangan terdiri atas neraca, laporan laba rugi, laporan arus kas, laporan perubahan ekuitas dan catatan atas laporan keuangan. Pemakai laporan keuangan dikelompokkan menjadi dua kelompok yaitu pihak internal dan pihak eksternal. Pihak internal yang berkepentingan terhadap laporan keuangan ialah manajemen. Pihak eksternal perusahaan terdiri dari investor, kreditor, pemerintah, karyawan perusahaan, dan masyarakat umum. Laporan keuangan menjadi sumber informasi bagi kedua pihak tersebut dalam pengambilan keputusan. Laporan keuangan dapat menggambarkan keadaan perusahaan, karena dalam laporan keuangan tersebut banyak mengandung informasi yang sangat dibutuhkan oleh pihak-pihak yang berkepentingan, terutama informasi mengenai laba perusahaan. Alat yang digunakan untuk mengukur kinerja manajemen salah satunya adalah laba. Tujuan informasi laba adalah untuk menilai kinerja manajemen, membantu mengestimasi kemampuan laba dalam jangka panjang, dan memperkirakan risiko-risiko investasi. Kemampuan dan nilai perusahaan dalam mengelola aset-asetnya dapat digambarkan dengan cara melihat bagaimana perusahaan dalam menghasilkan laba dalam operasinya. http://jurnal.umb.ac.id/index.php/JAKTA/index ABSTRAK: Penelitian ini bertujuan untuk mengetahui pengaruh profitabilitas, ukuran perusahaan, dan risiko keuangan terhadap perataan laba pada perusahaan manufaktur sub sektor makanan dan minuman yang terdaftar di Bursa Efek Indonesia periode 2015-2019.Populasi penelitian ini adalah perusahaan manufaktur sub sektor makanan dan minuman yang terdaftar di Bursa Efek Indonesia dengan jumlah sampel 13 perusahaan. Teknik pengambilan sampel dilakukan dengan teknik purposive sampling. Data yang digunakan dalam penelitian adalah data laporan keuangan tahunan perusahaan tahun 2015-2019. Analisis data dan pembahasan dilakukan dengan menggunakan metode deskriptif kuantitatif dengan menguji kebenaran hipotesis. Hasil penelitian secara parsial menunjukkan bahwa profitabilitas Return On Asset (ROA) didapatkan nilai signifikan sebesar 0,041 ˂ 0,05, ini berarti Ha ditolak dan Ho diterima, maka terdapat pengaruh signifikan antara Return On Asset terhadap perataan laba ukuran perusahaan didapatkan nilai signifikan sebesar 0,031 ˂ 0,05, ini berarti Ha ditolak dan Ho diterima, maka terdapat pengaruh signifikan antara ukuran perusahaan terhadap perataan laba dan risiko keuangan yang diukur dengan Leverage Ratio didapatkan nilai signifikan sebesar 0,390 ˃ 0,05, ini berarti Ha diterima dan Ho ditolak maka tidak terdapat pengaruh signifikan antara risiko keuangan terhadap perataan laba. Sedangkan hasil penelitian secara simultan menyatakan bahwa nilai Fsig sebesar 0,140 ˃ 0,05, ini berarti Ha diterima dan Ho ditolak maka profitabilitas, ukuran perusahaan, dan risiko keuangan, secara simultan tidak memiliki pengaruh signifikan terhadap perataan laba. Vol 3. No. 2. Desember 2022 645 http://jurnal.umb.ac.id/index.php/JAKTA/index PENDAHULUAN Oleh sebab itu, pengguna laporan keuangan terutama investor dan kreditor biasanya hanya terfokus pada laba perusahaan, tanpa mengetahui bagaimana prosedur yang dilakukan oleh manajemen untuk menghitung laba seperti yang tertulis di laporan keuangan. Laba merupakan salah satu informasi potensial yang terkandung di dalam laporan keuangan. Laba perusahaan berguna sebagai penghasil bagi investor dan orang-orang yang berkepeningan di dalamnya sehingga proses produksi dapat terus berjalan dan menghasilkan laba-laba berikutnya. Menurut Widyaningdyah, 2001 (Amanza, 2012), Standar Akuntansi Keuangan (SAK), memberikan fleksibilitas bagi manajemen untuk memilih kebijakan akuntansi yang lebih mempresentasikan keadaan perusahaan sesungguhnya. Fleksibilitas itulah yang terkadang dimanfaatkan oleh manajemen untuk melakukan manajemen laba (earnings management). Oleh karena itu, manajemen mempunyai kecenderungan untuk melakukan tindakan yang dapat membuat laporan keuangan menjadi baik. Salah satu bentuk dari tindakan ini adalah praktik perataan laba Vol 3. No. 2. Desember 2022 646 ISSN: 2723-1399 e-ISSN: 2723-1488 (income smoothing) yang pada dasarnya merupakan tindakan yang dinilai bertentangan dengan tujuan perusahaan. (income smoothing) yang pada dasarnya merupakan tindakan yang dinilai bertentangan dengan tujuan perusahaan. Menurut Fudenberg dan Tirole dalam Hasanah, 2007 (Pratama, 2012), perataan laba adalah proses manipulasi waktu terjadinya laba atau laporan laba agar laba yang dilaporkan terlihat stabil. Menurut Belkaoui, 2006:73 (Handayani, 2016), perataan laba adalah pengurangan fluktuasi laba dari tahun ke tahun dengan memindahkan pendapatan dari tahun-tahun yang tinggi pendapatannya ke periode-periode yang kurang menguntungkan. Lebih lanjut dijelaskan bahwa perataan laba sebagai fenomena proses manipulasi profil waktu dari pendapatan atau laporan laba menjadi kurang bervariasi, sambil sekaligus tidak meningkatkan pendapatan yang dilaporkan selama periode tersebut. Ada beberapa faktor yang telah mendorong perusahaan dalam menjalankan praktek perataan laba. Menurut Budiasih dan Igan, 2009:44-50 (Handayani, 2016), menemukan bukti bahwa perataan laba dipengaruhi oleh ukuran perusahaan, profitabilitas dan deviden pay out (DPR). Menurut (Agustianto, 2014), rasio keuangan juga diduga sebagai salah satu faktor yang mempengaruhi tindakan perataan laba, rasio leverage sebagai proksi atas risiko keuangan terhadap perataan laba digunakan dalam beberapa penelitian terdahulu. Hal ini diperkuat oleh pernyataan yang dikemukakan oleh Kustini dan Ekawati, 2004 dalam Sindi dan Etna, 2011 yang menyatakan bahwa perusahaan yang memiliki tingkat rasio yang tinggi mempunyai risiko yang tinggi pula, maka laba akan berfluktuasi sehingga perusahaan cenderung untuk melakukan perataan laba agar laba perusahaan terlihat stabil, karena investor cenderung mengamati fluktuasi laba suatu perusahaan. Perataan Laba Menurut Belkaoui, 2006:73 (Handayani, 2016), perataan laba adalah pengurangan fluktuasi laba dari tahun ke tahun dengan memindahkan pendapatan dari tahun-tahun yang tinggi pendapatannya ke periode-periode yang kurang menguntungkan. Lebih lanjut dijelaskan bahwa perataan laba sebagai fenomena proses manipulasi profil waktu dari pendapatan atau laporan laba menjadi kurang bervariasi, sambil sekaligus tidak meningkatkan pendapatan yang dilaporkan selama periode tersebut. Menurut Hasen dan Mowen, (Faisal, 2017) mengatakan bahwa perataan laba didefinisikan sebagai upaya yang secara sengaja dilakukan untuk memperkecil fluktuasi pada tingkat laba yang dianggap normal bagi perusahaan, dengan kata lain, perataan laba atau dikenal dengan istilah income smoothing didefinisikan sebagai sebuah praktik dengan menggunakan teknik-teknik akuntansi untuk mengurangi fluktuasi laba bersih setelah beberapa periode waktu. Perataan Laba diuji dengan indeks Eckel (1981) (Bestivano, 2013). Eckel menggunakan Coefficient Variation (CV) variabel penghasilan dan variabel penghasilan bersih. Untuk menentukan kelompok perusahaan yang melakukan tindakan perataan laba dan yang tidak melakukan perataan laba. Adapun perhitungan indeks eckel dirumuskan sebagai berikut : p p g Indeks Perataan Laba = Keterangan: Indeks Perataan Laba = Keterangan: Keterangan: I = perubahan laba dalam suatu periode. I = perubahan laba dalam suatu periode. S=perubahan pendapatan bersih dalam suatu periode. Cv=koefisien variasi dari variabel. Yaitu standar deviasi dibagi dengan nilai rata-rata yang diharapkan. =koefisien variasi dari variabel. Yaitu standar deviasi dibagi dengan nilai rata-rata yang diharapkan. PENDAHULUAN Return On Assets merupakan salah satu bentuk rasio profitabilitas yang digunakan untuk mengukur kemampuan perusahaan dengan keseluruhan dana yang ditanamkan dalam aktiva yang digunakan untuk operasi perusahaan dalam menghasilkan keuntungan. Perataan laba dapat dipengaruhi oleh beberapa faktor. Banyak penelitian empiris terdahulu yang telah menguji faktor-faktor tersebut. Faisal (2017) meneliti Net Profit Margin, Return On Asset, dan Leverage Ratio terhadap perataan laba. Penelitian tersebut dapat membuktikan bahwa faktor- faktor tersebut berpengaruh terhadap perataan laba. Penelitian lain mengenai perataan laba dilakukan oleh Hartanza Putra Hutamanjaya (2019). Hartanza Putra Hutamanjaya (2019) menguji pengaruh faktor-faktor Finance Leverage, ukuran perusahaan, profitabilitas dan kepemilikan manajerial terhadap praktik perataan laba. Hasil yang diperoleh adalah bahwa finance leverage dan ukuran perusahaan berpengaruh terhadap perataan laba sedangkan profitabilitas dan kepemilikan manajerial tidak berpengaruh terhadap perataan laba. Penelitian terhadap perataan laba di Indonesia masih sangat penting untuk diteliti, karena perataan laba sendiri dapat merugikan pihak-pihak yang berkepentingan terhadap perusahaan seperti investor maupun pemakai laporan keuangan. Oleh karena itu, jika perataan laba terdapat pada perusahaan publik di Indonesia, maka praktik itu akan menimbulkan kerugian yang semakin besar bagi pihak-pihak yang berkepentingan. Hal ini karena informasi yang terdapat di dalam laporan keuangan sangat mempengaruhi pihak-pihak yang berkepentingan dalam proses pengambilan keputusan. Belum konsistennya hasil penelitian satu sama lain dan juga alasan yang telah diuraikan diatas, membuat peneliti tertarik untuk melakukan penelitian lebih lanjut terhadap faktor-faktor yang berpengaruh terhadap perataan laba. Oleh karena itu, penelitian ini menguji pengaruh beberapa faktor antara lain, Profitabilitas, Ukuran Perusahaan dan Risiko Keuangan terhadap Perataan Laba. Berdasarkan latar belakang diatas, maka penelitian ini mengambil judul : “Analisis Faktor- Faktor Yang Berpengaruh Perataan Laba Pada Perusahaan Manufaktur Subsektor Makanan Dan Minuman Yang Terdaftar Di Bursa Efek Indonesia”. ehubungan dengan keterbatasan waktu dan biaya, maka penelitian ini perlu diberi batasan yaitu: 1. Peneliti hanya melakukan penelitian terhadap perataan laba pada perusahaan Perusahaa 1. Peneliti hanya melakukan penelitian terhadap perataan laba pada perusahaan Perusahaan Manufaktur Subsektor Makanan Dan Minuman Yang Terdaftar Di Bursa Efek Indonesia. Manufaktur Subsektor Makanan Dan Minuman Yang Terdaftar Di Bursa Efek Indonesia. 2. Peneliti membatasi faktor-faktor hanya pada profitabilitas, ukuran perusahaan dan risiko keuangan. Vol 3. No. 2. Desember 2022 http://jurnal.umb.ac.id/index.php/JAKTA/index 647 Sedangkan masalah yang akan dibahas dalam penelitian ini adalah: g y g p 1.Apakah profitabilitas berpengaruh terhadap perataan laba? 3. Apakah ukuran perusahaan berpengaruh terhadap perataan laba? 3. Apakah ukuran perusahaan berpengaruh terhadap perataan laba? 4. Apakah risiko keuangan berpengaruh terhadap perataan laba? 4. TINJAUAN PUSTAKA Laporan Keuangan Laporan Keuangan Menurut (Kasmir, 2008), laporan keuangan adalah laporan yang menunjukkan kondisi keuangan perusahaan pada saat ini atau dalam suatu periode tertentu. Maksud laporan keuangan yang menunjukkan kondisi perusahaan saat ini adalah merupakan kondisi terkini. M j L b Menurut National Association of Certified Fraud Examiners (Sulistyanto, 2004), manajemen laba adalah kesalahan atau kelalaian yang disengaja dalam membuat laporan mengenai fakta material atau data akuntansi sehingga menyesatkan ketika semua informasi itu dipakai untuk membuat pertimbangan yang akhirnya akan menyebabkan orang yang membacanya akan menggati atau mengubah pendapat atau keputusannya. g p p p y Menurut Syafrudin, 2011:09 (Faisal, 2017), manajemen laba adalah suatu proses yang dilakukan dengan sengaja dalam batasan general accepted accounting principles (GAAP) untuk mengarah pada tingkatan laba yang dilaporkan. PENDAHULUAN Apakah risiko keuangan berpengaruh terhadap perataan laba? http://jurnal.umb.ac.id/index.php/JAKTA/index Klasifikasi Perataan Laba arnea et al, (Agustianto, 2014), mengemukakan bahwa tindakan perataan laba dapat dilakukan 3 cara yaitu: y 1. Perataan melalui waktu terjadinya transaksi atau j y 2. pengakuan transaksi (smoothing through event strategic management occurance or recognition). Perataan laba dilakukan dengan cara mengatur waktu transaksi aktual sehingga dapat mengurangi fluktuasi pendapatan yang dilaporkan. 3. Perataan melalui alokasi waktu (smoothing through allocation over time) manajemen memiliki kewenangan untuk mengalokasikan pendapatan atau beban dalam periode keuangan yang berbeda dalam rangka melakukan perataan laba. 4. Perataan laba melalui klasifikasi (classificatory smoothing) manajemen perusahaan melakukan rataan laba dengan cara mengklasifikasikan item-item dalam laba erjadinya Perataan Laba j y Menurut Harahap, 2013:249 (Faisal, 2017), cara-cara yang dapat digunakan untuk melakukan perataan laba adalah: 1. Melalui kejadian-kejadian dan pengakuan. Maksudnya untuk mengurangi fluktuasi laba yang dilaporkan manajemen dapat mengatur suatu tindakan atas keputusan, misalnya yang berkaitan dengan pelaksanaan penelitian dan pengembangan. berkaitan dengan pelaksanaan penelitian dan pengembangan. 2. Melalui alokasi. Manajemen melakukan perataan dengan mengalokasikan pendapatan atau biaya selama beberapa periode pelaporan. biaya selama beberapa periode pelaporan. 3. Melalui klasifikasi. Manajemen melakukan perataan dengan mengklasifikasi laba sebagai ordinary atau extra ordinary item. 5. Ancaman pergantian manajer. 5. Ancaman pergantian manajer. http://jurnal.umb.ac.id/index.php/JAKTA/index ISSN: 2723-1399 e-ISSN: 2723-1488 JURNAL AKUNTANSI KEUANGAN DAN TEKNOLOGI INFORMASI AKUNTANSI Available online at: http://jurnal.umb.ac.id/index.php/JAKTA DOI: https://doi.org/10.36085/jakta.v2i1 4. Meningkatkan persepsi pihak eksternal terhadap kemampuan manajemen. Sasaran Perataan Laba Foster dalam Zuhro, 1996 (Putri, 2008), mengklasifikasikan unsur-unsur laporan keuanga seringkali menjadi sasaran untuk melakukan perataan laba (income smoothing) adalah: Foster dalam Zuhro, 1996 (Putri, 2008), mengklasifikasikan unsur-unsur laporan keuanga seringkali menjadi sasaran untuk melakukan perataan laba (income smoothing) adalah: 1. Unsur penjualan a. Saat pembuatan faktur, misalnya penjualan yang sebenarnya untuk periode yang akan datang, pembuatan fakturnya dilakukan pada periode ini dan dilaporkan sebagai penjualan periode ini. b. Pembuatan pesanan atau penjualan fiktif. b. Pembuatan pesanan atau penjualan fiktif. c. Penurunan (downgrading) produk, misalnya dengan cara mengklasifikasikan produk yang belum masuk ke dalam produk rusak dan selanjutnya dilaporkan telah terjual dengan harga yang lebih rendah dari harga yang sebenarnya. y g g y g y 2. Unsur biaya a.Memecah-mecah faktur, misalnya faktur untuk sebuah pembelian pesanan dipecah menjadi beberapa pembelian atau pesanan dan selanjutnya dibuatkan beberapa faktur dengan tanggal yang berbeda kemudian dilaporkan dalam beberapa periode akuntansi. b.Mencatat biaya dibayar dimuka (prepayment) sebagai biaya. Misalnya melaporkan biaya advertensi dibayar dimuka untuk tahun depan sebagai biaya advertensi tahun ini. Tujuan Perataan Laba Menurut Foster, (Bestivano, 2013), tujuan perusahaan melakukan perataan laba adalah sebagai j Menurut Foster, (Bestivano, 2013), tujuan perusahaan melakukan perataan laba adalah sebagai rikut: 1. Memperbaiki citra perusahaan dimata pihak luar, bahwa perusahaan tersebut memiliki resiko yang rendah. 2. Memberikan informasi yang relevan dalam melakukan prediksi terhadap laba di mas yang akan datang. 3. Meningkatkan kepuasan relasi bisnis. 3. Meningkatkan kepuasan relasi bisnis. Vol 3. No. 2. Desember 2022 648 Vol 3. No. 2. Desember 2022 648 http://jurnal.umb.ac.id/index.php/JAKTA/index Vol 3. No. 2. Desember 2022 648 http://jurnal.umb.ac.id/index.php/JAKTA/index 648 y p Faktor-Faktor yang Mempengaruhi Perataan Laba y g p g Budiasih, 2013:5 (Faisal, 2017), mengatakan bahwa perataan laba dilakukan oleh perusahaan dalam rangka promosi harga jual saham perusahaan pada investor (pemilik saham). Keberhasilan pemerataan laba bergantung pada faktor-faktor sebagai berikut: Budiasih, 2013:5 (Faisal, 2017), mengatakan bahwa perataan laba dilakukan oleh perusahaan dalam rangka promosi harga jual saham perusahaan pada investor (pemilik saham). Keberhasilan pemerataan laba bergantung pada faktor-faktor sebagai berikut: 1. Ukuran perusahaan baik atau tidak baik dimata pemegang saham dan pihak lain. 1. Ukuran perusahaan baik atau tidak baik dimata pemegang saham dan pihak lain. 2 Profitabilitas yaitu suatu ukuran dalam persentasi yang digunakan untuk menil 1. Ukuran perusahaan baik atau tidak baik dimata pemegang saham dan pihak lain. 2. Profitabilitas, yaitu suatu ukuran dalam persentasi yang digunakan untuk menilai sejauh mana perusahaan mampu menghasilkan laba pada tingkat yang dapat diterima angka profitabilitas dinyatakan dalam angka laba sebelum atau sesudah pajak, laba investasi, pendapatan persaham, dan laba penjualan. Vol 3. No. 2. Desember 2022 http://jurnal.umb.ac.id/index.php/JAKTA/index 649 3. Sektor industri, yakni suatu usaha yang bergerak dalam bidang pengelolaan bahan mentah menjadi bahan jadi, dan setengah jadi. j j g j 4. Rencana bonus, adalah rencana pemberian bonus yang diberikan pada periode tertentu. 4. Rencana bonus, adalah rencana pemberian bonus yang diberikan pada periode tertentu. 5. Kebangsawanan, yakni tingkatan masyarakat dalam suatu bangsa. ntungan Adanya Perataan Laba Budiasih, 2013:12 (Faisal, 2017), mengungkapkan alasan manajemen diuntungkan denga ya praktik perataan laba, yaitu: 1. Skema konvensasi manajemen dihubungkan dengan kinerja perusahaan yang disajikan dalam laba akuntansi yang dilaporkan, 2. Fluktuasi dalam kinerja manajemen dapat berakibat intervensi pemilik untuk mengganti manajemen dengan cara pengambialihan atau penggantian manajemen langsung. Profitabilitas Profitabilitas merupakan alat ukur kesuksesan sebuah perusahaan yang utama. Benar, profitabilitas penting untuk kelangsungan hidup sebuah perusahaan. Pengambilan atas ekuitas merupakan ukuran profitabilitas yang sering digunakan (Robert Libby, 2008). Profitabilitas menunjukkan kemampuan perusahaan memperoleh laba dalam hubungannya dengan penjualan, total aktiva, maupun modal sendiri (Winarni, 2005). T j d f t i fit bilit t (H 2015) k l h g y g p j p Tujuan dan manfaat rasio profitabilitas menurut (Hery, 2015) secara keseluruhan: 1. Untuk mengukur kemampuan perusahaan dalam menghasilkan laba selama periode tertentu. 2. Untuk menilai posisi laba perusahaan tahun sebelumnya dengan tahun sekarang. 2. Untuk menilai posisi laba perusahaan tahun sebelumnya dengan tahun se p p y 3. Untuk menilai perkembangan laba dari waktu ke waktu. k menilai perkembangan laba dari waktu ke wakt 4. http://jurnal.umb.ac.id/index.php/JAKTA/index y p Faktor-Faktor yang Mempengaruhi Perataan Laba Untuk mengukur seberapa besar jumlah laba bersih yang akan dihasilkan dari setiap rupiah dana yang tertanam dalam total aset. 5. Untuk mengukur seberapa besar jumlah laba bersih yang akan dihasilkan dari setiap rupia dana yang tertanam dalam total ekuitas. 6. Untuk mengukur margin laba kotor atas penjualan bersih. 7. Untuk mengukur margin laba operasional atas penjualan bersih. 8. Untuk mengukur margin laba bersih atas penjualan bersih. Jenis-jenis rasio profitabilitas menurut (Hery, 2015) sebagai berikut: 8. Untuk mengukur margin laba bersih atas penjualan bersih. 8. Untuk mengukur margin laba bersih atas penjualan bersih. Jenis jenis rasio profitabilitas menurut (Hery 2015) sebagai berikut: s-jenis rasio profitabilitas menurut (Hery, 2015) sebagai berikut: 1. Hasil Pengembalian Atas Aset (Return on Assets) Rumus : Return on Assets = x 100% 2. Hasil Pengembalian Atas Ekuitas (Return on Equity) Rumus: Return on Equity = 3. Margin Laba Kotor (Gross Profit Margin) Rumus: Gross Profit Margin = 4. Margin Laba Operasional (Operating Profit Margin) Rumus: Operating Profit Margin = 5. Margin Laba Bersih (Net Profit Margin) Rumus : Net Profit Margin = g ( f g ) Rumus : Net Profit Margin = Hipotesis p Adapun hipotesis penelitan sebagai berikut: Adapun hipotesis penelitan sebagai berikut: p p p g H1: Diduga profitabilitas berpengaruh positif terhadap perataan laba . H1: Diduga profitabilitas berpengaruh positif terhadap perataan laba . H2: Diduga ukuran perusahaan berpengaruh positif terhadap perataan laba H2: Diduga ukuran perusahaan berpengaruh positif terhadap perataan laba H : Diduga risiko keuangan berpengaruh positif terhadap perataan laba H3: Diduga risiko keuangan berpengaruh positif terhadap perataan laba METODELOGI PENELITIAN Objek dalam penelitian ini adalah Perusahaan Manufaktur Subsektor Makanan Dan Minuman Yang Terdaftar Di Bursa Efek Indonesia. Populasi dalam penelitian ini adalah seluruh perusahaan manufaktur sub sektor makanan dan minuman yang terdaftar di Bursa Efek Indonesia ,sedangkan penarikan sampelnya menggunakan dalam penelitian metode purposive sampling dengan kriteria tertentu. Adapun kriteria pengambilan sampel adalah sebagai berikut: Objek dalam penelitian ini adalah Perusahaan Manufaktur Subsektor Makanan Dan Minuman Yang Terdaftar Di Bursa Efek Indonesia. Populasi dalam penelitian ini adalah seluruh perusahaan manufaktur sub sektor makanan dan minuman yang terdaftar di Bursa Efek Indonesia ,sedangkan penarikan sampelnya menggunakan dalam penelitian metode purposive sampling dengan kriteria tertentu. Adapun kriteria pengambilan sampel adalah sebagai berikut: Yang 1. Perusahaan bidang manufaktur sub sektor makanan dan minuman yang terdaftar di bursa efek Indonesia dibawah tahun 2015-2020. 1. Perusahaan bidang manufaktur sub sektor makanan dan minuman yang terdaftar di bursa efek Indonesia dibawah tahun 2015-2020. 2. Menyediakan laporan keuangan yang berisi informasi lengkap, pada periode pengamatan tahun 2015-2020. 3. Perusahaan bidang manufaktur sub sektor makanan dan minuman yang selalu mendapatkan laba pada periode pengamatan. laba pada periode pengamatan. laba pada periode pengamatan. nik Analisis Data ik li i d l li i b i i d l h i li i b d P d l h Ukuran Perusahaan Ukuran perusahaan yang lebih besar cenderung akan lebih kritis mendapatkan perhatian baik dari para analisis, investor maupun pemerintah. Perusahaan besar akan menghindari fluktuasi laba yang drastis dengan melakukan tindakan perataan laba, karena perusahaan nantinya akan dibebani pajak yang besar dan meminimalisir resiko yang kemungkinan akan terjadi (Putra, 2015). Ukuran perusahaan merupakan salah satu variabel yang mempengaruhi perataan laba (income smoothing). Di Indonesia sendiri banyak berdiri perusahaan-perusahaan baik yang berukuran besar maupun kecil. Perusahaan besar terutama yang sudah go publik cenderung lebih berhati-hati dalam Vol 3. No. 2. Desember 2022 650 Vol 3. No. 2. Desember 2022 650 650 melakukan pelaporan keuangan. Hal tersebut berdampak pada semakin sedikit kemungkinan perusahaan tersebut menjalankan perataan laba. Perhatian yang besar dari masyarakat luas menyebabkan manajemen perusahaan bersikap hati-hati dalam melakukan pelaporan keuangan (Ina Setyaningtyas, 2014). melakukan pelaporan keuangan. Hal tersebut berdampak pada semakin sedikit kemungkinan perusahaan tersebut menjalankan perataan laba. Perhatian yang besar dari masyarakat luas menyebabkan manajemen perusahaan bersikap hati-hati dalam melakukan pelaporan keuangan (Ina Setyaningtyas, 2014). y g y Menurut (Agustianto, 2014), ukuran perusahaan dihitung dengan skala rasio dimana pengukuran menggunakan logaritma natural dari total asset yang didapat dari laporan posisi keuangan sebuah perusahaan. Risiko Keuangan Menurut Tarjo dan Sulistyowati, 2005 (Sudarsi, 2012), leverage merupakan perbandingan antara hutang dan aktiva yang menunjukkan berapa bagian aktiva yang digunakan untuk menjamin hutang. Sehingga secara sistematis leverage ratio (total debt to asset ratio) dapat dirumuskan sebagai berikut: Lev = x 100% Gambar Kerangka Pemikiran Hipotesis d hi i li b i b ik Perataan Laba (Y) Risiko Keuangan (X₃) Ukuran Perusahaan (X₂) Profitabilitas (X₁) Gambar Kerangka Pemikiran Profitabilitas (X₁) Perataan Laba (Y) Ukuran Perusahaan (X₂) Risiko Keuangan (X₃) Teknik Analisis Data eknik Analisis Data eknik analisis dalam penelitiab ini adalah regresi linier berganda . Persamaan umumnya adalah: eknik analisis dalam penelitiab ini adalah regresi linier berganda . Persamaan umumnya adalah: p Y = α + β₁X₁ + β₂X₂ + .... + βnXn Y = α + β₁X₁ + β₂X₂ + .... + βnXn Keterangan: Y = perataan laba Y = perataan laba Vol 3. No. 2. Desember 2022 651 Vol 3. No. 2. Desember 2022 651 Vol 3. No. 2. Desember 2022 http://jurnal.umb.ac.id/index.php/JAKTA/index Vol 3. No. 2. Desember 2022 651 http://jurnal.umb.ac.id/index.php/JAKTA/index 651 ISSN: 2723-1399 e-ISSN: 2723-1488 JURNAL AKUNTANSI KEUANGAN DAN TEKNOLOGI INFORMASI AKUNTANSI Available online at: http://jurnal.umb.ac.id/index.php/JAKTA DOI: https://doi.org/10.36085/jakta.v2i1 α = konstanta β = koefisien regresi X₁ = profitabilitas X₂ = ukuran perusahaan X₃ = risiko keuangan HASIL PENELITIAN Uji Normalitas Tabel 4.31 Uji Normalitas Sumber: data sekunder yang diolah dengan SPSS B d k t b l t t SPSS di t dik t h i b h il i i ifik i A t Si (2 t il d) L X1 One-Sample Kolmogorov-Smirnov Test Ln_X1 Ln_X2 Ln_X3 Ln_y N 65 65 65 65 Normal Parametersa Mean 1.8918 2.1720 3.5209 1.5844 Std. Deviation .79258 1.38268 .75218 1.39724 Most Extreme Differences Absolute .169 .222 .288 .135 Positive .119 .192 .183 .135 Negative -.169 -.222 -.288 -.128 Kolmogorov-Smirnov Z 1.364 1.787 2.318 1.092 Asymp. Sig. (2-tailed) .058 .063 .062 .184 a. Test distribution is Normal. Uji Normalitas Tabel 4.31 Uji Normalitas Sumber: data sekunder yang diolah dengan SPSS One-Sample Kolmogorov-Smirnov Test Ln_X1 Ln_X2 Ln_X3 Ln_y N 65 65 65 65 Normal Parametersa Mean 1.8918 2.1720 3.5209 1.5844 Std. Deviation .79258 1.38268 .75218 1.39724 Most Extreme Differences Absolute .169 .222 .288 .135 Positive .119 .192 .183 .135 Negative -.169 -.222 -.288 -.128 Kolmogorov-Smirnov Z 1.364 1.787 2.318 1.092 Asymp. Sig. (2-tailed) .058 .063 .062 .184 a. Test distribution is Normal. Tabel 4.31 Uji Normalitas Berdasarkan tabel output SPSS diatas, diketahui bahwa nilai signifikansi Astmp. Sig (2-tailed) Ln X1 sebesar 0,058 ˃ 0,05, Ln X2 sebesar 0,063 ˃ 0,05, Ln X3 sebesar 0,062 ˃ 0,05, dan Ln y sebesar 0,184 ˃ 0,05. Maka sesuai dengan dasar pengambilan keputusan dalam Uji Normalitas Kolmogorov- Smirnov Test diatas, dapat disimpulkan bahwa data berdistribusi normal. Dengan demikian, asumsi atau persyaratan normalitas dalam model regresi sudah terpenuhi. j Tabel Uji Autokorelasi Model Summaryb Model R R Square Adjusted R Square Std. http://jurnal.umb.ac.id/index.php/JAKTA/index Teknik Analisis Data Error of the Estimate Durbin-Watson 1 .292a .085 .040 1.36893 2.238 a. Predictors: (Constant), Ln_X3, Ln_X2, Ln_X1 b. Dependent Variable: Ln_y Sumber: data sekunder yang diolah dengan SPSS Berdasarkan tabel output SPSS diatas, diketahui bahwa nilai Durbin Watson (DW) sebesar 2,238. Selanjutnya nilai ini akan kita bandingkan dengan nilai tabel Durbin Watson signifikansi 5% dengan rumus (K;N). Adapun jumlah variabel independen adalah 3 atau “K” = 3, sementara jumlah sampel atau “N” = 65, maka (K;N) = (3;65). Angka ini kemudian kita lihat pada distribusi nilai tabel Durbin Watson. Maka ditemukan nilai dl sebesar 1,503 dan du sebesar 1,696. Tabel Uji Autokorelasi Model Summaryb Model R R Square Adjusted R Square Std. Error of the Estimate Durbin-Watson 1 .292a .085 .040 1.36893 2.238 a. Predictors: (Constant), Ln_X3, Ln_X2, Ln_X1 b D d V i bl L b. Dependent Variable: Ln_y p y Sumber: data sekunder yang diolah dengan SPSS y g g Berdasarkan tabel output SPSS diatas, diketahui bahwa nilai Durbin Watson (DW) sebesar 2,238. Selanjutnya nilai ini akan kita bandingkan dengan nilai tabel Durbin Watson signifikansi 5% dengan rumus (K;N). Adapun jumlah variabel independen adalah 3 atau “K” = 3, sementara jumlah sampel atau “N” = 65, maka (K;N) = (3;65). Angka ini kemudian kita lihat pada distribusi nilai tabel Durbin Watson. Maka ditemukan nilai dl sebesar 1,503 dan du sebesar 1,696. Berdasarkan tabel output SPSS diatas, diketahui bahwa nilai Durbin Watson (DW) sebesar 2,238. Selanjutnya nilai ini akan kita bandingkan dengan nilai tabel Durbin Watson signifikansi 5% dengan rumus (K;N). Adapun jumlah variabel independen adalah 3 atau “K” = 3, sementara jumlah sampel atau “N” = 65, maka (K;N) = (3;65). Angka ini kemudian kita lihat pada distribusi nilai tabel Durbin Watson. Maka ditemukan nilai dl sebesar 1,503 dan du sebesar 1,696. Vol 3. No. 2. Desember 2022 652 Vol 3. No. 2. Desember 2022 652 http://jurnal.umb.ac.id/index.php/JAKTA/index Vol 3. No. 2. Desember 2022 652 652 ISSN: 2723-1399 e-ISSN: 2723-1488 JURNAL AKUNTANSI KEUANGAN DAN TEKNOLOGI INFORMASI AKUNTANSI Available online at: http://jurnal.umb.ac.id/index.php/JAKTA DOI: https://doi.org/10.36085/jakta.v2i1 ISSN: 2723-1399 e-ISSN: 2723-1488 JURNAL AKUNTANSI KEUANGAN DAN TEKNOLOGI INFORMASI AKUNTANSI Available online at: http://jurnal.umb.ac.id/index.php/JAKTA DOI: https://doi.org/10.36085/jakta.v2i1 Nilai Durbin Watson sebesar 2,238 lebih besar dari batas atas (du) yakni 1,696 dan kurang dari (4-du) 4-1,696 = 2,304. Maka sebagian dasar pengambilan keputusan dalam uji Durbin Watson diatas, dapat disimpulkan bahwa tidak terdapat masalah atau gejala autokorelasi. Teknik Analisis Data Dengan demikian, maka analisis regresi linier berganda untuk uji hipotesis penelitian diatas dapat dilakukan atau dilanjutkan. Uji Multikolinearitas Nilai Durbin Watson sebesar 2,238 lebih besar dari batas atas (du) yakni 1,696 dan kurang dari (4-du) 4-1,696 = 2,304. Maka sebagian dasar pengambilan keputusan dalam uji Durbin Watson diatas, dapat disimpulkan bahwa tidak terdapat masalah atau gejala autokorelasi. Dengan demikian, maka analisis regresi linier berganda untuk uji hipotesis penelitian diatas dapat dilakukan atau dilanjutkan. Uji Multikolinearitas Tabel Uji Multikolineritas Coefficientsa Model Collinearity Statistics Tolerance VIF 1 (Constant) Ln_X1 .911 1.097 Ln_X2 .952 1.050 Ln_X3 .954 1.048 a. Dependent Variable: Ln_y Sumber: data sekunder yang diolah dengan SPSS Tabel Uji Multikolineritas Coefficientsa p y Sumber: data sekunder yang diolah dengan SPSS p y Sumber: data sekunder yang diolah dengan SPSS Berdasarkan besaran Tolerance dan Variance Inflaction Factor (VIF) batasan umum yang digunakan adalah Tolerance ˃ 0,10 dan nilai VIF ˂ 10, dari hasil output SPSS diatas besar Tolerance (Ln X1 = 0,911 , Ln X2 = 0,952 dan Ln X3= 0,954) ˃ 0,10 dan VIF (Ln X1 = 1,097 Ln X2 = 1,050 dan Ln X3 = 1,048) ˂ 10. Maka dapat disimpulkan bahwa antar variabel bebas tidak terjadi multikolinearitas. Uji Heterokedastisitas Uji Heterokedastisitas Gambar 4.1 Uji Heterokedastisitas Dari Scatterplot, terlihat pada titik-titik menyebar secara acak, tidak membentuk suatu pola tertentu yang jelas, serta tersebar baik diatas maupun dibawah. Titik-titik tidak mengumpul hanya diatas atau dibawah saja. Dengan demikian, dapat kita simpulkan bahwa tidak terjadi masalah heterokedastisitas, hingga model regresi yang baik dan ideal dapat terpenuhi. j Gambar 4.1 Uji Heterokedastisitas Gambar 4.1 Uji Heterokedastisitas Gambar 4.1 Uji Heterokedastisitas Gambar 4.1 Uji Heterokedastisitas Dari Scatterplot, terlihat pada titik-titik menyebar secara acak, tidak membentuk suatu pola tertentu yang jelas, serta tersebar baik diatas maupun dibawah. Titik-titik tidak mengumpul hanya diatas atau dibawah saja. Dengan demikian, dapat kita simpulkan bahwa tidak terjadi masalah heterokedastisitas, hingga model regresi yang baik dan ideal dapat terpenuhi. Dari Scatterplot, terlihat pada titik-titik menyebar secara acak, tidak membentuk suatu pola tertentu yang jelas, serta tersebar baik diatas maupun dibawah. Titik-titik tidak mengumpul hanya diatas atau dibawah saja. Dengan demikian, dapat kita simpulkan bahwa tidak terjadi masalah heterokedastisitas, hingga model regresi yang baik dan ideal dapat terpenuhi. http://jurnal.umb.ac.id/index.php/JAKTA/index Vol 3. No. 2. Teknik Analisis Data Desember 2022 653 Regresi Linier Berganda Tabel Regresi Linier Berganda Coefficientsa Regresi Linier Berganda Tabel Regresi Linier Berganda Coefficientsa Regresi Linier Berganda Tabel Regresi Linier Berganda Coefficientsa Tabel Regresi Linier Berganda Coefficientsa Coefficientsa Vol 3. No. 2. Desember 2022 Vol 3. No. 2. Desember 2022 http://jurnal.umb.ac.id/index.php/JAKTA/index 653 ISSN: 2723-1399 e-ISSN: 2723-1488 JURNAL AKUNTANSI KEUANGAN DAN TEKNOLOGI INFORMASI AKUNTANSI Available online at: http://jurnal.umb.ac.id/index.php/JAKTA DOI: https://doi.org/10.36085/jakta.v2i1 Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 (Constant) .703 .824 .852 .397 Ln_X1 .426 .223 .244 1.912 .041 Ln_X2 .024 .286 .123 .084 .031 Ln_X3 .385 .233 .109 .867 .390 a. Dependent Variable: Ln_y Sumber: data sekunder yang diolah dengan SPSS p y Sumber: data sekunder yang diolah dengan SPSS Berdasarkan tabel output SPSS diatas, diperoleh persamaan regresi sebagai berikut: Berdasarkan tabel output SPSS diatas, diperoleh persamaan regresi β β βn n Y = 0,703 + 0,426 + 0,024 + 0,385 Dari hasil persamaan regresi berganda tersebut, masing-masing variabel bebas dapat diinterprestasikan pengaruhnya terhadap perataan laba sebagai berikut: 1. Konstanta sebesar 0,703 menunjukkan bahwa meskipun tidak ada return on aset, ukuran perusahaan, dan leverage ratio tingkat perataan laba tetap ada sebesar 0,703. 1. Konstanta sebesar 0,703 menunjukkan bahwa meskipun tidak ada return on aset, ukuran perusahaan, dan leverage ratio tingkat perataan laba tetap ada sebesar 0,703. 2. Koefisien regresi return on aset sebesar 0,426 artinya setip pertambahan return on aset sebesar 1% jika variabel lainnya dianggap konstanta, maka akan menurukan tingkat perataan laba sebesar 0,426. 3. Koefisien ukuran perusahaan sebesar 0,024 artinya setiap pertambahan ukuran perusahaan sebesar 1% jika variabel lainnya dianggap konstanta, maka akan menurunkan tingkat perataan laba sebesar 0,024. 3. Koefisien ukuran perusahaan sebesar 0,024 artinya setiap pertambahan ukuran perusahaan sebesar 1% jika variabel lainnya dianggap konstanta, maka akan menurunkan tingkat perataan laba sebesar 0,024. 4. Koefisien leverage ratio sebesar 0,385 artinya setiap pertambahan leverage ratio sebesar 1% jika variabel lainnya dianggap konstanta, maka akan menaikkan perataan laba sebesar 0,385. 4. Koefisien leverage ratio sebesar 0,385 artinya setiap pertambahan leverage ratio sebesar 1% jika variabel lainnya dianggap konstanta, maka akan menaikkan perataan laba sebesar 0,385. j y Koefisien Determinasi (R²) Koefisien Determinasi (R²) Tabel Koefisien Determinasi Model Summaryb Model R R Square Adjusted R Square Std. Error of the Estimate 1 .292a .085 .040 1.36893 a. Predictors: (Constant), Ln_X3, Ln_X2, Ln_X1 b. Uji Simultan (Uji F) Uji Simultan (Uji F) p y Sumber: data sekunder yang diolah dengan SPSS Berdasarkan tabel output SPSS diatas, hasil uji F menunjukkan bahwa variabel return on aset, ukuran perusahaan dan leverage ratio mempunyai tingkat signifikansi lebih besar dari 0,05 (0,140 ˃ 0,05). Ini berarti bahwa Ha diterima dan Ho ditolak. Dengan demikian hasil analisis dalam penelitian ini menunjukkan bahwa variabel independen yaitu return on aset, ukuran perusahaan dan leverage ratio tidak terdapat pengaruh signifikansi secara simultan terhadap perataan laba pada perusahaan manufaktur subsektor makanan dan minuman yang terdaftar di bursa efek indonesia periode 2015- 2020. Teknik Analisis Data Dependent Variable: Ln_y Sumber: data sekunder yang diolah dengan SPSS Berdasarkan tabel output SPSS diatas, menunjukkan bahwa nilai signifikan Ln X1 (Return On Asset) terhadap Ln Y (perataan laba) adalah sebesar 0,041 ˂ 0,05 maka Ha ditolak dan Ho diterima, artinya terdapat pengaruh signifikansi antara Ln X1 (return on aset) terhadap Ln Y (perataan laba). Berdasarkan tabel output SPSS diatas, menunjukkan bahwa nilai signifikan Ln X2 (ukuran perusahaan) terhadap perataan laba adalah sebesar 0,031 ˂ 0,05 ini berari Ha ditolak dan Ho diterima, artinya terdapat pengaruh signifikansi antara Ln X2 (ukuran perusahaan) terhadap Ln Y (perataan aba). Berdasarkan tabel output SPSS diatas, menunjukkan bahwa nilai signifikan Ln X3 (leverage ratio) terhadap Ln Y (perataan laba) adalah sebesar 0,390 ˃ 0,05 ini berarti Ha diterima dan Ho ditolak, artinya tidak terdapat pengaruh signifikansi antara Ln X3 (leverage ratio) terhadap Ln Y perataan laba). p y Sumber: data sekunder yang diolah dengan SPSS Sumber: data sekunder yang diolah dengan SPSS Berdasarkan tabel output SPSS diatas, menunjukkan bahwa nilai signifikan Ln X1 (Return On Asset) terhadap Ln Y (perataan laba) adalah sebesar 0,041 ˂ 0,05 maka Ha ditolak dan Ho diterima, artinya terdapat pengaruh signifikansi antara Ln X1 (return on aset) terhadap Ln Y (perataan laba). y p p g g ( ) p (p ) Berdasarkan tabel output SPSS diatas, menunjukkan bahwa nilai signifikan Ln X2 (ukuran perusahaan) terhadap perataan laba adalah sebesar 0,031 ˂ 0,05 ini berari Ha ditolak dan Ho diterima, artinya terdapat pengaruh signifikansi antara Ln X2 (ukuran perusahaan) terhadap Ln Y (perataan laba). Berdasarkan tabel output SPSS diatas, menunjukkan bahwa nilai signifikan Ln X3 (leverage ratio) terhadap Ln Y (perataan laba) adalah sebesar 0,390 ˃ 0,05 ini berarti Ha diterima dan Ho ditolak, artinya tidak terdapat pengaruh signifikansi antara Ln X3 (leverage ratio) terhadap Ln Y (perataan laba). Uji Simultan (Uji F) Tabel Uji Simultan (Uji F) ANOVAb Model Sum of Squares df Mean Square F Sig. 1 Regression 10.634 3 3.545 1.892 .140a Residual 114.313 61 1.874 Total 124.947 64 a. Predictors: (Constant), Ln_X3, Ln_X2, Ln_X1 b. Dependent Variable: Ln_y Sumber: data sekunder yang diolah dengan SPSS KESIMPULAN Berdasarkan hasil penelitian maka dapat disimpulkan sebagai berikut: Teknik Analisis Data Dependent Variable: Ln_y Sumber: data sekunder yang diolah dengan SPSS Nilai R Square dari output SPSS diatas sebesar 0,017. Nilai R Square 0,085 ini berasal dari pengkuadratan nilai koefisien korelasi atau “R” yaitu 0,292 x 0,292 = 0,085. Besarnya angka koefisien determinasi (R Square) adalah 0,085 atau sama dengan 8,5%. Angka tersebut mengandung arti bahwa variabel independen yang terdiri dari ROA, ukuran perusahaan dan leverage ratio mampu menjelaskan variasi variabel dependen yaitu perataan laba sebesar 8,5%. Sedangkan sisanya (100%- 8,5% = 91,5%) dijelaskan oleh variabel lain yang tidak diteliti oleh peneliti lain. Uji Parsial (Uji t) Tabel Koefisien Determinasi Model Summaryb Model R R Square Adjusted R Square Std. Error of the Estimate 1 .292a .085 .040 1.36893 a. Predictors: (Constant), Ln_X3, Ln_X2, Ln_X1 b. Dependent Variable: Ln_y Sumber: data sekunder yang diolah dengan SPSS Tabel Koefisien Determinasi Model Summaryb Model R R Square Adjusted R Square Std. Error of the Estimate 1 .292a .085 .040 1.36893 a. Predictors: (Constant), Ln_X3, Ln_X2, Ln_X1 b. Dependent Variable: Ln_y Sumber: data sekunder yang diolah dengan SPSS Nilai R Square dari output SPSS diatas sebesar 0,017. Nilai R Square 0,085 ini berasal dari pengkuadratan nilai koefisien korelasi atau “R” yaitu 0,292 x 0,292 = 0,085. Besarnya angka koefisien determinasi (R Square) adalah 0,085 atau sama dengan 8,5%. Angka tersebut mengandung arti bahwa variabel independen yang terdiri dari ROA, ukuran perusahaan dan leverage ratio mampu menjelaskan variasi variabel dependen yaitu perataan laba sebesar 8,5%. Sedangkan sisanya (100%- 8,5% = 91,5%) dijelaskan oleh variabel lain yang tidak diteliti oleh peneliti lain. Uji P i l (Uji t) Uji Parsial (Uji t) 654 http://jurnal.umb.ac.id/index.php/JAKTA/index ISSN: 2723-1399 e-ISSN: 2723-1488 JURNAL AKUNTANSI KEUANGAN DAN TEKNOLOGI INFORMASI AKUNTANSI Available online at: http://jurnal.umb.ac.id/index.php/JAKTA DOI: https://doi.org/10.36085/jakta.v2i1 Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 (Constant) .703 .824 .852 .397 Ln_X1 .426 .223 .244 1.912 .041 Ln_X2 .024 .286 .123 .084 .031 Ln_X3 .385 .233 .109 .867 .390 a. Dependent Variable: Ln_y Sumber: data sekunder yang diolah dengan SPSS ISSN: 2723-1399 e-ISSN: 2723-1488 JURNAL AKUNTANSI KEUANGAN DAN TEKNOLOGI INFORMASI AKUNTANSI Available online at: http://jurnal.umb.ac.id/index.php/JAKTA DOI: https://doi.org/10.36085/jakta.v2i1 Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 (Constant) .703 .824 .852 .397 Ln_X1 .426 .223 .244 1.912 .041 Ln_X2 .024 .286 .123 .084 .031 Ln_X3 .385 .233 .109 .867 .390 a. URNAL AKUNTANSI KEUANGAN DAN TEKNOLOGI INFORMASI AKUNTA Available online at: http://jurnal.umb.ac.id/index.php/JAKTA DOI: https://doi.org/10.36085/jakta.v2i1 1. Hasil pengujian menunjukkan bahwa variabel Return On Asset (ROA) didapatkan nilai signifikan sebesar 0,041 ˃ 0,05, ini berarti bahwa Ha ditolak dan Ho diterima, maka terdapat pengaruh signifikan antara Return On Asset terhadap perataan laba. 1. Hasil pengujian menunjukkan bahwa variabel Return On Asset (ROA) didapatkan nilai signifikan sebesar 0,041 ˃ 0,05, ini berarti bahwa Ha ditolak dan Ho diterima, maka terdapat pengaruh signifikan antara Return On Asset terhadap perataan laba. p g g p p 2. Hasil pengujian menunjukkan bahwa variabel ukuran perusahaan didapatkan nilai signifikan sebesar 0,031 ˃ 0,05, ini berarti bahwa Ha ditolak dan Ho diterima, maka terdapat pengaruh signifikan antara ukuran perusahaan terhadap perataan laba. p g g p p 2. Hasil pengujian menunjukkan bahwa variabel ukuran perusahaan didapatkan nilai signifikan sebesar 0,031 ˃ 0,05, ini berarti bahwa Ha ditolak dan Ho diterima, maka terdapat pengaruh signifikan antara ukuran perusahaan terhadap perataan laba. g p p p 3. Hasil pengujian menunjukkan bahwa variabel Leverage Ratio didapatkan nilai signifikan sebesar 0,390 ˃ 0,05, ini berarti bahwa Ha diterima dan Ho ditolak, maka tidak terdapat pengaruh signifikan antara Leverage Ratio terhadap perataan laba. 3. Hasil pengujian menunjukkan bahwa variabel Leverage Ratio didapatkan nilai signifikan sebesar 0,390 ˃ 0,05, ini berarti bahwa Ha diterima dan Ho ditolak, maka tidak terdapat pengaruh signifikan antara Leverage Ratio terhadap perataan laba. 4. Hasil pengujian hipotesis secara simultan diperoleh bahwa nilai Fsig sebesar 0,140 ˃ 0,05, sehingga return on asset (roa), ukuran perusahaan dan leverage ratio secara simultan tidak terdapat pengaruh signifikan terhadap perataan laba. Berdasarkan hasil penelitian ini, maka saran yang dapat diberikan adalah sebagai berikut: Berdasarkan hasil penelitian ini, maka saran yang dapat diberikan adalah sebagai berikut: 1. Diharapkan perusahaan dapat menyajikan informasi keuangan dengan tidak me perataan laba. 1. Diharapkan perusahaan dapat menyajikan informasi keuangan dengan tidak me perataan laba. p Diharapkan investor lebih cermat dan teliti dalam mengambil keputusan sebelum berinvestasi. p 2. Diharapkan investor lebih cermat dan teliti dalam mengambil keputusan sebelum berinvestasi. 3. Diharapkan penilitian ini dapat menjadi referensi bagi peneliti selanjutnya dengan menambahkan variabel-variabel lain yang dapat mempengaruhi perataan laba. iharapkan penilitian ini dapat menjadi referensi bagi peneliti selanjutnya dengan menambahkan variabel-variabel lain yang dapat mempengaruhi perataan laba. 3. Diharapkan penilitian ini dapat menjadi referensi bagi peneliti selanjutnya dengan menambahkan variabel-variabel lain yang dapat mempengaruhi perataan laba. http://jurnal.umb.ac.id/index.php/JAKTA/index http://jurnal.umb.ac.id/index.php/JAKTA/index Vol 3. No. 2. Desember 2022 655 ISSN: 2723-1399 e-ISSN: 2723-1488 DAFTAR PUSTAKA gustianto, R. N. (2014). Analisis Faktor-Faktor Yang Berpengaruh Terhadap Perataan Laba. Amanza, A. H. (2012). Analisis Faktor-Faktor Yang Mempengaruhi Praktik Perataan Laba (Income Smoothing). Ardiansyah, G. (2020, November). GuruAkuntansi.co.id. Bestivano, W. (2013). Pengaruh Ukuran Perusahaan, Umur Perusahaan, Profitabilitas, dan Leverage Terhadap Perataan Laba pada Perusahaan yang Terdaftar di BEI. udi Susetyo, M. (2010). Statistika Untuk Analisis Data Penilitian. Bandung: PT Refika Aditama Darmawan, R. (2013). peran battra dalam penunukanngobatan tradisional pada komunitas dayak agabag di kecamatan lumbis kabupaten. e-jurnal sosiatri-sosiologi. Eko Budi Santoso, d. (2012). Pengaruh Profitabilitas, Financial Leverage, Dividen, Ukuran Perusahaan, Kepemilikan Institusional, dan Kelompok Usaha Terhadap Perataan Laba . Pengaruh Profitabilitas, Financial Laverage, 188. Ermitati. (2014). Pengungkapan budaya suku anak dalam melalui kosakata bahasa kubu. Kand Faisal. (2017). Analisi Faktor-Faktor Yang Mempengaruhi Praktik Perataan Laba (Income Smoothing) Pada Perusahaan Manufaktur Yang Terdaftar Di Bursa Efek Indonesia (BEI) Periode 2010-2014. Handayani, S. (2016). Analisis Faktor-Faktor Yang Berpengaruh Terhadap Perataan Laba. Jurnal Penelitian Ekonomi dan Akuntansi, 1(3), 226. Hans Kartikahadi, d. (2016). Akuntansi Keuangan Berdasarkan SAK Berbasis IFRS. Hery. (2015). Analisis Laporan Keuangan. Yogyakarta: CAPS (Center for Academic Publishing Service). Hugiono. (1992). Pengantar Ilmu Sejarah. jakarta: Rineka Cipta. Hugiono. (1992). Pengantar Ilmu Sejarah. jakarta: Rineka Cipta. Ina Setyaningtyas, B. H. (2014). Analisis Faktor-Faktor Yang Mempengaruhi Perataan Laba (Income Smoothing). Diponegoro Journal Of Accounting, 03(02), 1. g g j j p Ina Setyaningtyas, B. H. (2014). Analisis Faktor-Faktor Yang Mempengaruhi Perataan Laba (Income Smoothing). Diponegoro Journal Of Accounting, 03(02), 1. g p g f g Juarsih, D. d. (2014). kegiatan pembelajaran yang mendidik. jakarta: Rineka Cipta. g p j y g j p K, E. P. (2016). Olah Data Skripsi Dengan SPSS 22. Bangka Belitung: Lab Kom Manajemen FE UBB. Muhammad Rifky Santoso, W. (2014). Analisis Laporan Keuangan dan SPT. Nurdin, M. &. (2013). Kehidupan keagamaan suku anak dalam didusun senami Iii desa jebak kabupaten batang hari jambi. Kontekstualita, 152-153. Pohan, D. M. (2008). Pengaruh Laba. Vol 3. No. 2. Desember 2022 656 http://jurnal.umb.ac.id/index.php/JAKTA/index Vol 3. No. 2. Desember 2022 656 ISSN: 2723-1399 e-ISSN: 2723-1488 ISSN: 2723-1399 e-ISSN: 2723-1488 DOI: https://doi.org/10.36085/jakta.v2i1 Pratama, D. F. (2012). Profitabilitas, Resiko Keuangan, Nilai Perusahaan, Struktur Kepemilikan dan Dividend Payout Ratio Terhadap Perataan Laba. Jurnal Akuntansi & Investasi, 35. Putra, I. K. (2015). Pengaruh Profitabilitas, Leverage, Ukuran Perusahaan, Kepemilikan Publik, Dividend Payout Ratio dan Net Profit Margin pada Perataan Laba. Jurnal Akuntansi Universitas Udayana, 604. y H. A. (2008). Analisis Pengaruh Faktor Ukuran Perusahaan, Profitabilitas dan Leverage Terhadap Income Smoothing . Rahmawati, D., & Muid, D. (2012). Analisis Faktor-Faktor Yang Berpengaruh Terhadap Prakti Perataan Laba. Diponegoro Journal Of Accounting, 1(2), 1. Robert Libby, d. (2008). Akuntansi Keuangan. Yogyakarta : Andi. Robert Libby, d. (2008). Akuntansi Keuangan. Yogyakarta : Andi. (2008). Akuntansi Keuangan. Yogyakarta : And Saskara, I. K. (2016). seni pengobatan tradisional tradisional sebagai pariwisata alternstif di desa selat kecamatan abiansemal kabupaten bandung. e-jurnal. Sudarsi, L. K. (2012). Pengaruh Ukuran Perusahaan, Profitabilitas, Leverage, dan Kepemilikan Instutisional Terhadap Perataan Laba. Dinamika Akuntansi, Keuangan dan Perbankan, 1. S i (2011) t d liti k tit tif k lit tif d R&D B d Alf b t g y g sugiyono. (2015). metode penelitian pendidikan pendekatan kuantitatif, kualitatif, dan R&D. Jakarta: Alfabeta. Sugiyono. (2017). metode penelitian kuantitatif, kualitatif dan R&D. BANDUNG: Alfabeta. Sulistyanto, H. S. (2004). Manajemen Laba Teori dan Model Empiris. Semarang. Supriyanto. (2010). Iliran dan uluan dikotami dan dinamika dalam sejarah kultur palembang. Palembang: Eja Publiner. Triratnawati, A. (2010). pengobatan tradisional upaya meminimalkan biaya kesehatan masyarakat desa di jawa. e-jurnal, 69. Warsita, B. (2008). Teknologi Landasan Pembelajaran Dan Aplikasi. jakarta: Rineka Cipta. Winarni, E. W. (2018). Teori dan Praktik Penelitian Kuantitatif Kualitatif. Jakarta: Bumi Aksa Winarni, G. S. (2005). Manajemen Keuangan. Yogyakarta : Media Pressindo. Vol 3. No. 2. Desember 2022 657 http://jurnal.umb.ac.id/index.php/JAKTA/index http://jurnal.umb.ac.id/index.php/JAKTA/index http://jurnal.umb.ac.id/index.php/JAKTA/index
https://openalex.org/W2061285938	https://europepmc.org/articles/pmc3760854?pdf=render	English	null	Predicting Binding within Disordered Protein Regions to Structurally Characterised Peptide-Binding Domains	PloS one	2,013	cc-by	8,388	Abstract Disordered regions of proteins often bind to structured domains, mediating interactions within and between proteins. However, it is difficult to identify a priori the short disordered regions involved in binding. We set out to determine if docking such peptide regions to peptide binding domains would assist in these predictions.We assembled a redundancy reduced dataset of SLiM (Short Linear Motif) containing proteins from the ELM database. We selected 84 sequences which had an associated PDB structures showing the SLiM bound to a protein receptor, where the SLiM was found within a 50 residue region of the protein sequence which was predicted to be disordered. First, we investigated the Vina docking scores of overlapping tripeptides from the 50 residue SLiM containing disordered regions of the protein sequence to the corresponding PDB domain. We found only weak discrimination of docking scores between peptides involved in binding and adjacent non-binding peptides in this context (AUC 0.58).Next, we trained a bidirectional recurrent neural network (BRNN) using as input the protein sequence, predicted secondary structure, Vina docking score and predicted disorder score. The results were very promising (AUC 0.72) showing that multiple sources of information can be combined to produce results which are clearly superior to any single source.We conclude that the Vina docking score alone has only modest power to define the location of a peptide within a larger protein region known to contain it. However, combining this information with other knowledge (using machine learning methods) clearly improves the identification of peptide binding regions within a protein sequence. This approach combining docking with machine learning is primarily a predictor of binding to peptide-binding sites, and is not intended as a predictor of specificity of binding to particular receptors. Citation: Khan W, Duffy F, Pollastri G, Shields DC, Mooney C (2013) Predicting Binding within Disordered Protein Regions to Structurally Characterised Peptide- Binding Domains. PLoS ONE 8(9): e72838. doi:10.1371/journal.pone.0072838 Edit L k K U i it f Alb t C d Editor: Lukasz Kurgan, University of Alberta, Canada Received May 21, 2013; Accepted July 12, 2013; Published September 3, 2013 Received May 21, 2013; Accepted July 12, 2013; Published September 3, 2013 Copyright: 2013 Khan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Predicting Binding within Disordered Protein Regions to Structurally Characterised Peptide-Binding Domains 1 Complex and Adaptive Systems Laboratory, University College Dublin, Dublin, Ireland, 2 Hussain Ebrahim Jamal Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan, 3 Conway Institute of Biomolecular and Biomedical Science, University College Dublin, Dublin, Ireland, 4 School of Medicine and Medical Science, University College Dublin, Dublin, Ireland, 5 School of Computer Science and Informatics, University College Dublin, D bli I l d Abstract Funding: This work was supported by Science Foundation Ireland principal investigator grant [grant number 08/IN.1/B1864] to D.C. Shields. W. Khan gratefully acknowledges the award of a European Commission (EC) Erasmus Mundus Europe Asia (EMEA) Split-Doctoral Scholarship Scheme in University College Dublin (UCD), Ireland. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: denis.shields@ucd.ie Competing Interests: The authors have declared that no competing interests exist. * E-mail: denis.shields@ucd.ie * E-mail: denis.shields@ucd.ie September 2013 \| Volume 8 \| Issue 9 \| e72838 Citation: Khan W, Duffy F, Pollastri G, Shields DC, Mooney C (2013) Predicting Binding within Disordered Protein Regions to Structurally Characterised Peptide- Binding Domains. PLoS ONE 8(9): e72838. doi:10.1371/journal.pone.0072838 Results We evaluated if the Vina docking score could be used to identify peptide binding regions in protein sequences. Specifically, if we have the 3D structure of a peptide binding domain could we predict regions within an unstructured protein sequence that might interact with it? For each of our 84 ELM instances we selected a window of 50 residues around each ELM, where the ELM is in the centre of the region, unless it is found within the 25 residues closest to the N or C terminus, in which case the 50 N or C terminal residues are selected. We prepared sets of overlapping peptides of lengths three residues by sliding a window along these 84 regions of the protein sequence. We docked each tripeptide to the respective PDB receptors. The best Vina score for each peptide was normalised and the results displayed as ROC curves (Figure 1). The results (AUC of 0.58) show only very modest discrimination between peptides involved in binding and non-binding peptides. Given this poor performance we investigated if the docking scores could be used as input to a BRNN and if this would improve the predictive power. We used the same set of 50 residue sequence regions to train seven BRNN in ten-fold cross-validation using a similar approach to that used to train SLiMPred [30]. The predictors were trained using as input the 50 residue sequence, and either predicted secondary structure, predicted disorder or Vina score, or a combination of two, or all three of these features. We plotted ROC curves for the performance of the training set in ten-fold cross-validation for each of the seven predictors and the AUCs are shown in Table 1. The BRNN does in fact improve the predictive power, from an AUC of 0.58 when the Vina scores are assessed, to an AUC of 0.68 using only the Vina score and the sequence to train the BRNN. However, we found that the combination of all three features provided the highest AUC (0.72) (Figure 1), compared to using only secondary structure and disorder predictions without Vina (AUC 0.63). We refer to this method as PepBindPred (Peptide Binding-region Predictor). Computational docking is a technique that uses protein three- dimensional structural information to predict ligand binding poses. Predicting Binding in Disordered Protein Regions disordered interaction hubs [20,21] explaining the many func- tional roles for these regions. The small binding areas which SLiMs constitute result in weak binding [9] making them suitable for short-lived interactions [22]. However, regardless of their short length, these motifs bind their target protein with sufficient strength to establish a functional relationship [23]. molecules. Unconstrained peptides are flexible and tend to adopt several conformations by rotating within the given search space of the receptor site adding complexity to the docking protocol as the number of rotatable bonds increases. Despite this, peptide docking experiments have recently been used to successfully predict the interaction site of elastin-binding protein and an elastin peptide motif [36], confirming the potential of peptide docking. Several databases exist to capture instances of SLiMs, for example, Minimotif Miner [12] and the ELM server [14] and a variety of methods are available for novel SLiM discovery. Primarily, these methods which seek to identify over-represented, convergently evolved, motifs in protein sequences [24,25], however, the motif may be over-represented in a protein by chance [26]. To avoid identifying false positives many methods employ evolutionary information at the local and global level to filter potential SLiM instances [27]. Profile based methods may be able to improve on this but are unable to identify motifs that occur with low frequency or as single occurrences [28]. Recently, a number of de novo prediction methods that are not dependent on evolutionary information have been developed [29–31]. These methods use the physicochemical properties of the protein sequence and predicted structural features to predict protein binding regions, however, until now there has been no publicly available prediction method which exploits protein-peptide structures available in the Protein Data Bank (PDB) [32]. Here, we investigated if docking, with AutoDock Vina [37], can be used to identify protein-peptide interactions with the objective of evaluating if the docking score could be used to discriminate a peptide binding region from adjacent non-binding regions within a defined stretch of protein sequence. First, we generated a non- redundant dataset of protein receptor and SLiM containing peptide interacting structures from the ELM database [14]. We then performed high throughput docking of sets of overlapping peptides generated by moving a sliding window along a 50 residue region from the parent protein sequence which is centred around the SLiM containing peptide. Predicting Binding in Disordered Protein Regions We compared these results to those obtained by submitting the same sequences to SLiMPred [30], MoRFpred [29] and ANCHOR [31]. Finally, we trained a bidirectional recurrent neural network (BRNN) to predict the peptide binding region within a protein sequence, using as input the protein sequence, predicted secondary structure, predicted disorder and Vina docking score. At present, the estimated number of protein-peptide interac- tions in the proteome is not reflected in the number of protein- peptide structures available in the PDB. However, the rapid increase in protein structural data in the PDB does provide an excellent opportunity to investigate how this information might be exploited to predict potentially SLiM mediated protein-peptide interactions in a novel manner. As the number of PDB protein structures available is limited, methods that can provide useful information about protein-peptide interactions in the absence of structural information, for example in the case where one of the interactors is unstructured or disordered, are desirable. A possible solution is to use protein-peptide docking in order to infer interaction information, such as the likely binding regions, when a PDB structure is available for only one of the interacting pairs. Introduction comparatively small interface with an average size of 350 A˚ 2 [8]. An estimated 15–40% of all interactions in the cell are protein- peptide interactions [4,9]. These peptide regions are ideal for signalling transduction networks because they are specific, transient and have low-affinity (1–150 mM) [10]. Thousands of proteins expressed in cells carry out their specific intracellular and extracellular functions by interacting with each other (protein-protein interactions). These interactions have been acknowledged to play fundamental roles in almost every biological event. Significant biological processes such as protein signalling, trafficking and their synchronised degradation [1,2], DNA repairing, replication and gene expression [3,4] require interaction between protein-protein interfaces to perform their tasks. The extent of complexity, co-operativity and diversity for these interactions is enormous, and itself is coordinated by intricate regulatory networks that will ultimately determine the behaviour of biological systems. Many interactions are mediated between the two domains of globular proteins (domain-domain interactions) which tend to be stable (contact surfaces are flat) and require an average size of 1,500–3,000 A˚ 2 [5,6]. Others are intended for fast response to stimuli (domain-motif interactions (DMI)/domain- peptide interactions/peptide-mediated interactions) [7] that occur when a globular domain in one protein recognises a short linear peptide from its corresponding protein partner, creating a y ( ) [ ] Typically, the peptides involved in DMI or protein-peptide interactions are categorised by a simple sequence pattern, that is, a short linear motif (SLiM) [11], also referred to as linear motifs, minimotifs [12], pre-structured motifs (PreSMos) [13], Eukaryotic Linear Motifs (ELMs) [14] or molecular recognition features (MoRFs) [15]. These peptide regions can vary in their length from 3 to 12 amino acid, typical of SLiMs, through to much longer regions of up to 70 amino acids [16]. In general, SLiMs can be expressed as regular expressions, a consensus motif with specific conserved residues restricted to particular positions recognised by a binding domain, with a set of similar residues or even arbitrary ones at other locations [17]. Structurally, SLiMs are frequently found in disordered regions at protein termini or between domains [18] with the ability to adopt a variety of conformations [15,19]. SLiMs may also originate from loops within a structured domain, exposing them to potential binding partners including many of the 1 September 2013 \| Volume 8 \| Issue 9 \| e72838 PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e72838 Results ROC curves plotting the true positive rate of peptide binding residue identification against the false positive rate, with thresholds for a positive identification decreasing from 1 to 0, tested on the training set of 84 ELM containing examples (A) normalised Vina scores (B) ten-fold cross-validation PepBindPred predictions. doi:10.1371/journal.pone.0072838.g001 Table 1. AUC calculated from ROC curves for BRNN trained with seven different input options. BRNN Input AUC Sequence, secondary structure and disorder 0.63 Sequence and secondary structure 0.64 Sequence and disorder 0.64 Sequence and Vina 0.68 Sequence, secondary structure and Vina 0.71 Sequence, disorder and Vina 0.71 Sequence, secondary structure, disorder and Vina 0.72 Predictors trained with either secondary structure, disorder or Vina score along with the protein sequence, or a combination of two, or all three of these features. doi:10.1371/journal.pone.0072838.t001 Table 1. AUC calculated from ROC curves for BRNN trained with seven different input options. Predictors trained with either secondary structure, disorder or Vina score along with the protein sequence, or a combination of two, or all three of these features. d i /j l and Methods). We submitted these to PepBindPred, SLiMPred, MoRFpred and ANCHOR, and also compared the results to the Vina docking scores for overlapping tripeptides using the same method as described above. Again we see that the Vina docking scores do not discriminate well between binding and non-binding residues (AUC 0.53) (Figure 2 (A)). While the performances of ANCHOR is not strong (Figure 2 (B)), this is not surprising, since it is designed to identify larger scale regions involved in protein binding, rather than to identify specific residues within such binding regions that bind to peptide-binding domains. MoRFpred aims at shorter sequences (5–25 residues), which may explain why its performance is better (Figure 2 (C)), but less efficient than SLiMPred which targets shorter sequences (Figure 2 (D)). The results for these alternative methods are included as a reminder to use the appropriate computational tool when addressing a specific question, since PepBindPred substantially out performs AN- CHOR, MoRFpred, Vina and SLiMPred (AUC 0.75) (Figure 2 (E)). ( )) To test how specific the predictions for a binding region, or SLiM, are to a particular receptor we repeated the predictions for the 21 sequence in the independent test set but we ‘‘shuffled’’ the receptors. Results That is, we submitted each sequence to the Pep- BindPred server, however we selected a receptor other than the native receptor, making sure that the new receptor was not in the same class as the native receptor (i.e. care was taken not to submit a SH3 ligand containing sequence to another SH3 domain). The results were almost identical (AUC 0.75). Although the lack of receptor specificity is surprising in some respect, it is also important to remember that it is the nature of many SLiMs to bind with moderate rather than high affinity [38], and that all the receptors here are peptide binding domains, and therefore have some inherent similarity. In the light of this, we conclude that, PepBindPred is a peptide binding region predictor, not a receptor specificity predictor, and users must remember this when interpreting results. The docking information from Vina is providing an additional layer of information that is not available from the primary sequence alone, regarding the propensity of regions to bind to peptide-binding domains. The precise nature of this additional structural information is not clear, and may in future become clearer as the training sets become larger and more informative. Figure 1. ROC curves – Training set. ROC curves plotting the true positive rate of peptide binding residue identification against the false positive rate, with thresholds for a positive identification decreasing from 1 to 0, tested on the training set of 84 ELM containing examples (A) normalised Vina scores (B) ten-fold cross-validation PepBindPred predictions. d i 10 1371/j l 0072838 001 human proteome. 67 instances of the motif (L[ˆP]2,2[HI]I[ˆ- P]2,2[IAV][IL]) were found in 65 Proteins. The ELM server identified two of these as true positives (O75376 and Q9Y618), both of which were ranked highly by SLiMSearch, using conservation analysis. We investigated if PepBindPred would be able to suggest some other potential true positive candidates in this set of CORNR box motif instances. The PepBindPred server takes as input the UniProt [40] identifier of the sequence, the residue to start the search at (we set this to 25 residues before the start of the motif instance), the PDBID and the chain ID (for this example 3N00 and B respectively). We averaged the PepBindPred predictions over the nine motif residues for the 67 instances and have listed them in order in Table 2. Results The score distribution is skewed, with two of the true positives in the tail (Figure 3), which suggests that some of the other top ranked motif instances could be worth further investigation. The O75376 true positive instance is ranked highest, however this is not surprising as the sequence is in our training set. The second highest ranked sequence, P05160, we Results There are two main kinds of docking: protein-ligand docking, where a flexible small-molecule compound is ‘‘docked’’ to a known or suspected protein-binding pocket; and macromolecular dock- ing, where two rigid protein (or other biological macromolecule, such as DNA) structures are docked, with the goal of identifying protein-protein interaction interfaces. Both protein-ligand and macromolecular docking have been used successfully to predict binding poses. Protein-ligand docking is commonly used as an early step in small-molecule drug screening campaigns, where it has been shown to have the ability to accurately retrieve the known binding poses of diverse sets of small molecule-protein complexes, although this does depend somewhat on the protein family, and the characteristics of the binding site [33]. Recently, two high-throughput macromolecular docking experiments have been reported that demonstrated the use of a general docking method to detect interacting partners. Mosca et al [34] used docking to identify pairs of proteins that accurately interact with each other in the Saccharomyces cerevisiae interactome by sampling from a large set of alternative possibilities. Wass et al [35] successfully distinguished between interacting (native) and non- interacting (non-native) protein partners. A disadvantage of macromolecular docking is the requirement for structural data for both proteins. In general, docking programs are not optimised for peptide docking and are most successfully used with small In order to bench mark PepBindPred we generated an independent test set of 21 disordered sequence regions which are shown to interact with protein receptors in the PDB (see Materials September 2013 \| Volume 8 \| Issue 9 \| e72838 PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e72838 2 Predicting Binding in Disordered Protein Regions human proteome. 67 instances of the motif (L[ˆP]2,2[HI]I[ˆ- P]2 2[IAV][IL]) were found in 65 Proteins The ELM server Figure 1. ROC curves – Training set. ROC curves plotting the true positive rate of peptide binding residue identification against the false positive rate, with thresholds for a positive identification decreasing from 1 to 0, tested on the training set of 84 ELM containing examples (A) normalised Vina scores (B) ten-fold cross-validation PepBindPred predictions. doi:10.1371/journal.pone.0072838.g001 Methods). We submitted these to PepBindPred, SLiMPred, RFpred and ANCHOR, and also compared the results to the a docking scores for overlapping tripeptides using the same hod as described above. Results Again we see that the Vina docking es do not discriminate well between binding and non-binding dues (AUC 0.53) (Figure 2 (A)). While the performances of CHOR is not strong (Figure 2 (B)), this is not surprising, since it esigned to identify larger scale regions involved in protein ding, rather than to identify specific residues within such ding regions that bind to peptide-binding domains. MoRFpred s at shorter sequences (5–25 residues), which may explain why performance is better (Figure 2 (C)), but less efficient than MPred which targets shorter sequences (Figure 2 (D)). The lts for these alternative methods are included as a reminder to the appropriate computational tool when addressing a specific tion, since PepBindPred substantially out performs AN- OR, MoRFpred, Vina and SLiMPred (AUC 0.75) (Figure 2 o test how specific the predictions for a binding region, or M, are to a particular receptor we repeated the predictions for 21 sequence in the independent test set but we ‘‘shuffled’’ the ptors. That is, we submitted each sequence to the Pep- dPred server, however we selected a receptor other than the ve receptor, making sure that the new receptor was not in the e class as the native receptor (i.e. care was taken not to submit H3 ligand containing sequence to another SH3 domain). The lts were almost identical (AUC 0.75). Although the lack of ptor specificity is surprising in some respect, it is also ortant to remember that it is the nature of many SLiMs to d ith d t th th hi h ffi it [38] d th t ll th human proteome. 67 instances of the motif (L[ˆP]2,2[HI]I[ˆ- P]2,2[IAV][IL]) were found in 65 Proteins. The ELM server id ifi d f h i i (O d Q ) ble 1. AUC calculated from ROC curves for BRNN trained th seven different input options. NN Input AUC quence, secondary structure and disorder 0.63 quence and secondary structure 0.64 quence and disorder 0.64 quence and Vina 0.68 quence, secondary structure and Vina 0.71 quence, disorder and Vina 0.71 quence, secondary structure, disorder and Vina 0.72 dictors trained with either secondary structure, disorder or Vina score along h the protein sequence, or a combination of two, or all three of these tures. :10.1371/journal.pone.0072838.t001 Figure 1. ROC curves – Training set. PepBindPred Web Server As an example of the possible use of PepBindPred we used SLiMSearch [39] to identify CORNR box motif instances in the PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e72838 3 Predicting Binding in Disordered Protein Regions Figure 2. ROC curves – independent test set. ROC curves plotting the true positive rate of peptide binding residue identification against the false positive rate, with thresholds for a positive identification decreasing from 1 to 0, tested on the independent test set of 21 examples (A) Vina (B) ANCHOR (C) MoRFpred (D) SLiMPred (E) PepBindPred. doi:10 1371/journal pone 0072838 g002 doi:10.1371/journal.pone.0072838.g002 think is a false positive as the motif is found in a signal peptide. However the third ranked sequence we believe may be worth further investigation as a possible true positive (Figure 4). This protein, Melanoma-associated antigen C2 (Q9UBF1 (MAG- C2_HUMAN)), has a subcellular location of ‘‘Cytoplasm. Nucleus.’’ which is compatible with the function of the CORNR box motif binding to nuclear receptors. The ROC curve for the independent test set results (Figure 2 (E)) suggests that 0.4 is a good threshold to choose as a cut-off as the false positive rate is still low (approximately 3%), while allowing for the capture of more true positives (approximately 20%, compared to ,10% at a threshold of 0.5). PLOS ONE \| www.plosone.org There are many other biological application of this method beyond the example given. PepBindPred is computationally more intensive than ANCHOR, MoRFpred or SLiMPred, so we would suggest using one, or all, of these methods on your protein, or proteins, of interest first to predict peptide binding regions. PepBindPred can then be used to refine the regions identified by these methods, as it has been shown to be more accurate than any of these methods individually. PepBindPred is especially useful where some experimental information is available which would lead the user to believe that the sequence of interest binds to, or interacts with, a particular structured protein domain for which there is a PDB structure available. September 2013 \| Volume 8 \| Issue 9 \| e72838 Discussion Disordered regions of proteins often bind to structured domains, mediating interactions within and between proteins. We have presented a computational analysis of the performance of peptide docking with AutoDock Vina to assess if the Vina docking scores could be used to predict protein-peptide interactions. As Vina is designed for small-molecule docking with a restriction on the number of rotatable bonds (#32), it is generally assumed that it is not suitable for docking peptides which have many more internal degrees of freedom. Previously, however, we have shown that there is a correlation between the Vina docking scores of dipeptides with ACE and experimentally determined ACE inhibition (IC50) [41]. Extensive preliminary work [42] which attempted to dock the full peptide sequence to the peptide binding domain showed that there is a direct correlation between the peptide length and the number of rotatable bonds in the peptide. As the number of rotatable bonds increase so does the RMSD between the docked peptide pose and the native peptide pose. Following this analysis we attempted to dock much shorter peptide fragments (2–5 residues in length) to the peptide binding domain and determined that tripeptides gave the best results [42]. Here, we investigated the binding of known SLiM-containing peptides from within disordered protein regions to peptide binding domains and investigated the docking of adjacent overlapping peptides from the peptide’s protein sequence. We found that we were unable to discriminate between binding and non-binding peptide regions based on the Vina docking scores alone in this context. We then trained a BRNN using the peptide sequence, predicted secondary structure, predicted disorder and Vina score as inputs. Our analysis shows that when the Vina docking score was used as an input to our BRNN, in combination with predicted PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e72838 4 Predicting Binding in Disordered Protein Regions Table 2. PepBindPred predictions, averaged over the nine motif residues, and evolutionary conservation p-value for the motif instance. Table 2. PepBindPred predictions, averaged over the nine motif residues, and evolutionary conservation p-value for the motif instance. September 2013 \| Volume 8 \| Issue 9 \| e72838 Discussion doi:10.1371/journal.pone.0072838.t002 PepBindPred predictions, averaged over the nine motif residues, and evolutionary conservation p-value for the motif instance, calculated using SLiMSearch [39], for the 67 CORNR box motif instances in the human proteome. Scores closer to 1 indicate that PepBindPred is more confident that regions is peptide binding, whereas SLiMSearch p-values closer to 0 indicate that the motif is more likely to be a true positive due to conservation. Two of the sequences have two instances of the motif, O75376 and Q9Y618. True positives identified on the ELM server. doi:10.1371/journal.pone.0072838.t002 PepBindPred predictions, averaged over the nine motif residues, and evolutionary conservation p-value for the motif instance, calculated using SLiMSearch [39], for the 67 CORNR box motif instances in the human proteome. Scores closer to 1 indicate that PepBindPred is more confident that regions is peptide binding, whereas SLiMSearch p-values closer to 0 indicate that the motif is more likely to be a true positive due to conservation. Two of the sequences have two instances of the motif, O75376 and Q9Y618. True positives identified on the ELM server. doi:10.1371/journal.pone.0072838.t002 secondary structure and disorder, the AUC increased from 0.63 to 0.72, compared to using only predicted secondary structure and disorder. We have shown that this method, PepBindPred, performs better on an independent test set than three other publicly available ab initio methods, ANCHOR, MoRFpred and SLiMPred. This approach emphasises the potential for including structural information when developing methods for refining the location of peptide binding residues within disordered protein regions. Figure 3. Histogram showing the distribution of PepBindPred scores. The scores have been averaged over the 9 CORNR box motif residues, for each of the 67 instances. doi:10.1371/journal.pone.0072838.g003 In this study we have evaluated only one docking method, AutoDock Vina. The Vina docking program has many advantag- es, it is easy to install and run locally, it is extremely fast and is very suitable for high-throughput docking which is essential for implementation as a web server of this kind. It is possible, however, that other methods, for example, DynaDock [43], which has been developed more specifically for the docking of peptides into flexible binding sites, may provide better results in individual cases. Unfortunately, this method is slower than Vina, so it is not suitable for high-throughput docking in this context. Discussion UniProt AC Motif PepBindPred Score Evolutionary conservation p-value O75376* LADHICQII 0.715 0.048 P05160 LTFIIILII 0.680 0.969 Q9Y618* LEAIIRKAL 0.557 0.026 Q9UBF1 LLIIILSVI 0.543 1 Q96BZ9 LIDIILLIL 0.520 0.208 Q8NI22 LINIIDGVL 0.486 0.214 Q07325 LLGIILLVL 0.467 0.396 Q5SVZ6 LKLIIENIL 0.434 0.329 Q96AH8 LKLIIVGAI 0.425 0.75 Q9HAU8 LTFIISSIL 0.401 0.436 Q9NRU3 LEDIIEEII 0.384 0.393 P53618 LMTIIRFVL 0.363 0.321 O75376* LEDIIRKAL 0.362 0.044 Q8IWF6 LRTHIDAII 0.350 0.197 Q8NHV5 LFFIIMGII 0.341 1 Q9UPM8 LRLHIIEII 0.338 0.3 Q9Y618* LAQHISEVI 0.335 0.055 Q96N64 LDHIIEDAL 0.333 0.562 O95477 LSRIIWKAL 0.332 0.614 O00273 LASHILTAL 0.327 0.546 Q7Z3J2 LQLIIKKVI 0.325 0.096 Q09161 LNYHIVEVI 0.306 0.799 Q08AE8 LGIIIYKAL 0.294 0.164 Q8TDJ6 LNNHIHDIL 0.287 0.115 P07384 LYQIILKAL 0.283 0.518 Q5MIZ7 LYEIIRGIL 0.282 0.295 Q8TDR0 LHDIITEVI 0.282 0.43 Q96PN6 LKNIITVVI 0.276 0.645 Q8TCG5 LGQHIEDAL 0.274 0.294 Q6ZMV5 LYEIIKGIL 0.272 0.321 Q8IX04 LQYIITNVL 0.267 0.539 Q93100 LVIHIGWII 0.267 0.81 Q8TDL5 LKNIITEII 0.267 0.617 Q9C093 LVDIIVNAI 0.266 0.086 Q7RTX7 LARIIRVIL 0.262 0.345 Q9UIA9 LVYIIGAVI 0.257 0.104 Q8NEG5 LCKHICWVL 0.257 0.114 Q9Y6X3 LLGHIFYVL 0.232 0.885 Q8IZQ1 LAQIILDAI 0.221 0.708 P35556 LNNHIRYVI 0.216 0.145 Q14185 LLSHILEVL 0.209 0.067 O95801 LKAIIRGAL 0.199 0.732 A6NHC0 LYQIIRKAL 0.193 0.817 P56192 LGNIIGCVL 0.189 0.542 A6NES4 LTSIIVAVI 0.184 1 O95714 LCTHIGDIL 0.183 0.034 Q8NF50 LVGIILDAL 0.182 0.629 O95450 LGAHINVVL 0.174 0.364 September 2013 \| Volume 8 \| Issue 9 \| e72838 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 Predicting Binding in Disordered Protein Regions Table 2. Cont. UniProt AC Motif PepBindPred Score Evolutionary conservation p-value Q8N485 LRHIIAQVL 0.174 0.898 Q8TCG1 LKMHIAKIL 0.173 0.55 Q6R327 LDHIIQKAI 0.162 0.067 Q5T215 LCGIIRGAL 0.160 0.131 Q8WZ26 LSTHICVVL 0.159 1 P51124 LTFHIKAAI 0.158 0.658 Q99698 LNSIIDQAL 0.156 0.672 Q562E7 LSDITYYVY 0.156 0.94 Q9UJ70 LGRHIVAVL 0.153 0.136 Q0VDD8 LDKHIKSAI 0.152 1 Q8N1T3 LFGIIASVL 0.151 0.326 P17655 LFKIIQKAL 0.148 0.804 P52743 LHVIIDFIL 0.147 1 Q6PGP7 LEDIIGFAL 0.146 0.128 Q13572 LLNHIATVL 0.134 0.551 O15072 LGVHINVVL 0.128 0.362 Q8WXS8 LGVHINIAL 0.110 0.585 Q9UG01 LVEHITAAL 0.107 0.082 P30307 LGGHIQGAL 0.061 0.072 PepBindPred predictions, averaged over the nine motif residues, and evolutionary conservation p-value for the motif instance, calculated using SLiMSearch [39], for the 67 CORNR box motif instances in the human proteome. Scores closer to 1 indicate that PepBindPred is more confident that regions is peptide binding, whereas SLiMSearch p-values closer to 0 indicate that the motif is more likely to be a true positive due to conservation. Two of the sequences have two instances of the motif, O75376 and Q9Y618. *True positives identified on the ELM server. September 2013 \| Volume 8 \| Issue 9 \| e72838 Discussion Another method, the Rosetta FlexPepDock server [44], refines docking conformations against a given PDB file and an estimated peptide conformation, however, FlexPepDock is computationally intensive as the protocol samples a significant conformational space and therefore, similar to DynaDock, is also not suitable for our use. We are not entirely sure why the Vina docking score is unable to discriminate between binding and non-binding residues. The Vina scoring function (see [37] for details) uses a weighted sum of interactions to predict a binding pose. Values for the weights were determined by fitting the scoring function to the PDBBind refined dataset. It is possible that the dataset used to fit the scoring function is biased towards small molecules rather than peptides. This could possibly explain why the binding site cannot be predicted using only the Vina docking score. Furthermore, although there is no fixed limit, it has been shown that Vina can Figure 3. Histogram showing the distribution of PepBindPred scores. The scores have been averaged over the 9 CORNR box motif residues, for each of the 67 instances. doi:10.1371/journal.pone.0072838.g003 PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e72838 September 2013 \| Volume 8 \| Issue 9 \| e72838 6 Predicting Binding in Disordered Protein Regions Predicting Binding in Disordered Protein Regions Figure 4. PepBindPred output for Q9UBF1 (MAGC2_HUMAN). Melanoma-associated antigen C2; Motif: LLIIILSVI, residues 229–237. doi:10.1371/journal.pone.0072838.g004 Figure 4. PepBindPred output for Q9UBF1 (MAGC2_HUMAN). Melanoma-associated antigen C2; Motif: LLIIILSVI, residues 229–237. doi:10.1371/journal.pone.0072838.g004 d output for Q9UBF1 (MAGC2_HUMAN). Melanoma-associated antigen C2; Motif: LLIIILSVI, residues 229–237. 0072838.g004 All possible peptides were generated by scanning a sliding windows of three residues along the 50 residue section of the 84 ELM containing protein sequence. Peptides were converted into the SMILES format using CycloPs [48], and from SMILES to PDB format using Open Babel [49]. AutoDock 4.2 [47] was used to prepare the peptides as ligand files for Vina. handle ligands with up to 32 rotatable bonds [37], and tripeptides would be on the high end of this scale. Finally, even with small peptides, in this case tripeptides, the search space is large, and difficult to search, although we never exceed the maximum search space recommended (i.e 30\|30\|30A˚ ). Discussion p ( ) Docking of tripeptides was chosen by us as a compromise between computational efficiency and potential informativeness as tripeptides appeared to perform slightly better than other longer peptide fragments in preliminary experiments [42]. Using longer peptides would likely require alternative strategies starting with many more initial configurations of the peptides, in order to adequately sample the conformational space prior to seeking to identify minima. This would substantially increase the computa- tional burden of docking peptides along the sequence. It is also possible that alternative docking methods or scoring functions may contribute to improved performance, unfortunately this is beyond the scope of this work at present, however, they will be of interest for future work. While it is possible that the initial docking may be improved over that used, we consider it likely that the kind of machine learning step we introduced here is still likely to improve on the docking in discriminating between binding and non- binding adjacent regions along a protein sequence. An independent test set was generated in a similar manner using the sequences in the SLiMPred ‘‘SteinAloy’’ independent test set, which was derived from analysis of peptide-mediated interactions within PDB structure by Stein and Aloy [50], see [30] for more details. The original dataset was redundancy reduced to less that 30% sequence similarity to our training set leaving 46 sequences with a peptide region which is known to interact with a PDB structure, 21 of these are in regions which are predicted to be disordered. Anchor [31], MoRFpred [26] and SLiMPred [30] were used to predict protein binding regions, and Distill [51] was used to predict secondary structure, for the full protein sequence. The predictions in the 50 residue window around the ELM were then extracted. Methods where Vmax is the absolute value of the minimum Vina binding score (i.e. {10) and Vmin is zero. We measure the sensitivity or true positive rate (TPR) and specificity or false positive rate (FPR) as we increase the discrimination threshold from 0 to 1. We plot the TPR against the FPR as Receiver Operating Characteristic (ROC) curves, which are calculated as follows: Analysis of Docking Results PepBindPred is available as part of our web server for SLiM discovery and annotation. The user submits the protein sequence that they wish to predict peptide binding regions within, and the PDB ID of the protein structure they wish to predict interactions with. At present the PDB structure is required to have a bound peptide, and the user must provide the chain ID of the bound peptide so that the search space for Vina can be defined around it. Predictions take approximately one hour per protein. Our server is freely available for academic users at http://bioware.ucd.ie/ pepbindpred. The output generated by Vina for each peptide was processed and the binding conformation having the lowest binding affinity (i.e the top ranked Vina docking score) was selected for further investigation. To evaluate the ability of Vina to discriminate between binding peptides and adjacent non-binding peptides extracted from the same protein sequence, we measure the TPR and FPR. First, we normalise the Vina docking scores for each peptide so they fall between 0 and 1: Normalised (vi)~ vi{Vmin Vmax{Vmin ð1Þ ð1Þ September 2013 \| Volume 8 \| Issue 9 \| e72838 Datasets 1,358 true positive LIG (ligand binding) ELM instances were downloaded from the ELM server [14]. 208 of these were annotated as having a structure in the PDB. Using the FASTA sequence of these 208 ELM containing sequences we predicted the disorder using IUPred [45]. We retained 135 sequence which had an average IUPred score of .0.5 (i.e. disordered) over a 50 residue window around the ELM. We internally reduced this set to make it non-redundant using BLAST [46] with an e-value of 0.001, to less than 30% sequence similarity between any two sequences, leaving 122 sequences. In some cases the exact ELM motif was not found in the bound peptides of the PDB structures and these structures were removed. The PDB chain ID of the 84 matches that were found were noted and used to define the centre of search space for Vina, which was then extended to cover the bound peptide [37]. The peptide chain was then removed from the PDB structure in order to allow for docking to this site. AutoDock 4.2 [47] was then used to prepare the PDB files as ‘‘receptors’’ for Vina. TPR~ TP TPzFN FPR~ FP FPzTN ð2Þ TPR~ TP TPzFN FPR~ FP FPzTN ð2Þ TPR~ TP TPzFN FPR~ FP FPzTN ð2Þ ð2Þ R [52] was used to plot the curves, and calculate the AUC. The AUC is a number between 0 and 1 inclusive, where 0.5 indicates a random model and 1 is perfect, which is equivalent to the PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e72838 7 Predicting Binding in Disordered Protein Regions Predicting Binding in Disordered Protein Regions probability that a randomly chosen positive instance will rank higher than a randomly chosen negative instance [53]. i(T) j ~(i(T) j,1 ,i(T) j,t ) ð6Þ ð6Þ Implementation of BRNN BRNN have recently been used successfully for the prediction of peptide binding regions using the protein sequence, predicted secondary structure, structural motifs, solvent accessibility and disorder as inputs [30]. We use a similar model here using the protein sequence, predicted secondary structure, predicted disor- der and Vina score as input. See [54] for a detailed explanation of the BRNN model. Hence ij contains a total of ezt components. We use e~21: the 20 standard amino acids are considered, while the 21st input encodes the length of the sequence. We use t~5 for representing structural information. The first three structural input units contain the predicted three-class secondary structure representing the predicted probability of the j-th residue belonging to either helix, strand or coil and the final two inputs are the predicted disorder and Vina score. Hence the total number of inputs for a given residue is ezt~26. These networks take the form: These networks take the form: oj~N (O) ij,h(F) j ,h(B) j Acknowledgments The authors acknowledge the Research IT Service at University College Dublin and the SFI/HEA Irish Centre for High-End Computing (ICHEC) for providing high performance computing (HPC) resources that have contributed to the research results reported within this paper. The authors thank Kevin Rue for technical assistance. W. Khan gratefully acknowl- edges the award of a European Commission (EC) Erasmus Mundus Europe Asia (EMEA) Split-Doctoral Scholarship Scheme in University College Dublin (UCD), Ireland. Input ij associated with the j-th residue contains primary sequence information and predicted structural information (sec- ondary structure, disorder and Vina score): ij~(i(E) j ,i(T) j ) ð4Þ ð4Þ Training, Ensembling Training is conducted by ten-fold cross-validation, i.e. ten different sets of training runs are performed in which a different tenth of the overall set is reserved for testing. The ten fifths are roughly equally sized, disjoint, and their union covers the whole set. The training set is used to learn the free parameters of the network by gradient descent. Three models are trained indepen- dently for each fold and ensemble averaged to build the final predictor. Differences among models are introduced by varying the size of the input window considered by network from 7 to 9 to 11 residues. 10,000 epochs of training are performed for each model and the learning rate is halved every time we do not observe a reduction of the error for more than 50 epochs. h(F) j ~N (F) ij,h(F) j{1 h(B) j ~N (B) ij,h(B) jz1 j~1, . . . ,N ð3Þ ð3Þ where ij (respectively oj) is the input (respectively output) of the network in position j, and h(F) j and h(B) j are forward and backward chains of hidden vectors with h(F) 0 ~h(B) Nz1~0. We parametrise the output update, forward update and backward update functions (respectively N (O), N (F) and N (B)) using three two-layered feed- forward neural networks. References 1. Castro A, Bernis C, Vigneron S, Labbe´ J, Lorca T (2005) The anaphase- promoting complex: a key factor in the regulation of cell cycle. Oncogene 24: 314–325. 1. 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September 2013 \| Volume 8 \| Issue 9 \| e72838 PLOS ONE \| www.plosone.org 8 Predicting Binding in Disordered Protein Regions Stein A, Aloy P (2010) Novel peptide-mediated interactions derived from high- resolution 3-dimensional structures. PLoS Comput Biol 6: e1000789. 32. Berman H, Westbrook J, Feng Z, Gilliland G, Bhat T, et al. (2000) The protein data bank. Nucleic Acids Res 28: 235–242. 51. Bau´ D, Martin AJ, Mooney C, Vullo A, Walsh I, et al. (2006) Distill: a suite of web servers for the prediction of one-, two-and three-dimensional structural features of proteins. BMC Bioinformatics 7: 402. 33. Cross JB, Thompson DC, Rai BK, Baber JC, Fan KY, et al. (2009) Comparison of several molecular docking programs: Pose prediction and virtual screening accuracy. J Chem Inf Model 49: 1455–1474. 52. R Development Core Team (2008) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org. ISBN 3-900051-07-0. 34. Mosca R, Pons C, Ferna´ndez-Recio J, Aloy P (2009) Pushing structural information into the yeast interactome by high-throughput protein docking experiments. PLoS Comput Biol 5: e1000490. 53. Fawcett T (2006) An introduction to ROC analysis. Pattern Recogn Lett 27: 861–874. 35. Wass M, Fuentes G, Pons C, Pazos F, Valencia A (2011) Towards the prediction of protein interaction partners using physical docking. Mol Syst Biol 7: 469. 54. Baldi P, Brunak S, Frasconi P, Soda G, Pollastri G (1999) Exploiting the past and the future in protein secondary structure prediction. Bioinformatics 15: 937. September 2013 \| Volume 8 \| Issue 9 \| e72838 September 2013 \| Volume 8 \| Issue 9 \| e72838 PLOS ONE \| www.plosone.org 9
https://openalex.org/W2970461618	https://www.frontiersin.org/articles/10.3389/fonc.2019.00902/pdf	English	null	Impact of Metformin on Systemic Metabolism and Survival of Patients With Advanced Pancreatic Neuroendocrine Tumors	Frontiers in oncology	2,019	cc-by	3,736	Reviewed by: Reviewed by: Federica Recine, Romagnolo Scientiﬁc Institute for the Study and Treatment of Tumors (IRCCS), Italy Shilpa Thakur, National Institutes of Health (NIH), United States p p g In recent years, the following clinical achievements dramatically expanded the therapeutic armamentarium against advanced pNETs: (a) somatostatin analogs (SAs) signiﬁcantly prolonged patient progression free survival (PFS) when compared to the placebo in patients with advanced disease (3); (b) the mTOR inhibitor everolimus and the multi-tyrosine kinase inhibitor sunitinib demonstrated anticancer activity and prolonged median PFS in pNET patients pre-treated with SAs (4, 5); (c) peptide receptor radiotherapy (PRRT) showed impressive anticancer activity coupled with excellent tolerability proﬁles in patients with pre-treated advanced pNETs. Finally, cytotoxic chemotherapy or liver-directed treatments remain a valid option for patients with high-grade pNETs, as well as for neoplasms progressing on biological agents. Despite these improvements, most advanced pNETs remain almost invariably incurable, and many patients ﬁnally die of their disease. Indeed, while 5-year survival is 65–94% in patients with limited-stage disease, it is reduced to 44–76% in the presence of lymph node metastases, and to 27% in the case of distant metastases (6, 7). In the advanced disease setting, factors associated with poorer survival include the presence of liver and peritoneal metastases, not having undergone resection the primary (pancreatic) tumor, high-grade (G3) disease. Therefore, new treatment options are needed for patients with advanced pNETs, in particular those with poor prognostic factors. Correspondence: Claudio Vernieri claudio.vernieri@istitutotumori.mi.it Sara Pusceddu sara.pusceddu@istitutotumori.mi.it Correspondence: Claudio Vernieri claudio.vernieri@istitutotumori.mi.it Sara Pusceddu sara.pusceddu@istitutotumori.mi.it Specialty section: This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the journal Frontiers in Oncology Specialty section: This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the journal Frontiers in Oncology Received: 29 July 2019 Accepted: 30 August 2019 Published: 20 September 2019 In recent years, preclinical and retrospective clinical data have shown that the antidiabetic compound metformin may have antitumor activity against diﬀerent tumor types including pNETs. Potential mechanisms of metformin anticancer eﬀects include: (1) modiﬁcations of systemic metabolism, including a reduction of blood glucose and insulin, which sustain cancer cell growth by fueling cell metabolism; (2) direct, cell-autonomous anticancer eﬀects, which are mediated by the inhibition of mitochondrial metabolism and ATP production, with the consequent impairment of intracellular energetic status and inhibition of mTOR, protein and fatty acid biosynthesis. Impact of Metformin on Systemic Metabolism and Survival of Patients With Advanced Pancreatic Neuroendocrine Tumors Claudio Vernieri 1,2, Sara Pusceddu 1 and Filippo de Braud 1,3 1 Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy, 2 IFOM, The FIRC Institute of Molecular Oncology, Milan, Italy, 3 Oncology and Hematology-Oncology Department, University of Milan, Milan, Italy Claudio Vernieri 1,2, Sara Pusceddu 1 and Filippo de Braud 1,3 1 Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy, 2 IFOM, The FIRC Institute of Molecular Oncology, Milan, Italy, 3 Oncology and Hematology-Oncology Department, University of Milan, Milan, Italy Keywords: pancreatic neuroendocrine tumors, metformin, metabolism, glucose, lipid metabolism, diabetes mellitus OPINION published: 20 September 2019 doi: 10.3389/fonc.2019.00902 OPINION Reviewed by: However, the potential anticancer role of metformin in patients with advanced pNETs remains to be fully elucidated. INTRODUCTION Edited by: Alessandro De Vita, Romagnolo Scientiﬁc Institute for the Study and Treatment of Tumors (IRCCS), Italy Pancreatic neuroendocrine tumors (pNETs) represent a subgroup of neuroendocrine malignancies with speciﬁc biological and clinical characteristics, whose incidence has increased in the last four decades. The growth and proliferation of pNET cells is especially dependent on the IGF-1/IGF1-receptor/PI3K/AKT/mTOR signaling pathway, while the activation of somatostatin receptor axis exerts antiproliferative eﬀects [Figure 1; (1, 2)]. September 2019 \| Volume 9 \| Article 902 METHODS We searched in PubMed database using the following string: “(biguanides OR metformin) AND (pancreatic neuroendocrine tumors OR pNETs OR neuroendocrine tumors.)” Our search strategy produced a total number of 82 articles. Among them, we selected for this review article those original preclinical and clinical papers that investigated the metformin in preclinical pNET models or in patients with advanced pNETs. Nevertheless, the following issues strongly limit the clinical translatability of in vitro studies published so far: (1) metformin concentrations used in cell growth media are in the range of mM (usually 1–20 mM), i.e., by far higher than those that can be reached in patient blood with commonly-used and safe metformin dosages 4–15 µM (11). This limitation highlights the importance of repeating crucial experiments with more physiological metformin concentrations; (2) the contribution of metformin-induced Citation: Vernieri C, Pusceddu S and de Braud F (2019) Impact of Metformin on Systemic Metabolism and Survival of Patients With Advanced Pancreatic Neuroendocrine Tumors. Front. Oncol. 9:902. doi: 10.3389/fonc.2019.00902 September 2019 \| Volume 9 \| Article 902 Frontiers in Oncology \| www.frontiersin.org Metformin in Pancreatic Neuroendocrine Tumors Vernieri et al. FIGURE 1 \| Potential cell-autonomous and systemic antitumor mechanisms of action of Metformin in pNETs. FIGURE 1 \| Potential cell-autonomous and systemic antitumor mechanisms of action of Metformin in pNETs. Here we review and discuss preclinical and clinical studies supporting a potential role of metformin in the treatment of pNETs. We also discuss how ongoing trials could elucidate a potential role of metformin in combination with established anti-pNET therapies. and QGP-1 (non-hormone-secreting) pNET cell lines (8). In BON-1 but not in QGP-1 cells, metformin strongly inhibited the transcription of insulin receptor gene (INSR), and also reduced levels of phosphorylated ERK and AKT (8). In another study, metformin inhibited the mTORC1/S6K/S6 pathway when used in the 1–10 mM concentration range, and reduced cell viability without inducing apoptosis (9). Due to the central role of the INSR-IGFR1/PI3K/AKT/mTOR pathway in pNET cell growth and proliferation, the ability of metformin to inhibit this axis via AMPK activation may be responsible for its cell- autonomous anticancer eﬀects, as well as for a potentially synergistic antitumor activity between mTORC1 inhibitors and metformin. September 2019 \| Volume 9 \| Article 902 Clinical Data T2DM is characterized by the concomitancy of hyperglycemia, insulin resistance, and hyperinsulinemia (17). Furthermore, T2DM is frequently associated with metabolic syndrome, which is deﬁned by the presence of glucose intolerance, hypertriglyceridemia, low HDL cholesterol levels, obesity, and high blood pressure (18). Therefore, the presence of both glucose and lipid metabolism dysregulation is common in T2DM patients (17). Metformin is eﬀective in reducing hyperglycemia and insulin resistance occurring in T2DM patients, and also reduced blood triglyceride and cholesterol concentration in some studies (15). The ﬁrst indication of a potential impact of metformin on the outcome of patients with advanced pNET patients came from a small retrospective study that we conducted in 31 patients. In this study, we found that metformin use in diabetic patients was associated with signiﬁcantly longer PFS when compared to diabetic patients not receiving metformin, or to non-diabetic patients (Table 1)(12). Aiming to expand these preliminary data, we conducted a large retrospective, multicentric study involving 24 Italian centers and 445 patients (13). In this study, we found that diabetic patients treated with metformin had remarkably longer PFS (44.2 months) when compared to other diabetics (20.8 months) or to non-diabetic patients (15.1 months) (Table 1). Importantly, the positive impact of metformin was observed regardless of the concomitant anticancer treatment (SA or everolimus plus SA), and was independent from other known prognostic variables, including the presence of liver metastases or having undergone previous surgery of the primary tumor. In our study, patient glycemic status was not independently associated with PFS, thus suggesting that plasma glucose levels do not aﬀect treatment eﬃcacy (13). We also provided indirect arguments supporting the conclusion that blood insulin concentration is unlikely to have an eﬀect on patient outcomes. Therefore, we ﬁnally hypothesized that the anticancer role of metformin in advanced pNETs is more likely to be mediated by cell-autonomous antitumor eﬀects. At our Institution, the single-arm, open label MetNet1 trial (NCT02294006) is currently enrolling patients with advanced pNETs regardless of their diabetic status (19). Patients enrolled in this trial are prescribed upfront treatment with SAs plus everolimus plus metformin, up to a maximum daily dosage of 2,000 mg. The primary objective of the study is to evaluate the eﬃcacy of the experimental treatment, as deﬁned as median PFS. Other study objectives consist in testing the tolerability of the experimental treatment, as well as its eﬀects on systemic metabolism. Clinical Data Of note, metformin does not signiﬁcantly alter glucose and lipid metabolism in patients with normal baseline proﬁles. Therefore, if metformin anticancer eﬀects in pNET patients are mainly mediated through modiﬁcations of systemic metabolism, diabetic pNETs patients, who more frequently have deregulated glucose and lipid metabolism, may beneﬁt from metformin signiﬁcantly more than non-diabetic ones. Conversely, if metformin mainly acts through a cell-autonomous anticancer eﬀect, diabetic, and non- diabetic patients should beneﬁt from metformin treatment in a similar way. Discarding between these two possibilities will be crucial to properly select pNET patients who are the best candidates to receive metformin in combination with standard anticancer treatments. On the other hand, we recently published results of a study indicating that modiﬁcations of lipid metabolism could be implicated in metformin anticancer properties. Indeed, in a 58 patients with advanced pNETs treated with everolimus, we found that the precocious (within 3 months from treatment initiation) onset of hypertriglyceridemia, or increased cholesterol levels during the whole treatment course, are associated with signiﬁcantly lower PFS independently from metformin use (Table 1) (14). We also found that high intratumor levels of Acetyl-CoA Carboxylase 1 (ACC1) enzyme, the limiting-step enzyme in the fatty acid de novo biosynthesis pathway, correlate with lower everolimus eﬃcacy [Figure 1; (14)]. Since metformin not only aﬀects systemic glucose metabolism, but it is also capable of lowering plasma triglycerides (15) and/or of causing inhibition of ACC1 in AMPK-mediated manner in cancer cells (16), the observed association of metformin use and signiﬁcantly longer patient PFS could be mediated by metformin eﬀects on systemic/tumor lipid metabolism (Figure 1). Prospective studies are needed to test this hypothesis, as well as to distinguish between an indirect (i.e., mediated by modiﬁcations of systemic metabolism) and a direct, cell-autonomous anticancer eﬀect of metformin in pNETs. Ongoing Studies systemic metabolism modiﬁcations on its anticancer activity cannot be assessed in in vitro studies. Since metformin may inhibit cancer growth by modifying systemic metabolism, and in particular by lowering the blood concentration of glucose, insulin, and lipids, this limitation is especially important in the perspective of their clinical translation. While retrospective analyses clearly indicate that metformin use in diabetic patients with advanced pNETs is associated with better clinical outcomes, no prospective studies have investigated metformin activity/eﬃcacy in combination with standard antitumor treatments so far. Moreover, it is currently unclear if also pNET patients who are not diabetics ma beneﬁt from metformin treatment. Preclinical Evidence Metformin has demonstrated anti-pNET activity in preclinical studies (8–10). For instance, metformin impaired cell migration capacity and reduced the survival of BON-1 (serotonin-secreting) September 2019 \| Volume 9 \| Article 902 Frontiers in Oncology \| www.frontiersin.org 2 Metformin in Pancreatic Neuroendocrine Tumors Vernieri et al. Frontiers in Oncology \| www.frontiersin.org DISCUSSION Based on the available preclinical and retrospective clinical evidence, metformin administration promises to provide clinical advantage when used in combination with established anticancer treatments, such as SAs and everolimus, in patients with advanced pNETs (12–14). Since plasma glucose levels have not been found to be associated with pNET patient prognosis, it is unlikely that the major eﬀect of metformin is mediated by its ability to reduce patient glycemia. On the other hand, emerging data suggest that the eﬀect of metformin could be mediated through its impact on systemic lipid metabolism, especially in patients treated with mTOR inhibitors, which increase September 2019 \| Volume 9 \| Article 902 Frontiers in Oncology \| www.frontiersin.org 3 Metformin in Pancreatic Neuroendocrine Tumors Vernieri et al. TABLE 1 \| Published and ongoing studies of metformin in pNET patients. References Study design N. patients Status Study ﬁndings Pusceddu et al. (12) Retrospective 31 Completed Diabetic pNET patients taking metformin have signiﬁcantly longer PFS when compared with non-diabetics or diabetics pNET patients treated with other antidiabetic therapies Pusceddu et al. (13) Retrospective 445 Completed Diabetic pNET patients taking metformin have signiﬁcantly longer PFS (44.2 months) when compared with non-diabetics (15.1 months) or diabetics pNET patients treated with other antidiabetic therapies (20.8 months). The impact of metformin on patient PFS was independent of other clinically relevant variables Vernieri et al. (14) Retrospective 58 Completed Hypertriglyceridemia and hypercholesterolemia are associated with signiﬁcantly worse PFS in advanced pNET patients treated with everolimus Pusceddu et al. (NCT02294006) (14) Prospective 43 Ongoing N.A. PFS: progression-free survival; pNET: pancreatic neuroendocrine tumor. TABLE 1 \| Published and ongoing studies of metformin in pNET patients. PFS: progression-free survival; pNET: pancreatic neuroendocrine tumor. triglyceride and cholesterol concentration in a signiﬁcant proportion of patients [Figure 1; (14)]. However, these data need to be conﬁrmed in larger retrospective and, in case, in prospective studies. Moreover, a direct, cell-autonomous anticancer eﬀect of metformin against pNETs cannot be excluded, even though metformin concentrations that are active in in vitro studies can be hardly reached in patients’ blood (Figure 1). important: indeed, pNET patients receiving metformin for T2DM treatment could be selected for being exposed to SAs and/or everolimus for longer periods, or for having undergone previous pancreatic surgery, i.e., all clinical characteristics associated with better patient prognosis independently from metformin use. DISCUSSION Another crucial issue in the debate around the use of metformin as an anticancer agent consists in clarifying its potential antitumor activity in patients who are not diabetics. Since in our retrospective study the diabetic status was not associated with patient PFS independently from other prognostic factors, it is reasonable to hypothesize that metformin could improve patient prognosis independently from its impact on glucose metabolism but, more reasonably, through its eﬀects on other metabolic pathways or through cell-autonomous anticancer eﬀects. In both cases, we would expect similar anticancer activity from metformin in patients with and without diabetes. Prospective studies including both diabetic and non-diabetic patients, as well as correlative analyses between kinetics of blood triglyceride/cholesterol concentration and treatment eﬃcacy, will be crucial to clarify the role of metformin-induced metabolic modiﬁcations on its anticancer activity. On the other hand, preoperative, window-of-opportunity trials with single-agent metformin in patients candidate to surgery could represent the ideal context to explore potential metformin cell-autonomous antitumor properties, as well as to clarify if commonly used dosages of this compound are suﬃcient to reach therapeutic intratumor concentrations. Crucial advantages of metformin consist in low drug costs and excellent tolerability at dosages that are commonly used for the treatment of T2DM. However, the tolerability of metformin in combination with standard anticancer treatments needs to be established yet. For instance, the metformin-everolimus combination could increase the risk of everolimus-induced diarrhea. The ongoing NCT02294006 trial will clarify if metformin is a safe and well-tolerated drug when combined with SAs plus everolimus (19). While the risks of speciﬁc pharmacological metformin-including combinations cannot be ignored, metformin could also prevent or reduce alterations of glucose and lipid metabolism that are often detected in patients with advanced pNETs, especially those treated with everolimus (4, 14). While published clinical studies indicate a potentially relevant advantage from adding metformin to standard anti-pNET treatments, prospective studies are necessary before concluding that metformin might provide a true clinical beneﬁt. Indeed, retrospective studies have important limitations that may lead to incorrect conclusions. For instance, metformin use had been associated with longer survival in patients with advanced pancreatic exocrine adenocarcinomas in retrospective studies (20, 21); however, three recent randomized trials showed no beneﬁt from adding metformin to ﬁrst- or second-line chemotherapy in this patient population (22–24). Frontiers in Oncology \| www.frontiersin.org REFERENCES Ambe CM, Mahipal A, Fulp J, Chen L, Malafa MP. Eﬀect of metformin use on survival in resectable pancreatic cancer: a single-institution experience and review of the literature. PLoS ONE. (2016) 11:e0151632. doi: 10.1371/journal.pone.0151632 7. Noone AM, Howlader N, Krapcho M, Miller D, Brest A, Yu M, et al. SEER Cancer Statistics Review, 1975–2015. Bethesda, MD: National Cancer Institute (2018). Available online at: https://seer.cancer.gov/csr/1975_2015/ (accessed August 20, 2019). 22. Kordes S, Pollak MN, Zwinderman AH, Mathot RA, Weterman MJ, Beeker A, et al. Metformin in patients with advanced pancreatic cancer: a double-blind, randomised, placebo-controlled phase 2 trial. Lancet Oncol. (2015) 16:839–47. doi: 10.1016/S1470-2045(15)00027-3 8. Herrera-Martinez AD, Pedraza-Arevalo S, L-López F, Gahete MD, Galvez- Moreno MA, Castano JP, et al. Type 2 diabetes in neuroendocrine tumors: are biguanides and statins part of the solution? J Clin Endocrinol Metab. (2019) 104:57–73. doi: 10.1210/jc.2018-01455 23. Braghiroli MI, de Celis Ferrari AC, Pﬁﬀer TE, Alex AK, Nebuloni D, Carneiro AS, et al. Phase II trial of metformin and paclitaxel for patients with gemcitabine-refractory advanced adenocarcinoma of the pancreas. Ecancermedicalscience. (2015) 9:563. doi: 10.3332/ecancer. 2015.563 9. Vlotides G, Tanyeri A, Spampatti M, Zitzmann K, Chourdakis M, Spttl C, et al. Anticancer eﬀects of metformin on neuroendocrine tumor cells in vitro. Hormones. (2014) 13:498–508. doi: 10.14310/horm.2002.1517 10. Soares HP, Ni Y, Kisfalvi K, Sinnett-Smith J, Rozengurt E. Diﬀerent patterns of Akt and ERK feedback activation in response to rapamycin, active-site mTOR inhibitors and metformin in pancreatic cancer cells. PLoS ONE. (2013) 8:e57289. doi: 10.1371/journal.pone.0057289 24. Reni M, Dugnani E, Cereda S, Belli C, Balzano G, Nicoletti R, et al. (Ir)relevance of metformin treatment in patients with metastatic pancreatic cancer: an open-label, randomized phase II trial. Clin Cancer Res. (2016) 22:1076–85. doi: 10.1158/1078-0432.CCR-15-1722 11. Anisimov VN. Do metformin a real anticarcinogen? A critical reappraisal of experimental data. Ann Transl Med. (2014) 2:60. doi: 10.3978/j.issn.2305-5839.2014.06.02 25. Wei M, Liu Y, Bi Y, Zhang ZJ. Metformin and pancreatic cancer survival: real eﬀect or immortal time bias? Int J Cancer. (2019) 145:1822–8. doi: 10.1002/ijc.32254 12. Pusceddu S, Buzzoni R, Vernieri C, Concas L, Marceglia S, Giacomelli L, et al. Metformin with everolimus and octreotide in pancreatic neuroendocrine tumor patients with diabetes. Future Oncol. (2016) 12:1251– 60. doi: 10.2217/fon-2015-0077 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 13. REFERENCES 15. Wulﬀele MG, Kooy A, de Zeeuw D, Stehouwer CD, Gansevoort RT. The eﬀect of metformin on blood pressure, plasma cholesterol and triglycerides in type 2 diabetes mellitus: a systematic review. J Intern Med. (2004) 256:1–14. doi: 10.1111/j.1365-2796.2004.01328.x 1. Missiaglia E, Dalai I, Barbi S, Beghelli S, Falconi M, della Peruta M, et al. Pancreatic endocrine tumors: expression proﬁling evidences a role for AKT-mTOR pathway. J Clin Oncol. (2010) 28:245–55. doi: 10.1200/JCO.2008.21.5988 16. Vernieri C, Casola S, Foiani M, Pietrantonio F, de Braud F, Longo V. Targeting cancer metabolism: dietary and pharmacologic interventions. Cancer Discov. (2016) 6:1315–33. doi: 10.1158/2159-8290.CD-16-0615 2. Moreno A, Akcakanat A, Munsell MF, Soni A, Yao JC, Meric-Bernstam F. Antitumor activity of rapamycin and octreotide as single agents or in combination in neuroendocrine tumors. Endocr Relat Cancer. (2008) 15:257– 66. doi: 10.1677/ERC-07-0202 17. DeFronzo RA, Ferrannini E, Groop L, Henry RR, Herman WH, Holst JJ, et al. Type 2 diabetes mellitus. Nat Rev Dis Primers. (2015) 1:15019. doi: 10.1038/nrdp.2015.19 3. Caplin ME, Pavel M, Cwikla JB, Phan AT, Raderer M, Sedlackova E, et al. Lanreotide in metastatic enteropancreatic neuroendocrine tumors. N Engl J Med. (2014) 371:224–33. doi: 10.1056/NEJMoa1316158 18. Saklayen MG. The global epidemic of the metabolic syndrome. Curr Hypertens Rep. (2018) 20:12. doi: 10.1007/s11906-018- 0812-z 19. Pusceddu S, de Braud F, Concas L, Bregant C, Leuzzi L, Formisano B, et al. Rationale and protocol of the MetNET-1 trial, a prospective, single center, phase II study to evaluate the activity and safety of everolimus in combination with octreotide LAR and metformin in patients with advanced pancreatic neuroendocrine tumors. Tumori. (2014) 100:e286–9. doi: 10.1700/1778. 19298 4. Yao JC, Shah MH, Ito T, Bohas CL, Wolin EM, Van Cutsem E, et al. Everolimus for advanced pancreatic neuroendocrine tumors. N Engl J Med. (2011) 364:514–23. doi: 10.1056/NEJMoa1009290 5. Raymond E, Dahan L, Raoul JL, Bang YJ, Borbath I, Lombard-Bohas C, et al. Sunitinib malate for the treatment of pancreatic neuroendocrine tumors. N Engl J Med. (2011) 364:501–13. doi: 10.1056/NEJMoa1003825 20. Sadeghi N, Abbruzzese JL, Yeung SC, Hassan M, Li D. Metformin use is associated with better survival of diabetic patients with pancreatic cancer. Clin Cancer Res. (2012) 18:2905–12. doi: 10.1158/1078-0432.CCR-11-2994 6. Genc CG, Klumpen HJ, van Oijen MGH, van Eijck CHJ, Nieveen van Dijkum EJM. A nationwide population-based study on the survival of patients with pancreatic neuroendocrine tumors in the Netherlands. World J Surg. (2018) 42:490–7. doi: 10.1007/s00268-017-4278-y 21. DISCUSSION Diﬀerent factors may account for discrepancies between retrospective and prospective studies, including the reporting bias, immortal time bias, and the fact that metformin is only taken by patients with T2DM in retrospective studies (13, 25). In the case of pNETs, the impact of the immortal time bias could be especially To date, the strongest rationale exists for combining metformin with everolimus, which could synergize at a molecular (i.e., by strengthening inhibition of the PI3K/AKT/mTOR pathway and inhibiting cancer cell anabolism) and systemic (i.e., by reducing blood glucose, triglyceride, and cholesterol concentration) levels. However, future preclinical and, in case, clinical studies should investigate metformin in combination with other therapies that are standard-of-care in pNET patients, such as SSAs and PPRT. September 2019 \| Volume 9 \| Article 902 4 Metformin in Pancreatic Neuroendocrine Tumors Vernieri et al. FUNDING This paper was funded by scientiﬁc grant of medical oncology Unit 1, Fondazione IRCCS Istituto Tumori Milano. All authors (CV, FB, and SP) have contributed to conception or design of the paper, as well as to the writing of the manuscript and its critical revision. REFERENCES Pusceddu S, Vernieri C, Di Maio M, Marconcini R, Spada F, Massironi S, et al. Metformin use is associated with longer progression-free survival of patients with diabetes and pancreatic neuroendocrine tumors receiving everolimus and/or somatostatin analogues. Gastroenterology. (2018) 155:479– 89.e7. doi: 10.1053/j.gastro.2018.04.010 Copyright © 2019 Vernieri, Pusceddu and de Braud. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 14. Vernieri C, Pusceddu S, Fuca G, Indelicato P, Centonze G, Castagnoli L, et al. Impact of systemic and tumor lipid metabolism on everolimus eﬃcacy in advanced pancreatic neuroendocrine tumors (pNETs). Int J Cancer. (2019) 144:1704–12. doi: 10.1002/ijc.32042 September 2019 \| Volume 9 \| Article 902 Frontiers in Oncology \| www.frontiersin.org 5
https://openalex.org/W4280584852	https://link.springer.com/content/pdf/10.1007/s10639-022-11108-2.pdf	English	null	Smartphone applications for physical activity promotion from physical education	Education and information technologies	2,022	cc-by	9,420	ERROR: type should be string, got "https://doi.org/10.1007/s10639-022-11108-2\nEducation and Information Technologies (2022) 27:11759–11779 https://doi.org/10.1007/s10639-022-11108-2\nEducation and Information Technologies (2022) 27:11759–11779 Keywords Mobile phone app · Educational technology · Digital literacy · Mobile \ndevice · Secondary school Smartphone applications for physical activity promotion \nfrom physical education Francisco Javier Gil‑Espinosa1 · Adriana Nielsen‑Rodríguez2 · \nRamón Romance2 · Rafael Burgueño3 Received: 24 December 2021 / Accepted: 11 May 2022 / \n© The Author(s) 2022\nPublished online: 19 May 2022 Received: 24 December 2021 / Accepted: 11 May 2022 / \n© The Author(s) 2022\nPublished online: 19 May 2022 Extended author information available on the last page of the article \t Francisco Javier Gil‑Espinosa \n\t\njaviergil@uma.es 1 Introduction The digital technology (e.g., digital blackboard, tablets, smartphone apps) has been \nincorporated as key elements into every sphere of our society such as health, work \nand education (Organisation for Economic Cooperation and Development, 2019). The use of digital technology to support the teaching and learning process has \ngrown sharply in recent years (Sargent & Casey, 2020), and its implementation in \neducation system has been accentuated by the COVID-19 pandemic. Indeed, Cal-\nderón-Garrido et al. (2022) review concluded that the scientific production seems \nto bet on the use of smartphones in class, as it is beneficial for educational objec-\ntives. In physical education (PE), while most research has focused on reporting use-\nfulness and advantages derived from the use of digital technology in PE (Cushion \nand Townsend, 2019; Sargent & Casey, 2020), little attention has been paid to the \nquestion about whether there are smartphone apps that could be linked with PE \ncurriculum to promote leisure-time physical activity (PA) in adolescents from the \ncontext of the secondary school PE. There is thus a need to help PE teachers take \na curricular approach to the selection and implementation of smartphone applica-\ntions (apps) that contribute to addressing the curriculum. This would allow us to \nrecommend an improved use of smartphone apps for PE lessons, which would lead \nto improve the quality of the teaching and learning process with more meaningful \nlearning experiences for students, and to accomplish one of the main educational \ngoals, such as leisure-time PA promotion (SHAPE America– Society of Health and \nPhysical Educators, 2014). This research sought to carry out a systematic search \nfor smartphone apps focused on PA that are available in the Google Play. Next, the \ninterplay between every included smartphone app and the secondary PE curriculum \nwas further analysed. Abstract Smartphone applications (apps) are thought to be an adequate instructional strategy \nnot only to improve the quality of the teaching in physical education (PE), but also \nto effectively promote leisure-time physical activity (PA) of adolescent students in \nthis context. Although the use of smartphone apps has been generalized in PE, lit-\ntle is known about the curricular approach of smartphone apps to be implemented \nby teacher to teach specific curricular contents in PE lessons. Therefore, the aim \nof this research was threefold: a) to conduct a systematic search for smartphone \napps focused on PA and sport; b) to assess the features, content and quality of every \nincluded smartphone app; and c) to analyze the relationships between every selected \napp and the secondary PE curriculum. Systematic searches were completed on \nGoogle Play Store from January 2021 to March 2021. Apps were included when \nthey met: main goal focused on PA and sport; permitted use by underage; they are \nfree; user scores of at least 4. The app selection process was carried out by sev-\neral reviewers and concordance measures were estimated. Additionally, an app \nquality assessment was independently conducted by three reviewers. A total of 18 \napps focused on PA were included. Particularly, eight apps were suitable for fitness, \nhealth and quality of life curricular content; two for sports content; four for body \nexpression content; and four apps for outdoor PA content. The mean quality score \nwas 4.00. Apps could be helpful for teachers to implement the secondary PE cur-\nriculum and effectively promote PA among adolescent students. Keywords Mobile phone app · Educational technology · Digital literacy · Mobile \ndevice · Secondary school 012341 456789)\n3 Education and Information Technologies (2022) 27:11759–11779 11760 1.1 \u0007Secondary physical education curriculum In common with another national PE curricula (e.g., SHAPE America– Society of \nHealth and Physical Educators, 2014; Irish Proffessional Service for Teachers, 2022; \nBritish Council for the Curriculum, Examinations and Assessment, 2022), one the \nof the main curricular goals for PE, in Spain, is to develop students as physically \nliterate people capable of applying the knowledge, skills and attitudes needed for \nlifelong health-related PA (Orden 15 of January, 2021; Royal Decree 1105/, 2014). In order to structure the knowledge, skills and attitudes to be learnt by students \nwhen completing secondary education, the content blocks of Spanish curriculum for \nsecondary PE could be organized according four: a) fitness, health and life quality, \nwhich includes contents related to the development of basic physical capacities, the \npromotion of healthy lifestyles (e.g., regular PA, nutrition and adequate rest breaks) \nand avoidance of potential health-damaging behaviours (e.g., sedentarism, tobacco, \nalcohol and drugs), as well as the identification of health benefits derived from a \ngood level of fitness and regular leisure-time PA; b) games and sports, which com-\nprises contents referring to the understanding of games and sports as sociocultural 1 3 Education and Information Technologies (2022) 27:11759–11779 11761 phenomena, the knowledge of Spanish traditional games, as well as the development \nof technical and tactical skills, the knowledge and respect for the rules of individual \nand team sports; c) body expression, which brings together contents concerning cre-\native and artistic communication and emotional regulation using body (e.g., dance, \nchoreography, and dramatization) d) outdoor PA, which tackles contents regard-\ning the interaction between students and nature through PA (e.g., hiking, orientat-\ning, and outdoor challenges), and the understanding and appreciation of the natural \nenvironment. The Spanish curriculum for secondary PE states two 60-minute lessons per week, \ndespite the substantial body of evidence indicating PE helped students meet daily \nPA recommendations (Kerr et al., 2018; Kwon et al., 2020; Lee & Gao, 2020). Thus, \nthere is a need for teachers to develop and implement instructional strategies aiming \nto promote students’ levels of leisure-time PA from PE lessons. For this goal, the \nuse of digital technology, including smartphone apps, is recommended not only to \ndevelop key digital competence, but also to improve the comprehensive education of \nstudents in general, and promote leisure-time PA from the context of the secondary \nschool PE in particular (Orden 15 of January, 2021; Royal Decree 1105/, 2014). 1.3 \u0007Literature review on apps in physical education The digital technology could allow a greater variety of instructional strategies in PE \nwhile helping to develop in students attitudes, knowledge and behaviours for a more \nphysically active life. Therefore, teachers need to experiment with apps related to \nthe PE teaching-learning process (Yu et al., 2018). Accordingly, previous research \nabout the use of apps, conducted in the school context of PE, has evidenced advan-\ntages in motivation, knowledge of results, assessment, and the improvement of stu-\ndent autonomy, among others (Kerner & Goodyear, 2017; Klenk et al., 2017; Phil-\nlips et al., 2014; Vega-Ramírez et al., 2020). Schwartz and Baca (2016) point out \nthat many PA apps are based on behavioural theory and use elements of gamifica-\ntion for success, with personal goals and specific feedback. Lee (2018) recommends \nusing apps to facilitate students’ group activities, as well as the knowledge of results. Similarly, a growing body of research has pointed out that the implementation of \nsmartphone apps was an efficient instructional strategy to increase in-classroom PA, \nas well as to promote leisure-time PA among adolescent students in PE (Böhm et al., \n2019; Brickwood et al., 2019; Gil-Espinosa et al., 2020; Lau et al., 2011). Likewise, Mokmin and Jamiat (2021) designed a mobile application taking into \nconsideration the motor learning theory (Muratori et al., 2014) and Mayer’s cogni-\ntive theory of multimedia learning (Mayer, 2011), obtaining satisfactory results in \nmotivation and PA performance after its implementation with students. In turn, the \nresults of Papastergiou et al. (2021) research with primary school students conclude \nthat the use of apps improved their interest, enjoyment, and motivation, while teach-\ners had more time to provide individualised feedback. Similarly, research carried out \nby Yu (2020) in secondary education found that, through the use of mobile appli-\ncations, students improved their tactics and performance of badminton skills while \nallowing for more active learning and improved knowledge of the results, conclud-\ning that PE teachers can integrate the use of mobile applications in their teaching \nwork.i In any case, the educational benefits of using mobile applications in PE will be \ndeterminate by the design of the learning activities (Greve et al., 2022), which is \nwhy their link with the curriculum is crucial. PE teachers are recommended to align \nthe selection of apps with the PE learning goals (Lee & Gao, 2020). Krause et al. 1.2 \u0007Smartphone apps in physical education The use of digital technology in PE is considered as a relevant instructional recourse \ngiven its potential in supporting the teaching and learning process in a manner that \nfits the nature of PE (Casey et al., 2017). For instance, digital technology such as \ndigital blackboard, tablets, video cameras, electronic devices, and smartphone apps \nprovide students with opportunities to reinforce their learning, and to foster their \nautonomy (Hyeonho & Taemin, 2021). Recently, inquiring app-integrated PE in \nwhich several apps are utilized on the smartphone in PE has been on the rise to \nease the teaching learning process (Krause & Sanchez, 2014; Zhu & Dragon, 2016). PE experts have suggested smartphone apps perform different roles in improving \nthe quality of PE: a) they can serve as communicational tools such as scoreboard, \nwhiteboard or display platforms; b) they can serve as classroom management tools \nby being useful for timers, music displayers, and microphones; c) they can be used \nas tools for information delivery, feedback, lesson plans, assessment; and d) they \nshould be personalised based on every student’s need and skills (Goodyear et al., \n2019; Penney et al., 2012; Pyle & Esslinger, 2014; Sinelnikov, 2012). It is, thus, thought to develop students as physical literate people via smartphone \napps may represent an effective and innovative strategy to promote leisure-time PA \nfrom the context of the secondary school PE. This idea relied on the fact that most \nsecondary students have a generalised use of digital technology in their everyday \nlife through the utilisation of social networks, websites, blogs, and smartphones, and \nuser accounts for several apps (Böhm et al., 2019; Direito et al., 2015; Goodyear & \nArmour, 2021; Vega-Ramírez et al., 2020). 1 3 Education and Information Technologies (2022) 27:11759–11779 11762 1.3 \u0007Literature review on apps in physical education (2020) recommend increasing research on the use of technology in PE, as well as \nimproving the training of PE teachers in the integrated use of technology in the \nsubject. 1.4 \u0007The current research and research question Smartphone apps represent a potential means both to develop students as physical \nliterate, and to promote leisure-time PA from the context of the secondary school \nPE. In accordance with previous research, the number of smartphone apps, particu-\nlarly those related to PA and sports, has been on the Google Play (Arigo et al., 2020). This great quantity of smartphone apps represents a good opportunity to provide \nstudents with improved meaningful learning experiences, yet it also makes it quite 1 3 Education and Information Technologies (2022) 27:11759–11779 11763 difficult for teachers to select and implement the most suitable app in accordance \nwith curricular content to be taught in lessons. Although previous research has well \ndocumented the benefits from the use of smartphone apps on learning-related out-\ncomes in students such as increased levels of in-classroom and leisure-time PA, the \nquestion about the curricular approach to smartphone apps to be used by teachers to \nteach specific curricular contents in the classroom remains still to be examined. To \nthe best of our knowledge, no studies were found to analyse smartphone apps to pro-\nmote PA in the school context and its potential relationships to the curriculum and \npossible use by PE teachers. At the same time, Koekoek and van Hilvoorde (2018) \nthought over the need to understand the way teachers select digital technology, with-\nout losing educational goals. Further, this question becomes even more important \ndue to the great use of digital technology in general, and particularly smartphone \napps in PE to adapt the implementation of curriculum to the new instructional mod-\nels derived from the COVID-19 pandemic (López-Fernández et al., 2021). This research attempts to provide a response to the questions about which smart-\nphone apps on PA and sports are linked directly to secondary curriculum for PE, \nand how PE teachers might implement them to teach curricular content with the \npurpose of promoting leisure-time PA from the school context of PE. Therefore, the \naim of this research was threefold. The first objective was to carry out a systematic \nsearch for smartphone apps focused on PA and sport that are available in the Google \nPlay. The second objective included a quality assessment for every smartphone app \nincluded in this study. 1.4 \u0007The current research and research question The third objective consisted of the analysis of the relation-\nship between every smartphone app and the PE curriculum, as well as the provision \nof possible strategies for the implementation of each app. 2.1 \u0007Search strategy Systematic searches were individually completed by two researchers (R1 and R2) \nin Google Play Store between 12th January 2021 and 31st March 2021. The identi-\nfication of apps was performed using the following search terms: “physical educa-\ntion”, “physical activity”, “health”, “fitness”, “sport”, “body expression”, “corporal \nlanguage”, “dance”, “outdoor physical activity”. Search terms were entered into the \nGoogle Play Store in isolation or in different combinations based on Boolean logic \n(i.e., AND, NOT, OR). 2.3 \u0007Quality assessment In line with previous app reviews, an app quality assessment was completed through \nthe Mobile App Quality Ratings (MARS) (Stoyanov et al., 2015). It includes 23 \nitems divided into 5 dimensions: engagement (5 items, e.g., “interest”), functionality \n(4 items, e.g. “ease of use”), aesthetics (3 items, e.g. “Visual appeal: How well does \nthe app look?”), information quality (7 items, e.g. “accuracy of app description”), \nand subjective quality (4 items, e.g. “Would you recommend this app?”) (Stoyanov \net al., 2015). Every item is answered on a 5-point Likert scale ranging from 1 (inad-\nequate) to 5 (excellent). To gather quality evidence for each app, a global average \nscore from the mean of each MARS dimension was estimated (Stoyanov et al., \n2015). This assessment was independently carried out by three reviewers (R1, R3 \nand R6). Likewise, disagreements were solved by a consensus meeting between the \nreviewers and the main authors of this study. 2.2 \u0007Inclusion criteria and selection process In this phase, a second set of inclusion criteria was estab-\nlished to identify the apps that would be included in this study. They would be \nincluded when: a) their use is allowed to underage; b) they are free (Bearne et al., \n2020; Simões et al., 2018); c) user score of at least 4 (scale range 1 to 5) follow-\ning previous app reviews (Bearne et al., 2020; Simões et al., 2018); d) a number \nof user scores equal to 100 or higher (Bearne et al., 2020; Simões et al., 2018). Two reviewers (R5 and R6) individually evaluated the names and descriptions of \nthe apps against the established inclusion criteria using a checklist (answer: yes, \nor not). A consensus meeting was held to resolve disagreements between both \nreviewers with the help of the main author of this research. 2.2 \u0007Inclusion criteria and selection process The apps selection process was developed in two phases. In the first phase and \nafter removing duplicates, apps were retrained and registered in a database \nwhen they met the following criteria: a) they were written in Spanish or English \n(description and application), and b) they had the main goal focused on PA pro-\nmotion. Apps could be used in isolation or in combination with an external device 3 Education and Information Technologies (2022) 27:11759–11779 11764 (e.g., PA tracker) or a back-office system, for instance, to communicate with a PE \nor PA professional. This first phase was carried out by two reviewers (R3 and R4) \nwho individually evaluated the names and descriptions of the apps against these \ntwo criteria using a checklist (response: yes, or not). To solve disagreements, a \nconsensus meeting was held between both reviewers and the main author of this \nresearch. The apps identified in this first phase were, then, included in the sec-\nond selection phase. In this phase, a second set of inclusion criteria was estab-\nlished to identify the apps that would be included in this study. They would be \nincluded when: a) their use is allowed to underage; b) they are free (Bearne et al., \n2020; Simões et al., 2018); c) user score of at least 4 (scale range 1 to 5) follow-\ning previous app reviews (Bearne et al., 2020; Simões et al., 2018); d) a number \nof user scores equal to 100 or higher (Bearne et al., 2020; Simões et al., 2018). Two reviewers (R5 and R6) individually evaluated the names and descriptions of \nthe apps against the established inclusion criteria using a checklist (answer: yes, \nor not). A consensus meeting was held to resolve disagreements between both \nreviewers with the help of the main author of this research. (e.g., PA tracker) or a back-office system, for instance, to communicate with a PE \nor PA professional. This first phase was carried out by two reviewers (R3 and R4) \nwho individually evaluated the names and descriptions of the apps against these \ntwo criteria using a checklist (response: yes, or not). To solve disagreements, a \nconsensus meeting was held between both reviewers and the main author of this \nresearch. The apps identified in this first phase were, then, included in the sec-\nond selection phase. 3.1 \u0007App selection process Figure 1 shows a flow diagram for the app selection process. A total of 4650 apps \nin Google Play Store were initially identified. After removing duplicates (κ = 1.00) \nand out-of-scope apps (κ = 0.95), 432 app titles and descriptions were screened for \neligibility based on a set of inclusion criteria (κ ranging from 0.85 to 1.00). Once \nthis app selection process was completed, 18 apps were definitively included in this \nstudy. 2.4 \u0007Relationships between apps included and PE curriculum Once apps were included in this research, the authors individually downloaded the \ndifferent apps and proceeded to individually analyse them by taking into considera-\ntion the four content blocks (i.e., fitness, health and life quality; games and sports; \nbody expression; and outdoor physical activities), established after the analysis of \nthe Spanish secondary PE curriculum (Orden 15 of January, 2021; Royal Decree \n1105/, 2014), and its respective implementation in the classroom. Subsequently, \neach author proposed a series of possible practical applications for every app. Lastly, \na meeting was held to agree both points and to resolve disagreements among the \nauthors. 3 Education and Information Technologies (2022) 27:11759–11779 11765 2.5 \u0007Data analysis For categorical data from a checklist, the degree of agreement among reviewers was \ncomputed using the kappa (κ) index. Consistent with Landis and Koch (1977), there \nis an insignificant agreement with values below 0.20, small with values between \n0.21 and 0.40, moderate with values between 0.41 and 0.60, acceptable with values \nbetween 0.61 and 0.80, and excellent with values between 0.81 and 1.00. For con-\ntinuous data from MARS scores, the degree of agreement between reviewers was \nestimated by the intraclass correlation coefficient (ICC, model: bidirectional mixed \neffects, absolute agreement). According to Koo and Li (2016), there is a poor agree-\nment when values are less than 0.50, moderate when values are between 0.50 and \n0.75, good when values are between 0.75 and 0.90, and excellent when values are \ngreater than 0.90. 3.2 \u0007App quality The total MARS mean score was 4.00 (SD = 0.50) out of 5 (ICC = 0.90, \n95%CI = 0.80–1.00) for the totally of apps included in this research. The dimen-\nsion with the highest score was subjective quality (M = 4.30, SD = 0.50; \nICC = 0.89, 95%CI = 0.80–0.97), followed by functionally (M = 4.25, SD = 0.25; \nICC = 0.85 95%CI = 0.76–0.94), aesthetics (M = 4.20, SD = 0.20; ICC = 0.90, \n95%CI = 0.79–0.95), information quality (M = 3.75, SD = 0.70; ICC = 0.89, \n95%CI = 0.80–0.97), \nand \nengagement \n(M = 3.70, \nSD = 0.60; \nICC = 0.81, \n95%CI = 0.76–0.88). 3.3 \u0007Links with the secondary PE curriculum The 18 apps that met the inclusion criteria were organized according to the four \ncontent blocks present in the Spanish secondary PE curriculum: 1) fitness, health \nand life quality, 2) games and sports, 3) body expression, and 4) outdoor PA (Orden \n15 of January, 2021; Royal Decree 1105/, 2014). A description of each of them is 3 3 Education and Information Technologies (2022) 27:11759–11779 11766 Fig. 1 Flow diagram displaying the app selection process Fig. 1 Flow diagram displaying the app selection process shown, as well as proposals for possible applications to PE, which should be under-\nstood as ideas that teachers must adapt to variables such as the characteristics of \ntheir students and schools, among others. 3.3.1 \u0007Apps for fitness, health and life quality Table 1 yields eight apps to be used in PE for the curricular contents related to fit-\nness, health and life quality. These apps would allow students and/or teachers to \ncreate individual and collective physical challenges, to encourage them to prepare \nexercise for the group-class or to work with other subjects in an interdisciplinarity \nmanner. 3.3.2 \u0007Apps for games and sport Table 2 shows the two apps selected for games and sports contents in PE. Both apps \nwould enable students to organise competitions and to create teams for sports activi-\nties. In addition, the two apps would facilitate interdisciplinary work with other \nsubjects. 1 3 3.3.4 \u0007Apps for outdoor physical activities Table 4 shows the four apps that can be used in order to address outdoor physi-\ncal activities from a curricular perspective in PE. They would allow students to \norganise and conduct sports events in natural and/or urban environments, in addi-\ntion to facilitating work with other subjects in an interdisciplinary way. 1 3 1 3 Education and Information Technologies (2022) 27:11759–11779 11767 Table 1 Apps linked to the fitness, health and life quality curriculum content block\n() Based on (Cabrera Ramos et al., 2019; Cummiskey, 2011; Gil-Espinosa et al., 2020; Mokmin & Jamiat, 2021; Papastergiou et al., 2021; Vega-Ramírez et al., 2020)\nFitness, health and life quality\nApp\nDescription\nPossible practical applications in PE()\nAdidas Runtastic\nDevelops activity tracking apps and services such as train-\ning logs, data analysis, comparisons to other users, and \nother functions to help users improve their overall fitness\nCreation of individual or collective challenges. Interdisciplinarity with subjects such as Geography and history and biol-\nogy, among others. Encourage the autonomy of the students by facilitating them to prepare \nthe exercises and physical activity and then share it with the rest of the \ngroup-class. Gowod\nAllows to test and improve the mobility and flexibility\nMapmyrun\nKnow your distance, pace, calorie burn, elevation, and more\nRealfooding\nFood database, food scan, food log, user community\nSmartwod generator\nWorkouts to choose based on the equipment available\nStrava\nInternet service for tracking human exercise which incorpo-\nrates social network features. It is mostly used for cycling \nand running using GPS data\nStretching & Flexibility at home\nAllows you to know popular stretching exercises\nSworkit\nAllows to customize and play personalized video workouts Education and Information Technologies (2022) 27:11759–11779 11768 Table 2 Apps linked to the games and sports curriculum content block\n) Based on (Andre & Hastie, 2018; Cecchini and Carriedo, 2020; Díaz, 2020; Vega-Ramírez et al., 2020)\nGames and sports\nApp\nDescription\nPossible practical applications in PE()\nLeverade real play\nOrganisation of team sports activities\nAllows students to be empowered by organizing competitions. Interdisciplinarity with subjects such as mathematics. Team maker\nCreation of groups and teams Table 2 Apps linked to the games and sports curriculum content block 1 3 3 Education and Information Technologies (2022) 27:11759–11779 11769 3.3.3 \u0007Apps for body expression Table 3 displays four apps to address the curricular contents of body-expression \nin PE. These apps emphasise the possibility of creating choreographies, rhythms \nand characterisations autonomously. Furthermore, they would allow students to \nwork on creative projects. 4 \u0007Discussion The objective of this research was threefold. The first of them was to carry out a \nsystematic search for smartphone apps focused on PA and sport that are available \nin the Google Play. The second objective included a quality assessment for every \nsmartphone app included in this study. The third objective consisted of the analy-\nsis of the relationship between every smartphone app and the PE curriculum, as \nwell as the provision of possible strategies for the implementation of each app. The main results revealed a total of 18 smartphone apps included: eight apps \nwere suitable for fitness, health and quality of life curricular content; two for \ngames and sports content; four for body expression content; and four apps for \noutdoor PA content. The mean quality score was 4.00. This research contributes \nto the scientific literature a list of apps that, linked to curriculum blocks of PE \ncontent, could help to improve their educational use by PE teachers, consistent \nwith Almusawi et al. (2021), who concluded that PE teachers need technology to \nsave class time, making it more convenient for PE. A second contribution of the \nresearch is the design of possible practical applications in PE of the apps, linked \nto each of the curricular contents, based on the opinion of PE teachers and the \nreview of the scientific literature. This study expands on literature by shedding \nthe need to link the use of apps to the school curriculum and its contents. When we refer to the educational context, teachers must be very demanding \nwith the criteria for selecting the apps to be used. Of the 4650 apps initially iden-\ntified, only 18 were selected for possible application in the educational context. This suggests a proliferation and offer of apps that, although they might seem \nuseful in the educational process, for various reasons are not recommended. Of \nthe 18 selected apps, eight have been linked to the “fitness, health and life qual-\nity” content block, two to “games and sport”, four to “body expression” and four \nto “outdoor physical activities”. This could be motivated by the creation of these \napps for society in general and not specifically for the educational context and, 1 3 3 3 Education and Information Technologies (2022) 27:11759–11779 11770 Table 3 Apps linked to the body expression curriculum content block\n) Based on (Bodsworth & Goodyear, 2017; Gorman, et al. 4 \u0007Discussion 2019; Martínez, 2019; Phelps et al., 2021)\nBody expression\nApp\nDescription\nPossible practical applications in PE()\nAcrosport eps\nPre-made body figures and involve students in a creation process\nLearning application based on creative projects, choreographic compositions, \nrhythm, characterizations, among others. ust dance\nSongs and choreographies without the need for a game console\nTikTok\nIs a video-sharing social networking service\nYoucam makeup\nTry makeup trends with live camera. Edit selfies with retouching able 3 Apps linked to the body expression curriculum content block\n) Based on (Bodsworth & Goodyear, 2017; Gorman, et al. 2019; Martínez, 2019; Phelps et al., 2021)\nBody expression\nApp\nDescription\nPossible practical applications in PE()\nAcrosport eps\nPre-made body figures and involve students in a creation process\nLearning application based on creative projects, choreographic compositions, \nrhythm, characterizations, among others. ust dance\nSongs and choreographies without the need for a game console\nTikTok\nIs a video-sharing social networking service\nYoucam makeup\nTry makeup trends with live camera. Edit selfies with retouching Table 3 Apps linked to the body expression curriculum content block\n() Based on (Bodsworth & Goodyear, 2017; Gorman, et al. 2019; Martínez, 2019; Phelps et al., 2021)\nBody expression\nApp\nDescription\nPossible practical applications in PE()\nAcrosport eps\nPre-made body figures and involve students in a creation process\nLearning application based on creative projects, choreographic compositions, \nrhythm, characterizations, among others. Just dance\nSongs and choreographies without the need for a game console\nTikTok\nIs a video-sharing social networking service\nYoucam makeup\nTry makeup trends with live camera. Edit selfies with retouching Table 3 Apps linked to the body expression curriculum content block\n() Based on (Bodsworth & Goodyear, 2017; Gorman, et al. 2019; Martínez, 2019; Phelps et al., 2021)\nBody expression\nApp\nDescription\nPossible practical applications in PE()\nAcrosport eps\nPre-made body figures and involve students in a creation process\nLearning application based on creative projects, choreographic compositions, \nrhythm, characterizations, among others. Just dance\nSongs and choreographies without the need for a game console\nTikTok\nIs a video-sharing social networking service\nYoucam makeup\nTry makeup trends with live camera. Edit selfies with retouching 1 3 1 3 3 Education and Information Technologies (2022) 27:11759–11779 11771 Table 4 Apps linked to the outdoor PA curriculum content block\n() Based on (Gallego-Lema et al., 2017; Jansson et al., 2019; Michalakis et al., 2020; Ruiz et al. 4 \u0007Discussion 2020)\nOutdoor physical activity\nApp\nDescription\nPossible practical applications in PE()\nGeocaching\nIt is based on hiding and finding “treasures” (objects left by \nusers) with the help of GPS\nCarrying out tours on the natural or urban environment. Interdisciplinarity with other subjects such as geography and history, biology, mathematics, \nphysics and chemistry. Calculation of scales and mathematics. QR scanner\nDecode QR codes directly\nMaps\nNavigate with GPS in real time\nMunzee\nGame based on geolocation, that is, through locations via GPS Table 4 Apps linked to the outdoor PA curriculum content block\n) Based on (Gallego-Lema et al., 2017; Jansson et al., 2019; Michalakis et al., 2020; Ruiz et al. 2020)\nOutdoor physical activity\nApp\nDescription\nPossible practical applications in PE()\nGeocaching\nIt is based on hiding and finding “treasures” (objects left by \nusers) with the help of GPS\nCarrying out tours on the natural or urban environment. Interdisciplinarity with other subjects such as geography and history, biology, mathematics, \nphysics and chemistry. Calculation of scales and mathematics. QR scanner\nDecode QR codes directly\nMaps\nNavigate with GPS in real time\nMunzee\nGame based on geolocation, that is, through locations via GPS 1\nTable 4 Apps linked to the outdoor PA curriculum content block\n() Based on (Gallego-Lema et al., 2017; Jansson et al., 2019; Michalakis et al., 2020; Ruiz et al. 2020)\nOutdoor physical activity\nApp\nDescription\nPossible practical applications in PE()\nGeocaching\nIt is based on hiding and finding “treasures” (objects left by \nusers) with the help of GPS\nCarrying out tours on the natural or urban environment. Interdisciplinarity with other subjects such as geography and history, biology, mathematics, \nphysics and chemistry. Calculation of scales and mathematics. QR scanner\nDecode QR codes directly\nMaps\nNavigate with GPS in real time\nMunzee\nGame based on geolocation, that is, through locations via GPS Table 4 Apps linked to the outdoor PA curriculum content block\n()Based on (Gallego Lema et al 2017; Jansson et al 2019; Michalakis et al 2020; Ruiz et al 2020)\nOutdoor physical activity\nApp\nDescription\nPossible practical applications in PE()\nGeocaching\nIt is based on hiding and finding “treasures” (objects left by \nusers) with the help of GPS\nCarrying out tours on the natural or urban environment. Interdisciplinarity with other subjects such as geography and history, biology, mathematics, \nphysics and chemistry. Calculation of scales and mathematics. 4 \u0007Discussion QR scanner\nDecode QR codes directly\nMaps\nNavigate with GPS in real time\nMunzee\nGame based on geolocation, that is, through locations via GPS 1 3 Education and Information Technologies (2022) 27:11759–11779 11772 therefore, the most of them are related to physical condition, health and quality \nof life. Therefore, it would be advisable to involve and coordinate educational adminis-\ntrations in order to design apps for educational purposes, both in collaboration with \nteachers and with companies specializing in new technologies. In this way, com-\npliance with the inclusion criteria that are determined adequate would be guaran-\nteed and the work of the teaching staff would be facilitated. In this vein, Almusawi \net al. (2021) suggests a focus on readiness encourages collaboration between sectors \nand industries to bring out the best innovative solutions for PE, as well as the col-\nlaborations of different Ministries in the app design. Just as the administration, for \nexample, coordinates, controls and finances the textbooks to be used, one should \nstart thinking about a similar implication with new technologies. The quality of apps \ndesigned for children and adolescents (evaluation of commitment and quality of \ninformation) correlates with the number of techniques identified to change health \nbehaviors (Ng et al., 2019). Another interesting variable to be considered in the \ndesign of the apps is that the interest in the use of mobile devices can have a novel \neffect that later disappears (Ridgers et al., 2018). However, initial motivation could \nserve to establish individual awareness of PA levels. PE teachers must assess the \napps, prior to use with students, so that they comply with the provisions of Organic \nLaw 3/, 2018, on the Protection of Personal Data and guarantee of digital rights, \nwhile also adjusting to the proposed objectives and the students involved. In minors, \nrequest authorization from legal guardians for use by students. Teachers are ultimately responsible for making the decision regarding the selec-\ntion and use of the apps, recommending a group or collective involvement of the \nstudents, whenever possible, in order to avoid isolation. A possible strategy could \nbe to provide challenging activities such as defiant, seeking self-efficacy, motiva-\ntion and that stimulate social support (Martins et al., 2016), paying special attention \nto promoting the adolescent’s perception of competence and positive experiences \n(Martins et al., 2018). In fact, presenting collective challenges has proven to be an \neffective methodological strategy (Gil-Espinosa et al., 2020). 4 \u0007Discussion Thus, physical activi-\nties that promote social relationships or include team activities are more likely to be \nsuccessful (Johansson & Ruud, 2016). Furthermore, combining school interventions \nwith family or community participation appears to be an effective strategy (Parody \net al., 2019). It is important to consider variables such as whether or not they are \nfree, the minimum age for registration and use, the need for authorization from legal \nguardians, the authorization of the educational center for the use of personal mobile \ndevices or the existence of advertising in the apps, among others. In any case, it is essential that teachers frame the use of apps for educational pur-\nposes such as increasing the students’ motivation towards PA, the implementation \nof more students-centered models or the work of digital competence, as some inves-\ntigations have already concluded (Gil-Espinosa et al., 2020; Zhao et al., 2016). In \nthis vein, Lau et al. (2011), in their review, provide evidence supporting the pos-\nitive effects of the use of digital technology in PA interventions for children and \nadolescents. 3 3 11773 Education and Information Technologies (2022) 27:11759–11779 Undoubtedly, the integration of the use of new technologies by students should \nguide us to work towards a responsible, formative, safe and educational use of them. In the case of PE, it is essential to guide students towards apps based on adequate \nscientific and technical criteria. Therefore, teachers must seek to empower students \nin the digital information age and, for this, they must have sufficient training to inte-\ngrate the use of digital technology in learning (Goodyear & Armour, 2018; Pereira \net al., 2019). This is how the Spanish curriculum establishes it in secondary educa-\ntion (Royal Decree 1105/, 2014). Therefore, taking into consideration that PE time during school hours is insuf-\nficient, the increase of PA levels of students during non-school hours should be pro-\nmoted (Gil-Espinosa et al., 2020). In this context, the use of apps is presented as a \nresource to promote strategies that increase adolescents’ PA and health (Böhm et al., \n2019; Cummiskey, 2011; Dute et al., 2016; Simões et al., 2018). Likewise, Lee and \nGao (2020) concluded that teachers are recommended to align the use of apps with \nthe PE learning goals. 4 \u0007Discussion Indeed, Cheng and Chen (2018) argued that the combination \nof traditional and a mobile APP support learning system is an effective approach \nthat would help students to improve their health-related fitness achievements. i\nThe strengths of this study include the systematic search for apps from Google Play, \nthe use of the MARS instrument to assess quality and its relationships with the PE cur-\nriculum in secondary education. Another strength is the participation of PE teachers of \nSecondary Education and University. Further, app ratings were performed by review-\ners and other ones participated in different phases. One highlight of this research is \nthat digital technology can be integrated with the PE curriculum to improve the teach-\ning-learning process and promote PA among students. Future research should test the \noverall effectiveness of apps designed to promote PA among adolescents considering \nits link with the official curriculum. Finally, research examining the accuracy of app \ncontent and developer expertise should also be of high priority. The limitations of this study include the exclusion of apps with low interrater \nreliability for the scoring of app quality through the MARS scale. This may be due \nto the subjective nature of some sections of the scale. The temporal relevancy of the \nresults from this study may also be considered a limitation. Another limitation was \nthe short period used for the individual assessment of each app. Likewise, it would \nbe convenient to carry out similar research in the App store. Conflict of interest The author declares no conflict of interest. Conflict of interest The author declares no conflict of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, \nwhich permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as \nyou give appropriate credit to the original author(s) and the source, provide a link to the Creative Com-\nmons licence, and indicate if changes were made. The images or other third party material in this article \nare included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the \nmaterial. If material is not included in the article’s Creative Commons licence and your intended use is \nnot permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission \ndirectly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licen\nses/by/4.0/. 5 \u0007Conclusions The results suggest that popular apps for measuring and, potentially, promoting PA \nare of moderate quality if we look at its use in the school context. The app content \nquality, particularly the use of international guidelines on linking PA to the official \ncurriculum could be improved. Furthermore, based on the findings from this assess-\nment, we suggest that education administrators and teachers should be involved \nin the development of apps targeting student’s PA behaviors; that apps should be \ndeveloped considering the target group (aged, motivation…) and the respective 3 Education and Information Technologies (2022) 27:11759–11779 11774 PA recommendations established by the WHO (2020); and consider the official \ncurriculum.fl More effort is needed to incorporate components that are more likely to influ-\nence PA behavior change, which is ultimately what they were developed for, while \nmaintaining good functionality. Educational administration and developers should \ncollaborate to provide self-monitoring and social comparison in students to enhance \na comprehensive, competency-based education. In summary, this study seeks to \nadvance research on the use of apps in PE, focusing on its link with the curriculum \ncontents. Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Funding for open access charge: Universidad de Málaga / CBUA. Rafael Burgueño is, specifically, sup-\nported by a “Margarita Salas” postdoctoral fellowship (grant number: RR_A_2021_02) from the Spanish \nMinistry of Universities. Adriana Nielsen-Rodríguez is supported by “Formación del Profesorado Univer-\nsitario” grants from the Ministry of Education, Culture and Sport (Spain), grant number FPU17/01554. Data availability The dataset generated for this study is available on request to the corresponding author. Declarations Competing interests The authors report there are no competing interests to declare. References Almusawi, H. A., Durugbo, C. M., & Bugawa, A. M. (2021). Innovation in physical education: Teach-\ners’ perspectives on readiness for wearable technology integration. Computers and Education, 167, \n104185. https://doi.org/10.1016/j.compedu.2021.104185 p\ng\nj\np\nAndre, M. 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National stand-\nards & grade-level outcomes for K-12 physical education: Vol. Champaign, IL (Human Kinetics.). https://www.shapeamerica.org/standards/pe/upload/Grade-Level-Outcomes-for-K-12-Physical-\nEducation.pdf Simões, P., Silva, A. G., Amaral, J., Queirós, A., Rocha, N. P., & Rodrigues, M. (2018). Features, behav-\nioral change techniques, and quality of the most popular mobile apps to measure physical activity: \nSystematic search in app stores. JMIR mHealth and uHealth, 6(10), e11281. https://doi.org/10.2196/\n11281 Sinelnikov, O. A. (2012). Using the iPad in a sport education season. Journal of Physical Education, Rec-\nreation & Dance, 83(1), 39–45. https://doi.org/10.1080/07303084.2012.10598710 p\ng\nStoyanov, S. R., Hides, L., Kavanagh, D. J., Zelenko, O., Tjondronegoro, D., & Mani, M. (2015). Mobile \napp rating scale: A new tool for assessing the quality of health mobile apps. JMIR mHealth and \nuHealth, 3(1), e27. https://doi.org/10.2196/mhealth.3422 p\ng\nVega-Ramírez, L., Notario, R. O., & Ávalos-Ramos, M. A. (2020). The relevance of mobile applica-\ntions in the learning of physical education. Education Sciences, 10(11), 329. https://doi.org/10.3390/\neducsci10110329 WHO (2020) (9th May 2022). WHO Guidelines on physical activity and sedentary behaviour. In World \nHealth Organization. https://www.who.int/publications/i/item/9789240015128 Yu, H., Hodges P.H. &. Lorenz, K.A. (2018). An integration of Mobile applications into physical educ\ntion programs. Strategies, 31(3), 13–19, https://doi.org/10.1080/08924562.2018.1442275.f Yu. (2020). The effects of Mobile app technology on technique and game performance in physical educ\ntion. ProQuest Dissertations Publishing.l Zhao, J., Freeman, B., & Li, M. (2016). Can mobile phone apps influence people’s health behavior \nchange? An evidence review. Journal of Medical Internet Research, 18(11), e287. https://doi.org/\n10.2196/jmir.5692 Zhu, X., & Dragon, L. A. (2016). Physical activity and situational interest in mobile technology inte-\ngrated physical education: A preliminary study. Acta Gymnica, 46(2), 59–67. doi.org/10.2196/mhealth.9199 https://doi.org/10.\n5507/ag.2016.010 Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published \nmaps and institutional affiliations. 1 3 1 3 11779 Education and Information Technologies (2022) 27:11759–11779 Authors and Affiliations\nFrancisco Javier Gil‑Espinosa1 · Adriana Nielsen‑Rodríguez2 · \nRamón Romance2 · Rafael Burgueño3 \n\t\nAdriana Nielsen‑Rodríguez \n\t\nadriananielsen@uma.es\n\t\nRamón Romance \n\t\narromance@uma.es\n\t\nRafael Burgueño \n\t\nrmburgueno@ual.es\n1\t\nAndalucía Tech, Faculty of Educational Sciences, IBIMA, Researching in Sport Sciences (RSS) \nResearch Group, Universidad de Málaga, Campus de Teatinos s/n, 29010 Málaga, Spain\n2\t\nAndalucía Tech, Faculty of Educational Sciences, Department of Didactics of Languages, \nArts and Sports, Human Kinetics and Body Composition Laboratory, Universidad de Málaga, \nCampus de Teatinos s/n, 29010 Málaga, Spain\n3\t\nDepartment of Education, University of Almeria, Almeria, Spain Francisco Javier Gil‑Espinosa1 · Adriana Nielsen‑Rodríguez2 ·\nRamón Romance2 · Rafael Burgueño3 Adriana Nielsen‑Rodríguez \nadriananielsen@uma.es Ramón Romance \narromance@uma.es 1\t\nAndalucía Tech, Faculty of Educational Sciences, IBIMA, Researching in Sport Sciences (RSS) \nResearch Group, Universidad de Málaga, Campus de Teatinos s/n, 29010 Málaga, Spain 1\t\nAndalucía Tech, Faculty of Educational Sciences, IBIMA, Researching in Sport Sciences (RSS) \nResearch Group, Universidad de Málaga, Campus de Teatinos s/n, 29010 Málaga, Spain 2\t\nAndalucía Tech, Faculty of Educational Sciences, Department of Didactics of Languages, \nArts and Sports, Human Kinetics and Body Composition Laboratory, Universidad de Málaga, \nCampus de Teatinos s/n, 29010 Málaga, Spain 3\t\nDepartment of Education, University of Almeria, Almeria, Spain 1 3"
https://openalex.org/W4361938710	https://figshare.com/articles/journal_contribution/Supplementary_Data_from_Proliferation_and_AKT_Activity_Biomarker_Analyses_after_Capivasertib_AZD5363_Treatment_of_Patients_with_ER_sup_sup_Invasive_Breast_Cancer_STAKT_/22479237/1/files/39930741.pdf	English	null	Supplementary Data from Proliferation and AKT Activity Biomarker Analyses after Capivasertib (AZD5363) Treatment of Patients with ER<sup>+</sup> Invasive Breast Cancer (STAKT)	null	2,023	cc-by	38	Supplementary Figure 1. The PI3K/AKT/mTOR signaling pathway and proof-of- mechanism biomarkers for capivasertib Supplementary Figure 1. The PI3K/AKT/mTOR signaling pathway and proof-of- mechanism biomarkers for capivasertib pp y g g g p y p mechanism biomarkers for capivasertib
https://openalex.org/W2093069073	https://zenodo.org/records/1518863/files/article.pdf	English	null	LABORATORY AND PLANT: AN APPARATUS FOR DIGESTING CRUDE FIBER	The journal of industrial and engineering chemistry/Journal of industrial and engineering chemistry	1,916	public-domain	2,381	T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y Vol. 8, No. The water in falling to E sucks in a sample of the air to he tested. Next the valve M is closed and the platinum wire N electrically heated. The methane in the com- bustion chamber burns to carbon dioxide and water, thus: CH, i- 2 0 2 = COS + 2H20 , i. e., contraction in volume of the sample occurs corresponding to the amount of methane originally present in the sample. At the end of I'/~ min. the electric current is turned off and the instrument shaken t o cool the gases in the combustion space and bring them to the same temperature as the gases were a t the beginning of the test. The water in the combustion space will then rise to take the place of the burned-out gas and fall a corresponding distance in the glass tube B, i. e., fa!l to a point on the graduated scale that will show the per cent of methane originally in the sample. A previous calibration, once and for all time, fixes the proper graduations on this scale. The latter carries four graduation columns, one for methane and natural gas, one for hydrogen, one for gasoline vapor, and one for coal gas. FIO. I The electrical energy for heating the platinum wire is derived from a miner's electric cap lamp storage battery, thereby providing the electric cap lamp with a gas detector, something it doe's not possess at present and which has been an objection to the use of electric lamps in gaseous mines. A t Fig. 111 is shown another gas detector similar in operation except it contains two small dry cells, A and B, for supplying the electrical energy. These cells cost about 7 cents apiece and will operate the detector for a minimum of 20 determinations. When they are used up, they are as easily replaced (by un- screwing the cap D ) as the dry cells of the well-known Ever-Ready flashlight batteries. FIO. I about 7 . j cm., length (vertical) about 10 cm.; this tube is soldered to the tube (d) about 3 . 5 cm. above the cone.' The author is indebted t o several members of the Bureau of Mines for assistance in developing this in- strument. Dr. G. A: Hulett, Consulting Chemist, first suggested that an apparatus of this nature be con- structed, and 0. P. T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y Vol. 8, No. Hood, Chief Mechanical Engineer, rendered valuable assistance in designing it. THE DELIVERY TUBES-The cooling water is deliv- ered into the condensers from the water-main by branch tubes each provided with a stop-cock. These cocks are ordinary brass gas-cocks, in the nozzles of which are soldered copper tubes ahout 4 mm. inside diameter and 2 4 cm. long. The adjustment should be such that the lower ends of these tubes are about 4 mm. above the bottoms of the condensers when they are suspended and about I to 2 cm. below the top of the cone when the condensers rest on the beakers. The condensers are suspended from a little catch or hooks soldered to the delivery tubes. Whether the condensers he suspended above the beakers or he resting on them, the water circulates through them freely and spills into a trough back of the heakers- no rubber connections. A horizontal strip with holes in it, through which the overflow tubes pass, keeps the exit ends of these tubes centered over the trough. I t is important that these ends should always he visi- Patent claims have been filed by the Bureau of Mines on the above instruments and means for ex- ploiting it are being considered so the best interests of the public will be served. PITTSBUROH PITTSBUROH I Since writins the above. an ordinary glass flask. whore neck i s pro- vided with a suitable side-tube. has been used quite satisfactorily for several months instead of one of the metallic condensers. I TXIS JOUENAL, a (i910). 280. T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y Vol. 8, No. T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y Vol. 8, No. 366 THE CONDENSERS-In Fig. 1 eight of the condensers are shown resting on the beakers. Two are shown sus- pended above the beakers. One is suspended. giving a view of the tube, through which the cooling water passes out of the condenser. Another, lying on the heating plate, affords a top view. The parts of a condenser (see Fig. 11) are: (a) a zinc or copper-in this case it chances to be zinc-shell (hemisphere) 7 . 6 cm. in diameter; (b) a copper ring, similar to those used on water baths, about I O cm. in diameter, the circular opening in the ring being about 5 . 7 cm. in diameter, this ring is soldered to the shell; (c) a copper cone whose big end is about j . 7 cm. in diam- eter, small end about I cm. in diameter ( I . 5 cm. would be better) and vertical height ahout 4 . 5 cm., this cone is soldered to the ring; ( d ) a copper tube about I cm. in diameter (I. 5 cm. would be better). and about 11 cm. long, soldered to the cone; in the side of this tube near its top is a small hole, by means of which the condenser may be suspended on a catch or hook on the influx tube; ( e ) an elbow-shaped exit, or over- flow, tube, ahout 4 mm. in inside diameter (I cm. would he better), length (horizontal) to the elbow valve M. Next, the instrument is raised t o the place where the sample is to be collected and the water allowed to seek the former levels at D and E . The water in falling to E sucks in a sample of the air to he tested. Next the valve M is closed and the platinum wire N electrically heated. The methane in the com- bustion chamber burns to carbon dioxide and water, thus: CH, i- 2 0 2 = COS + 2H20 valve M. Next, the instrument is raised t o the place where the sample is to be collected and the water allowed to seek the former levels at D and E . AN APPARATUS FOR DIGESTING CRUDE FIBER By J. M. P~CBBL Received May 28, 1915 In an article'.by the author several years ago, it was stated that round-bottom flasks, through which cold water is passing, set on the beakers in which crude fiber is digesting, prevent evaporation and hold froth- ing in check. The apparatus pictured here is the out- growth of that idea. The distinctive feature of it is the round-bottom reflux condenser. T H E J O C R N A L OF I N D C S T R I A L A N D ENGINEERING C H E M I S T R Y 367 by the heat of one small Bunsen burner; and the ash is as nearly perfect as the writer and designer of the furnace has ever seen. ble so that one may at any time know that there is ample flow of water through the condensers. These tubes should not, therefore, dip into the trough. The trough (galvanized sheet iron) is about 3 . 5 cm. wide, wafer about 13 cm. deep and. of course, extends along the entire length of the “plant.” A piece of asbestos board 0 . 7 X 19 X 19 cm., in whose center is cut a circular opening Q cm. in diam- eter, is laid on an ordinary laboratory tripod. On this board is set a disc of wrought iron (cast iron would probably be better) about 2.5 mm. thick and 13.5 cm. in diameter (14. j cm. would be better since it would furnish space for several more incinerations). The disc is supported on three legs, I cm. long, screwed into it. On the disc are set 1 2 crucibles. alundum RA 98, 3 . 8 cm. high and 3.7 cm. in outside diameter, in which the fiber has been filtered, washed, dried and weighed. On the asbestos board is placed an as- bestos cylinder 15. j cm. in diameter and 6 . 5 cm. deep. The cylinder is covered with a piece of asbestos board of the same dimensions as the one previously described, but having in its center a hole only 3. j cm. in diameter. A small Bunsen burner, whose gas tip has been widened somewhat by inserting the point of a penknife blade, furnishes the heat. Received November 15, 1915 Received November 15, 1915 Occasion arose in this laboratory to make use of a gas-heated thermostat, but a great deal of incon- venience was experienced because of varying gas pressure. I n casting about for some simple pressure regulator, the apparatus described below and illus- trated in Fig. I was finally hit upon as a practical solution of the trouble. It will be noted that the apparatus is constructed from material readily ob- tainable in almost any laboratory; also that there is no great skill required for its assembly. Practically every chemist has the rudimentary knowledge of glass-blowing necessary to make the one “Y” re- quired. LABORASORY OF THE NORSH CAROLINA DEPARTMENT OF AGRICULSURE, RALEIGH LABORASORY OF THE NORSH CAROLINA DEPARTMENT OF AGRICULSURE, RALEIGH AN APPARATUS FOR DIGESTING CRUDE FIBER By J. M. P~CBBL Received May 28, 1915 The burner should be set o n a block so as to bring its top close to the iron disc, thus causing the flame to spread over the under surface of the disc. In a few minutes (6 or 8) the disc and the crucibles will be brought to a bright glow. wafer FIG. 11-REFLUX CONDENSE FOR USE ON BEAKERS IN CRUDE FIBER DESER- ( 1 1 4 Size) MINASION FIG. 11-REFLUX CONDENSER FOR USE ON BEAKERS IN CRUDE FIBER DESER- ( 1 1 4 Size) MINASION blast could, however, e t e e gt o t e p a t. These condensers prevent completely loss of liquid by evaporation, and suppress, or hold sufficiently in check, frothing. But the boiling must be started gently and conducted gently; violent ebullition is not at all neces- sary and is to be avoided. Frothing is due to bubbles filled with steam-and doubt- less to some extent with hot air; condense the steam and the bubbles or froth collapse. The upper region of the beakers is kept cool by the condensers; hence there is no need of a cold blast of air-it is the cold- ”ress, not the blast, that checks the frothing. A cold air- be easily introduced through these condensers. Such a condenser is shown on a beaker in the foreground of Fig. I. FIG. 11-REFLUX CONDENSE FOR USE ON BEAKERS IN CRUDE FIBER DESER- ( 1 1 4 Size) MINASION The cylinder is easily and quickly made. Strips of suitable width (about 6. j cm.) are cut from asbestos board of suitable thickness (about 7 mm.) and their ends beveled by shaving with a sharp knife. These strips are saturated with water, and, while wet, are wound, two or three thicknesses, around a suitable core (an empty 2-kilo ether can dr a piece of sheet- iron stovepipe), bound in place by two or three bands of wire and allowed to dry out at room temperature and finally on or near a steam radiator. The core is removed and the lapped ends of the strips riveted. Beakers of joo cc. capacity, 7 . j cm. inside diam- eter and 14 cm. high, Jena glass, are preferred; but beakers of 600 cc. capacity, 8 cm. diameter, I j . 5 cm. high, can be used interchangeably with the smaller ones. AN APPARATUS FOR DIGESTING CRUDE FIBER By J. M. P~CBBL Received May 28, 1915 The beakers can be given a rotary shaking without lifting them from the heating plate. After the boiling has been gotten under way and the beak- ers have been rotated a time or two, the apparatus may be left to itself. LABORATORY OF SHE NORTH CAROLINA DEPARSMENT OF AGRICULTURE, RALEIGH LABORATORY OF SHE NORTH CAROLINA DEPARSMENT OF AGRICULTURE, RALEIGH The heating plate (wrought iron) is about 4 mm. thick and has a top surface about 9 . j cm. wide. Along its entire length is turned a flange about 2. j cm. wide. The object of the flange is to give rigidity and prevent sagging or buckling. LABORATORY OF SHE NORTH CAROLINA DEPARSMENT OF AGRICULTURE, RALEIGH A FURNACE FOR CRUDE FIBER INCINERATION By J. M. PICKEL Received May 28, 1915 If the chemist have at his dispbsal the requisite electric current and $50 to $100, he will be apt to invest in an electric furnace. If he have but a dollar or two, he can, with that capital and with materials ready to hand in the laboratory, construct a wonderfully efficient incinerating furnace. Such a furnace, oc- cupying on the table a space 2 0 X 2 0 cm. (8 X 8 in.), is shown in Fig. I in the previous article (page 366), at the left uncovered, at the right as it appears when performing. Twelve crude fiber incinerations are made in I j to 2 0 min. on, or in, this furnace A and C are rigidly fastened, in the position shown, to a board which acts as a mounting for the whole apparatus. B is suspended inside of A on the end of E by a rubber stopper. E is so arranged that it may
W2897117717.txt	https://www.repo.uni-hannover.de/bitstream/123456789/4114/1/Dzelme_2018_IOP_Conf._Ser.__Mater._Sci._Eng._424_012083.pdf	en		Simulation of electrically induced vortical flows	IOP conference series. Materials science and engineering	2,018	cc-by	1,743	IOP Conference Series: Materials Science and Engineering PAPER • OPEN ACCESS Simulation of electrically induced vortical flows To cite this article: V. Dzelme et al 2018 IOP Conf. Ser.: Mater. Sci. Eng. 424 012083 View the article online for updates and enhancements. This content was downloaded from IP address 194.95.159.70 on 29/11/2018 at 11:29 9th International Symposium on Electromagnetic Processing of Materials (EPM2018) IOP Publishing IOP Conf. Series: Materials Science and Engineering 424 (2018) 012083 doi:10.1088/1757-899X/424/1/012083 1234567890‘’“” Simulation of electrically induced vortical flows V. Dzelme1, A. Jakovics1, A. Chudnovsky2, E. Baake3 1 Laboratory for Mathematical Modelling of Environmental and Technological Processes, Department of Physics, University of Latvia, 23 Zellu street, Riga, Latvia 2 BIS Global Ltd., 79/81-15 Kr. Valdemara street, Riga, Latvia 3 Institute of Electrotechnology, Leibniz University of Hannover, 4 Wilhelm Busch street, D-30167 Hannover, Germany Corresponding author : valters.dzelme@lu.lv Abstract In this work, we study electrically induced flows numerically using open-source software and experimentally. Two systems are considered - single and multiphase (free surface) flows driven by axisymmetric DC and AC current injection. We investigate characteristic velocity and free surface deformation dependence on the injected current and validate it with experimental data. Results show that maximum axial velocity is a linear function of injected current, but free surface deformations are approximately proportional to current squared. Keywords : magnetohydrodynamics, electrovortex, simulation Introduction Electrically induced flows are found in many applications – arc furnaces, welding, etc. [1]. The flow is driven by Lorentz force created by the interaction between injected current and its own magnetic field. Depending on specific electrode configuration different vortical structures can form. Electrovortical phenomena have been extensively studied theoretically and experimentally [1]. Numerical work usually considers simplified systems, such as hemispherical arc furnaces [2][3] using commercial software. Free and open-source software is not widely used mostly due to lack of user interface and complicated coupling between, for example, electromagnetic and fluid dynamics simulations. The recent development of EOF-Library [4], which is a coupler between Elmer [5] and OpenFOAM [6] and is an open-source library itself, simplifies the task of simulating complex coupled systems. The coupling library has been validated against commercial solutions [7]. The goal of this work is to apply and validate open-source software for electromagnetically induced vortical flows, as well as to study two specific systems with industrial applications in mind. The first (system A) is a small cylindrical container with electric current flowing through the melt between a small electrode on top and conducting bottom of container. It is a model system of typical DC arc furnaces. The second system (system B) is more complex – it consists of cylindrical container with 50Hz alternating current flowing between a small bottom electrode and conducting side wall. In this case, the current flows through a layer of liquid metal causing free surface deformations near the small electrode at the center. Possible application of this system can be slag removal from the melt in special applications – surface deformation is pushing the slag towards the side wall where it can be easily collected – this is demonstrated experimentally. Another application could be surface wave generation and flow intensification for improved melt purification via evaporation from the surface. Numerical model Software used for electromagnetics is Elmer, which solves the Maxwell’s equations in potential formulation using the finite element method, but for fluid dynamics – OpenFOAM, which solves the Navier-Stokes, k-ω SST turbulence model equations, and for system B also transport equation for volume fraction, using the finite volume method. The coupling is achieved with the EOF-Library. The schemes of system A and B are shown in Fig. 1 and 2, respectively. Relevant material properties are listed in Tab.1. Since the 2D axisymmetric solver in Elmer is not designed to solve for azimuthal component of magnetic flux density (or current in meridional plane), quasi 2D approach is used modelling 2° sector of full cylinder. Electromagnetics mesh for system A consists of 133k hexahedral elements (three layers in azimuthal direction), but system B – 180k hexahedral elements (two layers in azimuthal direction). Fluid flow is also solved in quasi 2D approximation as 2° sector of full cylinder, as OpenFOAM does not have pure 2D capabilities. Fluid dynamics mesh for system A is 22k hexahedral elements (one element thick wedge), but for system B – 250k hexahedral elements (one element thick wedge). The electromagnetic force is computed only once, and the flow simulation is performed with constant momentum source. Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 9th International Symposium on Electromagnetic Processing of Materials (EPM2018) IOP Publishing IOP Conf. Series: Materials Science and Engineering 424 (2018) 012083 doi:10.1088/1757-899X/424/1/012083 1234567890‘’“” Fig. 1: System A Fig. 2: System B Table 1. Material properties Melt density, kg/m3 Melt electric cond., S/m Electrode electric cond., S/m Melt viscosity, mPa·s Surface tension, N/m System A 13534 8.6·105 5.8·107 2.20 - System B 6440 3.46·106 5.8·107 2.40 0.534 Experiments Experimental results for system A are taken from [8], where liquid mercury flow velocity was measured using optical fiber sensor. Experiments for system B are performed at the Institute of Electrotechnology of the Leibniz University of Hannover. The picture of the setup is shown in Fig. 3. The two side electrodes are of the same polarity; the other pole is a small electrode at the bottom not seen here; dimensions as shown in Fig.2. Bottom wall of the container is plastic, so that the current is flowing from the bottom electrode to the side wall only through the melt. The current is provided by a 50Hz voltage source. At 50Hz, the skin depth in liquid galinstan is approximately 4cm. Fig. 3: System B experimental setup Results System A Example of current and velocity distributions are shown in Fig. 4 and 5, respectively. The highest current density is near the edge of the top electrode, and this is also where the Lorentz force concentrates, pushing the melt away from the electrode. Axial velocity distribution on axis for various injected current values is shown in Fig. 6. In Fig. 7, we plot maximum axial velocity (20mm from the top electrode) as a function of injected current. The theoretical analysis provided in [8] says that axial velocity in such system is proportional to the injected current and it is seen in simulation and experimental results. 2 9th International Symposium on Electromagnetic Processing of Materials (EPM2018) IOP Publishing IOP Conf. Series: Materials Science and Engineering 424 (2018) 012083 doi:10.1088/1757-899X/424/1/012083 1234567890‘’“” Fig. 4: Example current distribution Fig. 5: Example velocity distribution, I=1kA Fig. 6: Velocity on axis Fig. 7: Max. axial velocity vs current System B Example of current distribution (without surface deformation) is shown in Fig. 8 and velocity and surface deformation at Irms=1000A is shown in Fig. 9. Free surface deformation height depending on current squared is shown in Fig. 10. In Fig. 11, we plot maximum axial velocity as a function of injected current. Fig. 8: Example current distribution, h0=20mm Fig. 9: Example velocity and surface deformation, h0=20mm, Irms=1kA Fig. 10: Surface deformation vs current squared Fig. 11: Max. axial velocity vs current 3 9th International Symposium on Electromagnetic Processing of Materials (EPM2018) IOP Publishing IOP Conf. Series: Materials Science and Engineering 424 (2018) 012083 doi:10.1088/1757-899X/424/1/012083 1234567890‘’“” In this system, no velocity measurements were performed at this stage, but are planned in the future. Deformation height disagreement between experiments and simulations can be due to many reasons. First, the surface height measurements were not done using precise equipment – they were manual measurements using simple tools. Second, the material properties (electric conductivity, viscosity etc) were not exactly known – values taken from literature may not be completely the same as in the experiments. Third, even the slightest imperfections (e.g., electrode centering) can cause different flow patterns and surface deformations. The numerical model, of course, is an idealization, especially being axisymmetric which does not allow the jet movement away from the axis. Nevertheless, the results of both simulation and experiment are the same order of magnitude and show the same tendency – in the considered range of currents surface deformation is approximately proportional to the square of injected current. Finally, to study possible slag removal towards container walls by free surface deformation, we use small (d=2mm) plastic particles on the surface. Particle distribution without current and with Irms=1000A is shown in Fig. 12 and 13, respectively. It is clearly seen that the surface deformation moves the “slag” away from the zone above the small bottom electrode. To move the particles even closer to the side wall, larger or several electrodes, or higher current could be used. Fig. 13: Particle “slag” distribution; h0=20mm, Irms=1000A Fig. 12: Particle “slag” distribution; h0=20mm, no current Conclusions Firstly, numerical model of single-phase electrically induced flow in a cylindrical container was validated using experimental data – maximum axial velocity is in a good agreement to the measurements. The proportionality of axial velocity to the injected current is also a result agreeing to theoretical considerations [8]. An experimental device was created to study liquid metal free surface deformations induced by current flowing between small bottom electrode and conducting side wall. It was found that the deformation height is approximately proportional to square of the injected current in the considered range. Like in the first system, the maximum axial velocity is proportional to current. The goal of applying open-source software for electrovortical flow simulations is achieved. The coupling solution EOFLibrary is a very efficient interface between Elmer and OpenFOAM, which enables virtually any variable transfer and interpolation between different meshes of electromagnetic and fluid flow simulation. Further experimental and numerical work will include different bottom electrode configurations to control the free surface deformations, as well as studying the process instabilities, as the deformation height was observed to be quite unstable, which can be of even more importance in industry-scale equipment. References 1. V. Bojarevics, J. A. Freibergs, E.I. Shilova, E.V. Scherbinin, Electrically Induced Vortical Flows, Kluwer Academic Publishers, 1989 2. O. Kazak, Magnetohydrodynamics, Vol. 50 (2014), pp. 193-205 3. Y. Ivochkin et al., Magnetohydrodynamics, Vol. 51 (2015), pp. 337-343 4. EOF-Library, https://eof-library.com 5. ElmerFEM, https://www.csc.fi/web/elmer 6. OpenFOAM, https://openfoam.org 7. J. Vencels et al., Magnetohydrodynamics, Vol. 53 (2017), pp. 643–652 8. V.G. Zhilin et al., Magnetohydrodynamics, Vol. 22 (1986), pp. 110-116 4
https://openalex.org/W3112768262	https://uia.brage.unit.no/uia-xmlui/bitstream/11250/2733119/4/Peck.pdf	English	null	Small pelagic fish in the new millennium: A bottom-up view of global research effort	Progress in oceanography/Progress in Oceanography	2,021	cc-by	49,866	* Corresponding author. E-mail address: myron.peck@nioz.nl (M.A. Peck). Small pelagic fish in the new millennium: A bottom-up view of global research effort a Department of Coastal Systems (COS), Royal Netherlands Institute for Sea Research (NIOZ), PO Box 59, 1790 AB Den Burg, Texel, The Netherlands b Leibniz Institute for Baltic Sea Research Warnemünde, Seestr. 15, 18119 Warnemünde, Germany c Institut de Recherche pour le D´eveloppement (IRD), MARBEC, Univ Montpellier, CNRS, Ifremer, IRD, Av. Jean Monnet, 34203 S`ete, France d Institut Mediterrani d’Estudis Avançats, IMEDEA (UIB-CSIC), Miquel Marqu´es 21, 07190 Esporles, Illes Balears, Spain e Portuguese Institute for Marine and Atmospheric Science (IPMA), Rua Alfredo Magalh˜aes Ramalho 6, 1449-006 Lisbon, Portugal f Centre for Coastal Research, University of Agder, Universitetsveien 25, 4630 Kristiansand, Norway g NOAA National Marine Fisheries Service, Pacific Islands Fisheries Science Center, 1845 Wasp Blvd., Honolulu, Hawaii 96818, USA h Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan i Department of Environment, Forestry and Fisheries (DEFF), Private Bag X2, Vlaeberg 8000, Cape Town, South Africa j Marine Research Institute and Department of Biological Sciences, University of Cape Town (UCT), Private Bag X3, Rondebosch 7700, Cape Town, South Africa k Department of Oceanography, University of Hawaiʻi at M¯anoa, 1000 Pope Rd., Honolulu, Hawaiʻi 96822 USA A B S T R A C T Small pelagic fish (SPF) play extremely important ecological roles in marine ecosystems, form some of the most economically valuable fisheries resources, and play a vital role in global food security. Due to their short generation times and tight coupling to lower trophic levels, populations of SPF display large boom-and-bust dynamics that are closely linked to climate variability. To reveal emerging global research trends on SPF as opposed to more recently published, ecosystem- specific reviews of SPF, we reviewed the literature published in two, 6-year periods in the new millennium (2001–2006, and 2011–2016) straddling the publica tion of a large, global review of the dynamics of SPF in 2009. We explored intrinsic and extrinsic (bottom-up) factors influencing the dynamics of SPF such as anchovies, sardines, herrings and sprats within the sub-order Clupeidae. Published research efforts within 16 different biogeographic ocean regions were compiled (more than 900 studies) and compared to identify i) new milestones and advances in our understanding, ii) emerging research trends and iii) remaining gaps in knowledge. Studies were separated into 5 categories (field, laboratory, mesocosms, long-term statistical analyses and spatially-explicit modelling) and discussed in relation to 10 bottom-up categories including 5 abiotic factors (temperature, salinity, pH, dissolved oxygen, density), 3 physical processes (advection, turbulence, turbidity) and 2 biotic factors (prey quantity and quality). The peer-reviewed literature reflects changes in the number of studies between the two time periods including increases (Mediterranean Sea, Humboldt Current) and decreases (Australia, Benguela Current). Our review highlights i) gaps in ecological knowledge on young juveniles and, in general, on the impacts of hypoxia and heatwaves on SPF, ii) the utility of paleo studies in exploring population drivers, iii) the continued need to develop spatially-explicit, full life-cycle models, iv) the importance of exploring how density-dependent processes impact vital rates (growth, survival, reproduction), and v) the benefits of international collaboration for knowledge transfer and building unifying hypotheses on the role of bottom-up factors and processes that regulate SPF populations. 1. Introduction anchoveta (Engraulis ringens) were landed by the largest single-species fishery in the world, while another six species of SPF were within the top 25 major global fisheries (FAO, 2017). Stocks of SPF such as Peru vian anchoveta are notorious for their rapid, large-scale changes in distribution and/or productivity in response to climate-mediated changes in bottom-up forcing (Lluch-Belda et al., 1992; Schwartzlose et al., 1999; Tourre et al., 2007; Salvatteci et al., 2018). For example, the large harvest of Peruvian anchoveta in 2015 was a 37% increase compared to 2014. These fishes are also emblematic of the relatively anchoveta (Engraulis ringens) were landed by the largest single-species fishery in the world, while another six species of SPF were within the top 25 major global fisheries (FAO, 2017). Stocks of SPF such as Peru vian anchoveta are notorious for their rapid, large-scale changes in distribution and/or productivity in response to climate-mediated changes in bottom-up forcing (Lluch-Belda et al., 1992; Schwartzlose et al., 1999; Tourre et al., 2007; Salvatteci et al., 2018). For example, the large harvest of Peruvian anchoveta in 2015 was a 37% increase compared to 2014. These fishes are also emblematic of the relatively y y y g a Department of Coastal Systems (COS), Royal Netherlands Institute for Sea Research (NIOZ), PO Box 59, 1790 AB Den Burg, Texel, The Netherlands b Leibniz Institute for Baltic Sea Research Warnemünde, Seestr. 15, 18119 Warnemünde, Germany c Institut de Recherche pour le D´eveloppement (IRD), MARBEC, Univ Montpellier, CNRS, Ifremer, IRD, Av. Jean Monnet, 34203 S`ete, France d Institut Mediterrani d’Estudis Avançats, IMEDEA (UIB-CSIC), Miquel Marqu´es 21, 07190 Esporles, Illes Balears, Spain e Portuguese Institute for Marine and Atmospheric Science (IPMA), Rua Alfredo Magalh˜aes Ramalho 6, 1449-006 Lisbon, Portugal f Centre for Coastal Research, University of Agder, Universitetsveien 25, 4630 Kristiansand, Norway g NOAA National Marine Fisheries Service, Pacific Islands Fisheries Science Center, 1845 Wasp Blvd., Honolulu, Hawaii 96818, USA h Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan i Department of Environment, Forestry and Fisheries (DEFF), Private Bag X2, Vlaeberg 8000, Cape Town, South Africa j Marine Research Institute and Department of Biological Sciences, University of Cape Town (UCT), Private Bag X3, Rondebosch 7700, Cape Town, South Africa k Department of Oceanography, University of Hawaiʻi at M¯anoa, 1000 Pope Rd., Honolulu, Hawaiʻi 96822 USA Progress in Oceanography 191 (2021) 102494 Progress in Oceanography 191 (2021) 102494 Available online 16 December 2020 0079-6611/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.pocean.2020.102494 Received 19 July 2020; Received in revised form 22 November 2020; Accepted 7 December 2020 Available online 16 December 2020 0079-6611/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). * Corresponding author. E-mail address: myron.peck@nioz.nl (M.A. Peck). https://doi.org/10.1016/j.pocean.2020.102494 Received 19 July 2020; Received in revised form 22 November 2020; Accepted 7 December 2020 1.1. Importance of small pelagic fish Catches of anchovies, sardines, herrings and other small pelagic clupeoid fishes are not only important for direct human consumption but also critical for fishmeal and fish oil used in agri- or aqua-feeds, making small pelagic fish (SPF) significant for global food security (FAO, 2016). In 2015, 4.3 million metric tonnes (MMT) of Peruvian Progress in Oceanography 191 (2021) 102494 M.A. Peck et al. rapid (regime) shifts (dramatic and abrupt changes in the abundance and relative dominance of SPF that are persistent in time) documented in many marine ecosystems worldwide (e.g., Alheit and Niquen, 2004; Alheit et al., 2005; Watanabe, 2009). The high inter-annual variability of SPF biomass, their sensitivity to climate variability, and the occur rence of productivity regimes that are difficult to predict in terms of onset and duration all pose challenges to the sustainable management and efforts to prevent overfishing of SPF populations. These fish also play a pivotal role in marine food webs by acting as conduits of energy from lower to upper trophic levels (Pikitch et al., 2012). Thus, from both an ecological and a societal point of view, it is essential that we gain a full understanding of the factors and processes that drive changes in the productivity of populations and management units (stocks) of these fishes. This knowledge can buttress science-based advice to fisheries management (e.g., Kaplan et al., 2016) as well as the robustness of longer-term projections of future impacts of climate change (MacKenzie et al., 2012; Peck et al., 2013). ancillary manner, or iv) focused on predators of SPF (seabirds, marine mammals and fish). The majority of excluded studies was within the latter category. At this point, the list contained 374 papers published from 2001 to 2006 and 376 papers published from 2011 to 2016. Some papers were not found by our search string and, based on our collective knowledge and expertise in specific regions, a further 195 studies were added. The final list contained 945 studies (434 in 2001–2006 and 511 in 2011–2016). We recognize that there is insightful research that has or is being conducted that may not be included here, and we do not intend to be insular in our assessment. Rather the approach taken is an analysis of trends in the mass literature, not an analysis of the most important (or most all inclusive) research on planktivorous fishes. 1.3. Categories of studies The following search was performed on ISI Web of Science: “Sprattus” OR sprat OR “Engraulis encrasicolus” OR “European an chovy” OR “Cape anchovy” OR “Northern anchovy” OR “Sardina pil chardus” OR “European sardine” OR “European pilchard” OR “European sprat” OR “Engraulis japonicus” OR “Japanese anchovy” OR “Sardinops melanostictus” OR “Sardinops sagax” OR “Japanese sardine” OR “Pacific sardine” OR “South American pilchard” OR “Engraulis mordax” OR “Californian anchovy” OR “Engraulis mitchilli“ OR “Anchoa mitchilli” OR “Bay anchovy” OR “Clupea harengus“ OR “Atlantic herring” OR “Pacific herring” OR clupei* OR “small pelagic”) AND (temperature OR “climate change” OR climat OR acidi* OR pH OR oxygen OR hypox* OR hypercapn* OR O2 OR salinity OR freshening OR stress* OR thermal OR tolera* OR limit* OR critic* OR lethal OR threshold* OR growth OR weight OR mass OR diameter OR develop* OR mortality OR surviv* OR metaboli* OR respir* OR oxygen consumption OR prefer* OR thermal window OR aerobic scope OR metabolic scope OR sensitivity OR matur* OR spawn* OR feed* OR prey OR predator OR produc* OR feed OR egg* OR “yolk sac” OR larv OR juvenil* OR light OR density OR recruit ment) NOT (chem* OR enginee* OR technology). Based on the search (and expert knowledge), the two time periods contained papers on 33 species (note, the search was limited to fish in the family Clupeidae and Engraulidae). The most research effort in the first period was on Atlantic herring (Clupea harengus), whereas in the second period most studies targeted European anchovy (Engraulis encrasicolus) (Fig. 3). Compared to the first period, the number of studies published on European anchovy almost doubled (78 to 145). Similarly, work published on Peruvian anchoveta (Engraulis ringens) tripled (10 to 30), and the first studies (based on our search terms) were published in the second period on Falkland sprat (Sprattus fuegensis) and Argentine anchovy (Engraulis anchoita) (Fig. 3). Some species seem to remain largely understudied (1 study in one of the two periods), such as scaled This search string resulted in 1420 and 1700 studies from 2001 to 2006 and 2011 to 2016, respectively. Studies outside the scope of this review were excluded after reading the title, and additional studies were excluded after reading the abstract and/or complete paper. 1.3. Categories of studies During the period between 2001 and 2006, field studies, statistical analyses/reviews, spatially-explicit modelling, laboratory experiments, and mesocosm research accounted for 70, 20, 10, 9 and 0.5%, respec tively, of the studies published on SPF (some studies were in multiple categories) (Fig. 1A). Between 2011 and 2016, these same five cate gories accounted for 67, 20, 12, 10 and 1%, respectively, of the pub lished studies (Fig. 1A). Studies were also categorized by life stage, separating work conducted on eggs (20%), larvae (35%), juveniles (32%) and adults (58%) (average for the two periods, papers often in more than one category (Fig. 1B)). Research specifically devoted to ju veniles was scarce (7–8% of all studies). Moreoever, the five categories of methods were not equally applied across all life stages (Fig. 2). During the 1st and 2nd periods, 55 and 71% of all field studies, respectively, and 62 and 64% of all time series analyses, respectively, was conducted on both juveniles and adults (mostly adults). On the other hand, 55 (1st period) and 57% (2nd period) of all laboratory studies and 62 and 64% of all spatially-explicit modelling studies was conducted on early life stages (eggs and larve). Less than one-third of the laboratory studies included work on eggs and none of the studies that used mecososm was conducted on eggs or larvae. Out of the 945 studies reviewed across the two, 6-yr time peiods, only 19 (1st period) and 9 (2nd period) examined all four life stages and these tended to be statistical time series analysis (10 in 2001–2006) or full life-cycle modelling approaches (6 in 2011–2016). The emphasis of this review was on how bottom-up processes and abiotic environmental factors regulate SPF populations or stocks. The review included peer-reviewed research on vital rates (e.g., feeding, growth, reproduction, survival) and ecophysiology, as well as studies exploring how key physical and biological features impact on habitat suitability. 1.1. Importance of small pelagic fish Although it is likely that some studies were missed during the two, 6-yr time periods, in our opinion, the 945 papers compiled here have captured trends during the new millennium in basic (fundamental) as well as emerging research themes on SPF among 16 regions (large marine ecosystems) around the world. i We also reviewed the types of studies including specific methods / themes (Section 2) and compared research conducted on SPF in different regions (see Section 3). For these and other sections, we integrated the findings of studies published before, between and after the two 6-yr time periods that were used to compare trends. For example, many studies were published from 2012 to 2015 and more than 150 references in this 4-yr period were used in this review. The 2017 symposium on SPF produced 38 publications in two special volumes (Alheit and Peck, 2019; Alheit et al., 2019b) and some of those and other recent studies are also included here. 1.2. Scope of this review The present study reviewed research conducted on SPF using a semi- structured review of literature published during 2001–2006 and 2011–2016. We compared the two time periods to reveal emerging research trends globally and to complement recently published, ecosystem-specific reviews of SPF (e.g., Takasuka, 2018). The first time period was chosen to capture research finalized at the start of the new millennium (e.g., published in 2001) that appeared in the peer-reviewed literature prior to a key, global review of the dynamics of SPF prepared in 2008 (Checkley et al., 2009) that arose from the GLOBEC Small Pe lagics and Climate Change (SPACC) collaborative research programme, and two special issues on upwelling systems (Bertrand et al., 2008b; Fr´eon et al., 2009). The second period represented the same number of years after the SPACC book but just prior to a large, international symposium on SPF that was convened in March 2017 (Alheit and Peck, 2019; Alheit et al., 2019b). 1.3. Categories of studies Studies were excluded because they either i) explored other topics (e.g., food science), ii) examined other small pelagic fish species (e.g., shads, mackerels), iii) reported on broad ecosystem overviews which included SPF in only an 2 Progress in Oceanography 191 (2021) 102494 Fig. 1. Summary of 945 studies published on small pelagic fish in two time periods that examined bottom-up factors and/or processes impacting on small pelagic fish (Clupeids and Engraulids) Panel A: Type of study conducted. Panel B: Life stage examined. M.A. Peck et al. M.A. Peck et al. Progress in Oceanography 191 (2021) 102494 Fig. 1. Summary of 945 studies published on small pelagic fish in two time periods that examined bottom-up factors and/or processes impacting on small pelagic fish (Clupeids and Engraulids) Panel A: Type of study conducted. Panel B: Life stage examined. Fig. 2. Summary of the five types of studies published in each of two time periods on different life stages of small pelagic fish (Clupeids and Engraulids). The thick line separates studies conducted on early (eggs and larvae) versus later (juveniles and adult) life stages. Note, many studies were conducted on multiple life stages (e.g., juveniles and adults). and 7% of the studies in the earlier and later 6-yr periods, respectively. Species of SPF are known to shift their spatial distribution in response to changes in bottom-up forcing, and considerable research effort has been focused on reporting and understanding these changes. Examples of this research include studies documenting the (re-) appearance of European anchovy at its high latitudinal limit in the North Sea (Alheit et al., 2012; Petitgas et al., 2012), the basin-scale distribution of Japanese anchovy (Engraulis japonicus) in the Pacific (Murase et al., 2012) and projecting shifts in distribution in Pacific sardine (Sardinops sagax) in the California Current (Kaplan et al., 2016). These studies, covering relatively large spatial scales, were complimented by a myriad of field work at specific locations relating patterns of spatial distribu tions to habitat characteristics (e.g., Nev´arez-Martínez et al., 2001; Islam et al., 2006; Song et al., 2012; Bonnano et al., 2014; Niu et al., 2014; Valencia-Gasti et al., 2015). That distribution shifts are a response to bottom-up forcing is supported by observations of consistent simi larities and differences in the strategies of space occupation by E. encrasicolus and S. herring (Harengula jaguana) or sind sardinella (Sardinella sindensis). herring (Harengula jaguana) or sind sardinella (Sardinella sindensis). herring (Harengula jaguana) or sind sardinella (Sardinella sindensis). 1.3. Categories of studies sagax at low and high biomass levels in the Southern Benguela (Barange et al., 2005) which suggested that spatial habiat utilization was density-independent. y p Determining the meta-population structure is fundamental to un derstanding the ecologies (e.g., habitat utilization, life cycle strategies) of and distinguishing between, and adequately managing stocks. During the two time periods reviewed here, most of the research focused on identifying mixtures of stocks was performed on herring in either the Pacific (Clupea pallasi) or Atlantic (C. harengus). Atlantic and Pacific herring exhibit spawning site fidelity and, hence, more complex stock structure with management implications. A variety of techniques were employed to distinguish unique reproductive units of herring in the Pacific or Atlantic including genetic analyses (Siple and Francis, 2016), otolith shape or microchemistry differences (Geffen et al., 2011; Gr¨ohsler et al., 2013; Vergara-Solana et al., 2013) or using parasites as natural tags (Unger et al., 2014). Otolith morphometrics and/or genetic analyses have also been employed to differentiate stocks of European anchovy (e.g., Magoulas et al., 2006; Vi˜nas et al., 2014) and European sardine (Jemaa et al., 2015) across Mediterranean and Eastern Atlantic waters. Body and otolith shapes have been used to discriminate stocks of Pacific sardine in the California Current ecosystem (Vergara-Solana et al., 2013), and parasite biotags in particular but also some life history and meristic and morphometric characteristics have been used for sardine stock identification in the southern Benguela Current System (Reed et al., 2012; van der Lingen et al., 2015; Weston et al., 2015; Idris et al., 2016). The advancement of biochemical and genetic techniques allowing the identification of allozyme loci (in the 1980 s) or micro satellites (in the 1990 s), has revealed differences in the relatedness of Fig. 2. Summary of the five types of studies published in each of two time periods on different life stages of small pelagic fish (Clupeids and Engraulids). The thick line separates studies conducted on early (eggs and larvae) versus later (juveniles and adult) life stages. Note, many studies were conducted on multiple life stages (e.g., juveniles and adults). 2.1.1. Productivity and distribution i Thus, spawning times, spawning habitats and links between oceanographic conditions and SPF productivity have been revealed by collecting the data needed to apply the DEPM (Somarakis et al., 2002, 2006; Ettahiri et al., 2003; Twatwa et al., 2005; Amenzoui et al., 2006; van der Lingen et al., 2006a, Fissel et al., 2011; Bernal et al., 2012; Haslob et al., 2012a, 2013; Tsikliras and Koutrakis, 2013; Bouhali et al., 2015; Mhlongo et al., 2015; Rajasilta et al., 2015). i g j Beyond field studies describing the basic features of reproduction in SPF, recent research has explored resource partitioning including po tential trade-offs between allocating energy to spawning or somatic growth. As opposed to some capital breeders such as gadoids that accumulate lipid reserved in the liver more than 6 months prior to spawning, SPF fuel reproduction based on relatively small lipid reserves in muscle tissue, and some SPF supplement energy requirements for spawning by feeding shortly before and during the spawning season. These fish depend less on the input of energy from feeding during their protracted spawning season (e.g., Hunter and Leong, 1981; Ganias et al., 2009; Nunes et al., 2011). Other SPF are more strictly income breeders, as shown for the Japanese anchovy, for which changes in the ratio of δ13C to δ15N in eggs closely follow the isotope ratios of the prey consumed by adult fish (Tanaka et al., 2016). The partitioning of re sources by females to their eggs and offspring is not fixed, therefore, but is relatively dynamic, depending on recent feeding conditions. Garrido et al. (2007b, 2008b) demonstrated how the concentration and composition of essential fatty acids (FAs) in the muscle of female sardine (Sardina pichardus) corresponded to that in hydrated oocytes and how this maternal provisioning of FAs changed both spatially and during the spawning season with implications for changes in egg quality. Research on Atlantic herring populations identified energy trade-offs between somatic growth, fecundity and migratory behaviour: migrants had significantly higher somatic growth rate and lower relative fecundity compared to females in non-migratory populations (Silva et al., 2013). It is important to establish baseline patterns in abundance (e.g., size of specific populations) and compile evidence on changes in the his torical spatial distributions of SPF to better understand ongoing changes or to better project what may happen in the future. 2.1.1. Productivity and distribution i 2.1.1. Productivity and distribution i The prominence of “field work” on SPF compared to other types of research was expected. Field surveys are routinely conducted to examine the status of these fisheries resources (changes in spawning stock biomass (SSB) or distribution), to identify nursery, feeding or spawning habitats and, to a lesser extent, to examine bottom-up processes that influence rates of mortality, growth and reproduction. In the majority of studies, multiple life stages were examined (e.g., juveniles with adults, eggs with larvae). Field research effort, therefore, appeared to be evenly distributed across these four life stages in the 1st period with a greater proportion targeting later life stages in the 2 ns period (as depicted in Fig. 2). Research focused solely on juveniles, however, formed only 8% 3 M.A. Peck et al. Progress in Oceanography 191 (2021) 102494 Fig. 3. The number of studies published on various species of small pelagic fish within two, 6-year time periods. Note, the 22 species shown are those investigated in at least 2 studies in either time period. In total, 33 species appeared in at least 1 study (see supplementary material). Fig. 3. The number of studies published on various species of small pelagic fish within two, 6-year time periods. Note, the 22 species shown are those investigated in at least 2 studies in either time period. In total, 33 species appeared in at least 1 study (see supplementary material). Fig. 3. The number of studies published on various species of small pelagic fish within two, 6-year time periods. Note, at least 2 studies in either time period. In total, 33 species appeared in at least 1 study (see supplementary material conspecifics at small spatial scales. The application of these techniques to SPF such as Pacific sardine, northern anchovy and Atlantic herring has also identified ‘chaotic patchiness’ (Hedgecock, 1994), in which inter-annual genetic variation within spawning aggregates was often larger than the genetic variation among nearby populations (see Olsen et al., 2002 and references therein). A review by Eldon et al. (2016) identified four processes contributing to such patchniness in populations of marine fish and other taxa, including sweepstakes reproductive suc cess and differences in phenology, larval dispersal and patterns of selection. ovulatory follicles based on the ground-breaking work of Hunter and Goldberg (1980). 2.1.1. Productivity and distribution i The pioneering research in the late 1960s examining scales preserved in anoxic sedi ments (e.g., Soutar and Isaacs, 1969) led to important publications reporting on long-term fluctuations in small pelagic fish populations (e. g., Baumgartner et al., 1992) as reviewed by Field et al. (2009). Recent studies have continued to document these decadal to centennial patterns such as the 150-yr time series generated for Japanese anchovy (Engraulis japonicus) in the Yellow Sea (Huang et al., 2016) and the work by Kuwae et al. (2017) in Japanese waters. These long-term time series are crucial in providing an historical perspective on the current abundances and trends and for revisiting models and paradigms based on historic data sets. In the northern Humboldt Current System, for instance, centennial to multi-millennial reconstructions of the abundance of Peruvian anchoveta suggested that the commercial fishery developed during a period of relatively high abundance of this stock respective to the last millennia (Salvatteci et al., 2019). That study also refuted some important aspects such as anchovy-sardine alternation or the role of the Pacific Decadal Oscillation (PDO) as a direct driver of SPF dynamics (Guti´errez et al., 2009; Salvatteci et al., 2019). 2.1.3. Feeding and diet Due to the tight coupling of SPF populations to bottom-up processes impacting on lower trophic levels, it is important to understand which prey are preferred and how much are needed to cover energy re quirements of SPF. Furthermore, as most SPF are income breeders, the richness of the feeding environment can have important and immediate consequences on reproductive output (Garrido et al., 2015). During the two periods reviewed here, a considerable amount of new information was published on the diets of various SPF species and the number of studies focusing on SPF diet almost tripled in the second period compared to the first (15 vs 42 studies). In the first period, a compre hensive comparison of the trophodynamics of sardine (Sardinops sagax) and anchovy (Engraulis encrasicolus) in the southern Benguela (van der Lingen et al., 2006b) underlined the trophic dissimilarity between the two species and indicated size-based resource partitioning of their zooplankton prey. That study led to the hypothesis that alternations in relative species dominance between the two species may be troph odynamically mediated. Between the periods assessed here, a compre hensive review that assembled available information on the feeding morphology, behaviour and diet of SPF from several regions (van der Lingen et al., 2009) supported the trophic dissimilarity hypothesis for sardine/anchovy species pairs in some (e.g., Ay´on et al., 2011) but not all (e.g., Costalago et al., 2012) ecosystems where adequate data exist. During the second period, a review of the diet and feeding behaviour of sardine and anchovy species was published (Garrido and van der Lingen, 2014). That work also tested the trophodynamic hypothesis with more data including results from new techniques to study trophic ecology such as analyses of stable isotope ratios. These new data indicated support for the trophic dissimilarity hypothesis in most systems. Within many ecosystems, large schools of SPF have the capacity to locally deplete zooplankton (e.g., Ay´on et al., 2008b; Koslow, 1981) and, in exceptional cases, exert top-down control of the zooplankton com munity (Arrhenius and Hansson, 1993). Juveniles likely exert particu larly strong top-down pressure on standing stocks of zooplankton because they are relatively abundant and they have relatively high mass- specific feeding rates. Intensive predation may lead to density- dependent prey limitation during the juvenile period as was suggested by various studies conducted on European sprat (Spattus sprattus) (Car dinale et al., 2002; Baumann et al., 2007; Hawkins et al., 2012). 2.1.3. Feeding and diet In one study, Hawkins et al. (2012) obtained estimates of sprat abundance from hydroacoustic measurements and zooplankton abundance from pump samples and reported rapid rates of prey depletion by large schools in an embayment in Ireland. In a second study, Baumann et al. (2007) used trajectories of otolith growth of field-caught individuals and laboratory starvation-refeeding experiments to demonstrate that large schools of young juvenile sprat were in starving condition within warm, nearshore waters of the southwestern Baltic Sea. In both Atlantic herring and sprat in the Baltic Sea, more than 90% of the long-term variability in fish condition (weight-at-length) was explained by fish abundance, high lighting the importance of density-dependent competition for prey (Casini et al., 2006; Casini et al., 2014). van der Lingen et al. (2006a) analysed a 52-year time series of biological data on Pacific sardine (Sardinops sagax) in the southern Benguela and reported density- dependent effects on condition factor and length-at-maturity. Simi larly, density-dependent growth and maturation of this species off Cal ifornia were suggested by Dorval et al. (2015) who compared the stock characteristics for fish born in three periods (1986–1993, 1996–2003 and 2004–2008). A plethora of studies has also documented the role of SPF as potential competitors with other groups of fish such as the overlap in zooplankton in the diets of Pacific herring and walleye pollock (Theragra chalcogramma) in Prince William Sound, Alaska (Sturdevant et al., 2001). y y y During the two time periods reviewed here, about three-fold more studies were published on the diet and feeding of juveniles and adults compared to larvae. Diet studies on larvae filled gaps in knowledge on rarely-studied species such as Falkland sprat (Sprattus fuegensis) and Araucarian herring (Strangomera bentincki) in fjords and channels in southern Chile (Contreras et al., 2014), and on well-studied species in specific habitats such as Atlantic herring in the Gulf of Riga in the Baltic Sea (Arula et al., 2012), European sardine in the Mediterranean Sea (Tudela et al., 2002; Fernandez and Gonz´alez-Quir´os, 2006) and Japa nese sardine and anchovy in the Kii Channel in Japan (Yasue et al., 2011). During the two periods, a similar (small) number of laboratory studies was conducted. During the first period most laboratory studies focused on the condition and growth of larvae (or post-larvae) in rela tion with feeding densities (e.g., Kono et al., 2003; Baumann et al., 2005). 2.1.3. Feeding and diet During the second period, most laboratory studies focused on the importance of protists for the diet of early larvae (Friedenberg et al., 2012; Illing et al., 2015). Field studies on larval feeding more than doubled in the second compared to the first period. In the first period, these studies mainly focused on gut content analysis to define changes in prey composition, size and preference with increasing larval length such as work by Llanos-Rivera et al. (2004) on Peruvian anchovy and Pacific sardine from Concepcion Bay, Chile. In the second period, a more ho listic approach was often taken in which larval diet and feeding was studied in relation to environmental factors influencing prey availability or larval feeding success such as wind speed, temperature, turbulence and freshwater discharge (e.g., Arula et al., 2012; Landaeta et al., 2012). These holistic studies have been important to the development of sub- routines for larval foraging in biophysical individual-based models (e. g., Hufnagl and Peck, 2011; Urtizberea and Fiksen, 2013). i Long-held beliefs concerning the diet of some SPF are changing due to recent research, particularly studies estimating prey energy as opposed to prey number and those studying the trophic web by means of stable isotopic composition. For example, for decades, the Peruvian anchoveta (Engraulis ringens) was considered to feed directly on primary producers (Ryther, 1969; Rojas de Mendiola, 1989; Alamo and Espinoza, 1998) leading to the belief that the large populations of anchoveta were supported by an unusually short and efficient food chain (Ryther, 1969; Walsh, 1981; Pauly and Christensen, 1995). Recent work estimating prey carbon content refutes this assumption by demonstrating that Peruvian anchoveta forage mainly on macrozooplankton, in particular euphausiids and large copepods (Espinoza and Bertrand, 2008, 2014). The reliance of anchoveta on macrozooplankton was confirmed by analysis of stable isotopes indicating a trophic level between 3.4 and 3.7 (Espinoza et al., 2017). These results suggest an ecological role for forage fish that challenges most current trophic models for the Hum boldt Current System (HCS), which are parameterized such that forage fish rely largely on diatoms. Fish are continuous samplers of the ecosystem. Since many species of SPF display a high degree of opportunistic feeding, changes in diet can reveal fundamental shifts in marine ecosystems. 2.1.2. Reproduction and Oregon, USA (McFarlane and Beamish, 2001; Emmett et al., 2005) and the southern Benguela EBUS off South Africa (van der Lingen, 2002) in the first period. Examples of diet overlap studies comparing two or more species of SPF in the second period include analyses in the NE Atlantic (Bachiller et al., 2013; Langøy et al., 2012; Raab et al., 2012), Bay of Biscay (Chouvelon et al., 2015; Bachiller and Irigoien, 2015) and in the Mediterranean Sea (Costalago et al., 2012, 2014; Bourg et al., 2015). 2.1.2. Reproduction Recent research in the NW Mediterranean investigated energetic consequences of environmental conditions unfavourable for growth (Brosset et al., 2015) and energetic trade-offs associated with lipid re serves, size-at-maturity, batch fecundity, spawning duration and egg quality (dry mass) of European anchovy and sardine (Brosset et al., 2016). Besides significant decreases in the length-at-maturity, both species displayed preferential allocation toward reproduction, even in fish that were in relatively poor condition (weight-at-length and length- at-age). This preferential allocation of energy to gonadal maturation and A considerable amount of information on the reproductive charac teristics of SPF has been gained by using the daily egg production method (DEPM) to estimate spawning stock biomass (SSB) (Lasker, 1985; Alheit, 1993). This calculation requires ecologically relevant in formation on key reproductive characteristics such as the in situ con centration of eggs and the mean weight, batch fecundity, and spawning frequency of females during the peak period of reproduction whereby spawning frequency is determined by histological ageing of post- 4 M.A. Peck et al. Progress in Oceanography 191 (2021) 102494 spawning in females was also observed in Atlantic herring in the Baltic Sea (Rajasilta et al., 2015). and Oregon, USA (McFarlane and Beamish, 2001; Emmett et al., 2005) and the southern Benguela EBUS off South Africa (van der Lingen, 2002) in the first period. Examples of diet overlap studies comparing two or more species of SPF in the second period include analyses in the NE Atlantic (Bachiller et al., 2013; Langøy et al., 2012; Raab et al., 2012), Bay of Biscay (Chouvelon et al., 2015; Bachiller and Irigoien, 2015) and in the Mediterranean Sea (Costalago et al., 2012, 2014; Bourg et al., 2015). spawning in females was also observed in Atlantic herring in the Baltic Sea (Rajasilta et al., 2015). and Oregon, USA (McFarlane and Beamish, 2001; Emmett et al., 2005) and the southern Benguela EBUS off South Africa (van der Lingen, 2002) in the first period. Examples of diet overlap studies comparing two or more species of SPF in the second period include analyses in the NE Atlantic (Bachiller et al., 2013; Langøy et al., 2012; Raab et al., 2012), Bay of Biscay (Chouvelon et al., 2015; Bachiller and Irigoien, 2015) and in the Mediterranean Sea (Costalago et al., 2012, 2014; Bourg et al., 2015). 2.1.3. Feeding and diet These diet studies are fundamental to understanding resource partitioning by co-occurring SPF in various ecosystems around the globe. Difference in the sizes of prey preferred by anchovy and sardine have been well-known for decades based on gill raker morphology (e.g., King and Macleod, 1976; van der Lingen et al., 2009; Rykaczewski (unpublished data)) and the “trophic-dissimilarity” hypothesis (van der Lingen et al., 2006b) forms one of the hypotheses attempting to explain the large-scale oscillations of populations of an chovy and sardine observed in the California, Peruvian and Benguela Eastern Boundary Upwelling Systems (EBUSs). In non-upwelling eco systems, less research has been conducted on the potential diet differ ences between sardine and anchovy, although considerable work was done in the second, 6-yr period. In one example, stable isotope analyses of plankton and fish muscle in the Bay of Biscay revealed differences in the diets and a lack of competition between co-existing anchovy and sardine during spring, when prey resources were high, but greater diet overlap during autumn, a period of lower plankton productivity (Chouvelon et al., 2015). Importantly, seasonal changes in diets and trophic niche breadth have been documented in response to changes in hydrography (stratification and mixing periods) and prey availability. In a second example, Nikolioudakis et al. (2014) reported that relatively large copepods were the main prey for both anchovy and sardine in the eastern Mediterranean Sea, although adult sardine (but not anchovy) consumed phytoplankton. Finally, competition for prey has also been reported for other species of co-existing SPF such as European sprat and Atlantic herring in the Baltic Sea (M¨ollmann et al., 2004). In SPF, particularly in sardines and anchovies, the diets and potential intra- guild competition appears to depend on the ecosystem-specific pro cesses regulating plankton community production and composition as discussed by Espinoza et al. (2009) who compared diets of sardines across three EBUS. Living organisms follow non-random yet non-uniform distributions and tend to aggregate in patches (Margalef, 1979; Legendre and Fortin, 1989). Both physical forcing (e.g., frontal zones (Munk et al., 1999)) and organismal behaviour initiate and maintain patchiness (Kotliar and Wiens, 1990). Bottom-up, physical processes structure pelagic habitats (Legendre and Fortin, 1989), introducing turbulence and/or creating convergence zones of nutrients and passive plankton. Trophodynamic coupling can transmit these spatial structures through the food web (Bertrand et al., 2008a, c). 2.1.4. Intra-guild competition and predation Although the present review is focused on the impacts of bottom-up processes, diet studies clearly indicate that intra-guild predation and cannibalism can be important controls of early life stage survival of SPF. For example, field work from the mid-1980s in the Benguela system indicated that 56% and 6% of the total mortality of anchovy eggs resulted from sardine predation and anchovy cannibalism, respectively, although mortality rates can be higher when SPF forage within dense patches of eggs (Vald´es-Szeinfeld et al., 1987; Vald´es-Szeinfeld, 1991). The proportions of egg cannibalism by anchovy were even higher in the Humboldt (21%) and the California (28%) Currents (Alheit, 1987). More recent work by Bachiller et al. (2015) reported that up to one-third of the total annual egg mortality of European anchovy was the result of pre dation by European sardine in the Bay of Biscay. Although population- level impacts were not quantified, Karaseva et al. (2013) documented substantial numbers of sprat eggs and larvae in the diets of Atlantic herring in the south-eastern Baltic Sea but little to no predation of sprat on herring early life stages. In the NW Pacific, Takasuka et al. (2004) reported that cannibalism of larvae by juveniles of Japanese anchovy was observed in 26% of fish analysed, which appeared to select slower- growing larvae. Finally, Garrido et al. (2015) estimated that cannibalism was responsible for 30% of the natural mortality of sardine eggs spawned off southern Portugal and laboratory experiments confirmed that eggs were one of the preferred prey items for the species (Garrido et al., 2007a). 2.2. Statistical analyses and reviews Given the importance of SPF to local economies, long-term records of catch often exist that can be exploited to understand historical fluctua tions in the sizes of stocks and potential environmental drivers (e.g., Alheit and Hagen, 1997). In specific ecosystems, contemporary surveys of specific species of SPF have now amassed more than 25 years of field data that have been harnessed to explore the strength of association between changes in climate-driven, bottom-up processes (e.g., changes in temperatures and/or prey fields) and changes in the distribution or productivity of SPF. In some ecosystems, time series data are currently not available. For example, the absence of long-term, large-scale sam pling programs in the Canary Current EBUS has hampered the analysis of historical trends in SPF dynamics, as well as management efforts (Braham et al., 2014). Efforts are underway to integrate national archived datasets and newly collected field data (Ba et al., 2016) together with the implementation of large international projects to in crease the capacity to understand trends and drivers of SPF in that EBUS. 2.1.3. Feeding and diet Over the last decade, advances in hydro acoustics have increased our understanding of the schooling dynamics of SPF and how the environment structures the distribution of clusters of schools. Primary ecosystem interactions were believed to occur at the mesoscale but acoustic data on SPF in the Humboldt Current revealed that the dynamics of upper ocean waters at sub-mesoscales (less than10 km) played the foremost role in shaping the seascape from zooplankton to predators and that fish behaviour (foraging in schools) magnifies the physically induced spatial structuring (Bertrand et al., 2014). Work on Peruvian anchoveta, thus, suggests that patchiness at spatial scales greater than that of schools (e.g., clusters) is also driven by physical forcing such as ocean turbulence at meso- and sub-mesoscales and not only by the total abundance of SPF in a region (Bertrand et al., 2008a, 2014). 2.1.4. Intra-guild competition and predation 2.1.3. Feeding and diet In Peru, for example, no clear relationship was observed between the prey composition of Peruvian anchoveta and inter-annual variability in oceanographic fea tures associated with El Ni˜no and La Ni˜na periods (Espinoza and Ber trand, 2008). However, at longer, decadal scales, Espinoza and Bertrand In terms of diet studies performed on juveniles and adults, in the first period, most of these were single-species analyses, while studies comparing diets of two to several SPF in the same ecosystem were more common in the second period. For example, the diet of adult Pacific sardine (Sardinops sagax) was examined in coastal waters off California 5 5 Progress in Oceanography 191 (2021) 102494 M.A. Peck et al. ‘‘an essential life unit in which fish feed, breed, rest, and flee” (Aoki, 1980). In SPF, larvae first start to display schooling behaviour directly after notochord flexion (e.g., Cotano et al., 2008; Somarakis and Niko lioudakis, 2010 and references cited therein). In Engraulis species, for example, schooling starts when larvae are between 11 and 17 mm in length (Hunter and Coyne, 1982; Masuda, 2011). This transition to schooling is correlated with the start of strong vertical migration and changes in diet. This shift is a ‘game-changer’ as it involves escaping the viscous flow regime (Müller and Videler, 1996), modifying the effects of bottom-up forcing (i.e., modifying the capacity to reach prey, interact with currents, etc.) as reviewed by Peck et al. (2012c). Since the development of multi-beam sonars (Gerlotto et al., 2000), knowledge on SPF school dynamics and behaviour has considerably increased (e.g., Misund et al., 2003; Handegard et al., 2017). School size, shape, and location depend on (i) local habitat characteristics, in particular the prey dynamics (e.g., Nøttestad et al., 1996; Mackinson et al., 1999; Bertrand et al., 2008a), hydrodynamic characteristics or the presence of predators (e.g., Axelsen et al., 2001; Nøttestad et al., 2004; Gerlotto et al., 2006; Bertrand et al., 2014), and (ii) intrinsic behaviour such as spawning (e. g., Axelsen et al., 2000). Once formed, these large school play a central role in how SPF respond to bottom-up (and top-down) processes and may contribute, through intra-guild competition, in the rapid shifts in dominance of SPF as described in “School Trap” hypothesis (Bakun and Cury, 1999). (2014) reported a shift in the proportion of euphausiids in the diet of Peruvian anchoveta that tracked changes in the size of dominant zooplankton (Ay´on et al., 2011). 2.3. Laboratory / mesocosm studies Controlled laboratory experiments were a relatively small proportion (less than 10%, Fig. 1) of the studies published on SPF during the two periods reviewed here and the majority of that work was conducted on larvae (Fig. 3). This is not surprising given that, for most species examined in this review, it is very challenging to maintain and spawn adults and rear their eggs and larvae in the laboratory. These practical challenges have led to a paucity of research directly examining the ef fects of abiotic factors using appropriate experimental designs across most taxa of fish, particularly SPF (Catal´an et al., 2019). The case is worse in clupeid fish which are relatively sensitive to handling and require large volumes of water to comfortably swim, school, feed and reproduce in captivity. Some species are more easily obtained and amenable to laboratory rearing such as Atlantic and Pacific herring which is the likely reason why these two species accounted for 61% and 42% of all laboratory work published in 2001–2006 and 2011–2016, respectively. Atlantic herring was also the only species to have been studied using mesocosms during the two time periods reviewed here. Laboratory studies on Atlantic herring have been performed on all life stages and have explored a variety of facets including the ecophysiology of development, growth, feeding and swimming as impacted by various factors such as temperature, oxygen and/or pH (pCO2) (e.g., Domenici et al., 2002; Geffen, 2002; Fox et al., 2003; Utne-Palm, 2004; Frommel et al., 2014; Maneja et al., 2015). Similarly, recent studies on Pacific herring have examined natural mortality rates of eggs of different sub- populations (Shelton et al., 2014), the strength of the protozooplankton-ichthyoplankton link (Friedenberg et al., 2012) and the sensitivity of larvae to suspended sediments (Griffin et al., 2012). Work on larvae and juveniles has examined the acute and chronic effects of a viral disease (Lovy et al., 2012). Long-term data sets have also shed light on the potential (bottom-up) processes limiting year-class success (year-to-year variability) of specific species in specific regions such as Atlantic herring in the North Sea (e.g., Corten, 2013) and European sprat in the Baltic Sea (Voss et al., 2012). Broader analyses including the whole fish community have been con ducted in some systems (Ralston et al., 2014; Norton and Mason, 2005). 2.1.5. Schooling and behaviour In the field, the organisation/patchiness of SPF occur across a variety of scales (Gerlotto and Paramo, 2003; Fr´eon et al., 2005). At spatial scales from the individual (10s of mm), to the school (10s of m), self- organisation mechanisms are likely to be dominant and drive the for mation and characteristics of schools. For gregarious fish, the school is Within “data-rich” EBUS, recent studies have provided new insights 6 Progress in Oceanography 191 (2021) 102494 M.A. Peck et al. on the well-documented oscillations between anchovies and sardines in various marine ecosystems including the California Current (Deyle et al., 2013; Lindegren et al., 2013). Based on late 20th century fish landings, the paradigm was that anchovy and sardine alternate periodically and that fluctuations in the size of their populations were associated with large- (basin-) scale changes in climate variability as captured from indices such as the PDO (e.g., Schwartzlose et al., 1999; Chavez et al., 2003). As previously mentioned, this paradigm has been challenged by a variety of paleoceanographic studies conducted in the Humboldt and California Current systems (e.g., Vald´es et al., 2008; Field et al., 2009; Guti´errez et al., 2009; McClatchie et al., 2017; Salvatteci et al., 2018). Given prehistorical records of population sizes inferred from ocean sediments, alternations between sardine and anchovy appear circum stantial and do not follow a consistent pattern (MacCall, 2009; Field et al., 2009; Salvatteci et al., 2018, 2019). Indeed, periods when both species are either rare or abundant have occurred in the past. Further more, fluctuations occur across a variety of timescales (Salvatteci et al., 2018) apart from the 50- to 60-years periodicity derived from fishery landings (Schwartzlose et al. 1999; Chavez et al., 2003). large fluctuations in SPF stocks is to correlate changes in stock biomass to abiotic factors such as wind strength and/or water temperature. Sudden shifts in the size of SPF populations have been linked to changes in physical forcing. Examples include the changes in the biomass of European sardine and prevailing winds off the coast of Portugal (Borges et al., 2003) or annual variability in satellite-derived spatial gradients in sea surface temperature (an upwelling index) in the Canary Current (Santos et al., 2005a,b), and how intra-seasonal variability in upwelling strength (Roy et al., 2001) can impact recruitment strength of European (Cape) anchovy in the southern Benguela. 2.1.5. Schooling and behaviour The rapid increase in Euro pean anchovy in the North Sea in the late 1990s was related to physi ologically favorable warming allowing over-winter survival of juveniles (Petitgas et al., 2012). Time series analysis of Pacific sardine and large- scale changes in temperature and winds in the northern Benguela Cur rent system highlighted how intensive (over-) exploitation of fish stocks and reductions in population size may change life history strategies (e. g., changes in spawning area) that can alter the strength (or can even reverse the sign) of correlations between environmental factors and stock productivity (Daskalov et al., 2003). Similarly, Essington et al. (2015) reported on collapses in 15 SPF populations and indicated that only 4 populations would have collapsed from natural productivity de clines alone. g The availability of long-term data sets has allowed explorations of the impacts of climate variability on population fluctuations of SPF. In the 1990s, biologists focused on the impact of the North Atlantic Oscillation (NAO), the differences in the centers of low and high at mospheric pressure between Iceland and the Azores, on marine eco systems and populations. Research in the late 1990s and early 2000s correlated changes in the NAO index and changes in the productivity of herring and sardines populations in NE Atlantic waters (Alheit and Hagen, 1997) and sprat in the Baltic Sea (Alheit et al., 2005). A decade later, the focus shifted to studying the impact of the Atlantic Multi decadal Oscillation (AMO) on ecosystems (Alheit et al., 2014). The dy namics of populations of anchovy, sardine, sprat and sardinellas in the Baltic, North Sea, Bay of Biscay, the Mediterranean and the Canarian upwelling were shown to be associated with AMO variability (Alheit et al., 2012, 2014, 2019a; Edwards et al., 2013; Montero-Serra et al., 2015; Tsikliras et al., 2019). In the NW Pacific, the SST in the Kuroshio and the Tsushima Warm Current (Japan/East Sea) markedly declined between 1968/71 and the late 1980 s. This appears to be related to climatic shifts associated with decadal dynamics of the East Asian Winter Monsoon and the Arctic Oscillation. Indices of these atmospheric conditions exhibited obvious changes in the late 1960s and the late 1980 s, the timing of which corresponded well to changes in catches of sardine and anchovy in these regions (Tian et al., 2008, 2014; Alheit and Bakun, 2010). 2.5. Summary of bottom-up factors and processes The development of biophysical models depicting the transport of passive particles (e.g., fish eggs and larvae) in the early 1990s was an important step forward in fisheries science as it allowed hypotheses on processes controlling in situ productivity to be generated and, in some cases, tested (Werner et al., 2001). Given the tight coupling between SPF populations and environmental (bottom-up) drivers such as changes in wind forcing (Cury and Roy, 1989) or aberrant drift (Iles and Sinclair, 1982), the number of studies utilizing 3-d particle tracking models to explore the transport dynamics (e.g., advection, dispersion, retention) of eggs and larvae of SPF continues to grow. Studies reporting on the re sults of biophysical model simulations of the transport of eggs and larvae of SPF comprised more than 40% of the studies classified as “biophysical modelling” during the two, 6-yr periods reviewed here. Some of the earliest applications of biophysical individual-based models (IBMs) to examine the transport dynamics of early life stages of SPF were devel oped during the first review period for European (Cape) anchovy (Engraulis encrasicolus) and Pacific sardine in the southern Benguela (see review by Lett et al., 2015). The growing availability of high-resolution hydrodynamic models in areas such as the Humboldt Current, Canary Current and Mediterranean Sea have allowed explorations of bottom-up factors influencing habitat connectivity and potential survival of SPF in these systems both in retrospective simulations and in future climate projections during the second review period (e.g., Brochier et al., 2011, 2013; Soto et al. 2012; Catal´an et al., 2013; Ospina-´Alvarez et al. 2015). This spatially-explicit modelling has been expanded to include elements of growth physiology such as foraging dynamics (Hufnagl and Peck, 2011) and food availability (Kone et al., 2013), as well as energy allo cation between somatic and reproductive tissues in adults (Pethybridge et al., 2013). These 5 methods of study (field, laboratory, mesocom, spatially- explicit modelling, and statistical analyses/reviews) have been applied to investigate various aspects of bottom-up control of SPF. To compare across regions and identify gaps in knowledge, we organized studies by drivers and responses examined (Fig. 4). For drivers, we used 10 cate gories including six abiotic factors (i.e., temperature, salinity, dissolved oxygen, pH and water density), two biotic factors (prey quantity and quality) and/or important physical processes (advection, turbulence and turbidity) (Fig. 4). 2.3. Laboratory / mesocosm studies The availability of long-term data derived from ocean sediments has revealed population fluctuations occurring at long-term (multi-decadal to millennial) scales (e.g., Guti´errez et al., 2009; Salvatteci et al., 2018, 2019). Adult biomass and year-class success (recruitment) often exhibit rapid changes and this so-called “boom-and-bust” phenomenon is emblematic of SPF. Time series data of sufficient length have allowed researchers to explore the potentially density-dependent changes in carrying capacity causing these rapid shifts (e.g., Tanaka, 2003). For example, Jacobson et al. (2001) calculated annual surplus production (ASP), the biological production required to maintain the previous year’s biomass in the absence of fishing, and used this to identify regime shifts in eight anchovy and nine sardine stocks around the world. Those authors exploited survey time series collected until 1997 and suggested that ≥30 years of data may be needed, especially for sardine, to adequately capture the full range of density-dependent changes in ASP (Jacobson et al., 2001). i Historically, when captive broodstocks of SPF have been established, this had led to a plethora of seminal studies advancing our mechanistic understanding of how species of SPF develop, feed and grow. An example includes studies on adult sardine in the Benguela region in the 1990s (e.g., van der Lingen 1995, 1998). Recent success at maintaining spawning brood stocks of species never before reared in captivity in Europe such as the European anchovy and European sardine has pro vided new information on changes in prey selection during the larval ontogeny of sardine (e.g., Caldeira et al., 2014; Garrido et al., 2016) and aspects of the bioenergetics (e.g., rates of feeding, growth, swimming and metabolism) of sardine or anchovy larvae (e.g., Garrido et al., 2007a, 2012, 2016; Iglesias and Fuentes, 2014; Moyano et al., 2014; A first step towards understanding the bottom-up processes causing 7 Progress in Oceanography 191 (2021) 102494 M.A. Peck et al. Silva et al., 2014). In California, laboratory trials have been used to calibrate oxygen isotopic values of otoliths so that this measure can estimate water temperatures utilized by juveniles (Dorval et al., 2011). Using a variety of biochemical measures made on blood and tissue samples, Pribyl et al. (2016) defined physiological optimal and sub- optimal temperatures for 1- to 1.5-year old Pacific sardine. 2.5. Summary of bottom-up factors and processes In terms of responses, we used nine categories, dis tinguishing between population- (e.g., changes in productivity and/or distribution) and individual-level responses such as vital rates (mortal ity, growth, feeding, reproduction) and energetic costs and losses (e.g., various aspects of internal physiology, swimming behaviour, impacts of disease / parasites). Papers often examined multiple drivers and re sponses. Some factors were examined a few times in specific regions that were not included in this overall scheme such as the impacts of pollution (e.g., sound, chemical). Nevertheless, these categories provided a useful graphical representation (Fig. 4) allowing one to visualize how the 5 categories of studies have been applied to factors, processes and re sponses across regions and/or species. Although it is compelling to make cross-hemisphere comparisons of the results of similar types of studies Fig. 4. The focus of peer-reviewed papers on SPF were categorized into 10 different bottom-up drivers (factors) and processes (forcing) and 9 different responses. Note, some research examined only responses (especially field sur veys). This diagram serves as a template to map and compare research effort across regions and /or species. 2.3. Laboratory / mesocosm studies Recent lab oratory work on Japanese anchovy included measurement of the escape response of adults at two temperatures and two pCO2 levels (Nasuchon et al., 2016) as well as a series of studies exploring how temperature and feeding affect aspects of maturation and spawning (Yoneda et al., 2014, 2015). These and previous laboratory studies have advanced conceptual models of environmental impact and helped parameterize mechanistic, physiology-based models to explore how the environment regulates the early life stage survival and life cycle closure of SPF (discussed in the next section). can be projected forward for many generations. These models include the interaction between various bottom-up (temperature, mesoscale turbulence, prey field) and top-down (predators and fisheries) drivers of SPF stocks. They are complex, parameter-rich models designed for ecosystem-level, management strategy evaluation. The reason that these state-of-the-art tools have been advanced is because of the unique, tight coupling of the population dynamics of SPF to changes in lower trophic levels, the importance of SPF as prey in food webs and the commercial importance of SPF to human communities. An important step in the development of these end-to-end models for SPF was the transfer of knowledge derived from field data on the trophodynamic relationships of SPF into mass-balance trophic models such as Ecopath. Although not included in this review, these and other 0-D (time only) food-web models have been extensively applied to SPF as exemplified by work in the Benguela system in the early 2000s (Shannon and Cury, 2003; Shannon et al., 2003; Heymans et al., 2004; Shannon et al., 2004a,2004b). 2.4. Spatially-explicit modelling studies 3.1. The Humboldt Current System The research on SPF in various regions around the globe has focused on specific processes thought to be important in driving changes in the distribution and productivity of various populations. Global coverage in the research conducted on SPF is clear from a map showing the distri bution of published studies (Fig. 5). There are differences between the two time periods in the number of studies published on species in certain habitats. Three regions had a considerable decline (NW Atlantic, Ben guela Current System, Australia) and three had a considerable increase (Humboldt Current System, Mediterranean Sea, Baltic Sea). For example, some studies were published in the first 6-yr period in SW Australia but no studies were published in the second period. Similarly, far fewer studies were published on species in the Benguela EBUS in the second (19) compared to the first 6-yr period (43). A similar decrease was apparent in the NW Atlantic (36 vs. 15 published studies). Increases in publications were noted in the Humboldt EBUS and Mediterranean Sea. There are various reasons for these differences including changes in the strength of international collaboration, shifts in national funding schemes (e.g., as a response to understand large shifts in SPF resources, particularly collapses of populations). l Although the four EBUS have similar levels of primary productivity, the northern Humboldt Current System (HCS) has 5- to 10-times more productivity of SPF and this region has produced more fish per surface area than any other marine system in the most recent decades (Chavez et al., 2008). Alongshore winds are present year-round in northern Chile and along most of the Peruvian coast, but these upwelling-favourable winds are limited to spring, summer, and early autumn in central and southern-central Chile (30◦–41◦S). The HCS is subject to large fluctua tions in climate, biological characteristics (e.g., community structure) and fisheries, from intra-seasonal to inter-annual (e.g., El Ni˜no Southern Oscillation, ENSO) and secular to millennial time scales (Chavez et al., 2008; Guti´errez et al., 2009; Salvatteci et al., 2014). El Ni˜no, La Ni˜na events, and climate variability in general, have had some of the largest documented impacts in this region (Keefer and Moseley, 2004; Chavez et al., 2008). A key feature of the HCS is the presence of a large, sub surface oxygen minimum zone (OMZ) located a few tens of meters below surface waters which vertically compresses the ecosystem. 3.1. The Humboldt Current System The absence of oxygen in continental shelf sediments preserves organic matter and the remains of organisms, creating a record of ecosystem components and climatic variability over tens of thousands of years (Guti´errez et al., 2009, 2011; Salvatteci et al., 2018, 2019). It is clear that periods of high and low SPF abundance have occurred long before the development of the commercial fishery in the HCS (Sandweiss et al., 2004; Guti´errez et al., 2009). The next sections briefly summarize research in 10 regions, where the majority of the SPF research reviewed here (899 of 945 studies) was conducted. During the two time periods, the most studies (18%) were published on SPF in the Mediterranean Sea followed by the NW Pacific, Baltic Sea, California Current and NE Atlantic (10 to 12% each). Studies on SPF in the Barents - Norwegian and Canary - Iberian systems accounted for 8 and 9%, respectively. Studies within the Northeast Atlantic, and Humboldt and Benguela Current systems were between 5 and 6% of those reviewed here. Research effort compared in this manner ignores the vast differences in the number of countries (EEZs) in these regions. For example, two countries are associated with the Humboldt Current System while more than 7 countries border regions such as the Mediterranean Sea, NE Atlantic and Baltic Sea. There are also large differences in the economic resources (e.g., GDP) potentially available for research and the economic (and cultural/societal) importance of SPF across all 17 regions. A relative view of research effort (normalized by the number of EEZs or GDP) would change this world-wide map, particularly with regard to the amount of research conducted in the Humboldt Current System relative to European and North American Although work published on SPF in the HCS represented about 5% of studies reviewed here (Fig. 5), arguably, the HCS is one of the most intensively studied EBUS as monitoring has occurred here for more than 50 years including two to four field surveys per year since the early 1980 s. This effort has advanced understanding of i) the trophodynamics of sardine and anchovy (Espinoza et al., 2009; Espinoza and Bertrand, 2014), (ii) long-term changes in SPF prey field characteristics such as zooplankton size distributions (Ay´on et al., 2011), (iii) the magnitudes of spawning activity of E. 2.4.2. End-to-end models One of the most notable developments in the new millennium has been the demonstration and application of fully coupled, end-to-end spatially-explicit biophysical models. This type of modelling repre sented roughly 10% of all the biophysical modeling studies published on SPF during the two time periods. SPF are ideal candidates for this type of modelling because of their dependency on the dynamics and standing stocks of phytoplankton and zooplankton populations. Initial steps in creating these tools were to couple feeding requirements from bioenergetics-based growth models for Pacific herring as mortality terms on phytoplankton and zooplankton groups within lower-trophic- level, biogeochemical models (e.g., Rose et al., 2007, 2008). These tools now include movement sub-routines, dynamic predator–prey feedbacks and top-down forcing from predatory fish and fishing fleets (Fiechter et al., 2015; Politikos et al., 2015; Rose et al., 2015). These end-to-end models not only include life-stage-specific physiology and behaviour but also close the life cycle (from eggs to adults to eggs) and Fig. 4. The focus of peer-reviewed papers on SPF were categorized into 10 different bottom-up drivers (factors) and processes (forcing) and 9 different responses. Note, some research examined only responses (especially field sur veys). This diagram serves as a template to map and compare research effort across regions and /or species. 8 M.A. Peck et al. Progress in Oceanography 191 (2021) 102494 on “anchovies” or “sardines”, important physiological and/or morpho logical differences occur within these groups. For example, adult body size is smaller in the genus Sardina (Atlantic) compared to the Sardinops (Pacific) (e.g., 27.5 versus 39.5 cm). regions. Moreover, more recent research programmes have been funded to advanced knowledge on the dynamics of SPF in developing regions such as sub-Saharan west Africa (e.g., Thiaw et al., 2017; Diankha et al., 2018) that were not included in this review. 3.2. The Canary/Iberian Current System , g The Iberian subregion has two main components, related to the topography and orientation of the coastline. Productivity in the north western Iberian Peninsula is highly influenced by summer upwelling and by river runoff (Arístegui et al., 2009). In the southern Iberia, upwelling is weaker and more intermittent, mainly occurring during late spring/ summer. The European sardine is the most landed fish in the Iberian sub- region, representing approximately 40% of the total catch (DGRM, 2016). Mean annual catches of the Atlanto-Iberian sardine have declined three-fold in the last decade, with severe socio-economic consequences for Portuguese and Spanish fishing communities. The European anchovy has a stable population in the sheltered Gulf of Cadiz and intermittent bursts of recruitment off the NW coast likely linked to occasionally favorable environmental conditions. In this sub-region both SPF are often found in mixed shoals with other abundant coastal pelagic fish such as Atlantic chub mackerel (Scomber colias) and horse mackerel (Trachurus trachurus). In the Moroccan subregion (21 −32◦N), coastal upwelling (strongest in June-August) creates a complex array of meso scale features (front, filaments and eddies) which interact with those generated in the nearby Canary archipelago to influence the life history dynamics of several species of SPF (see Arístegui et al., 2006; Rodríguez et al., 2009). Catches of SPF are dominated by European sardine (with peak abundance in Moroccan waters) followed by round sardinella (Sardinella aurita) (FAO, 2018a). European anchovy occurs in low abundance and, unlike other EBUS, inter-decadal shifts in dominance between sardine and anchovy (e.g., Alheit et al., 2009; Alheit and Bakun, 2010), have not been observed. Changes in the SPF community have been associated with episodic warming and hypoxia (Arístegui et al., 2009). For example, after a warming event in 1995/97, the abundance of round sardinella began to gradually increase while Eu ropean sardine drastically decreased in the north of this subregion. The Mauritanian-Senegalese subregion (12 – 21 ◦N) is the most productive of the three sub-regions mainly due to the presence of nutrient-rich North Atlantic Central Waters and deposition of Saharan dust (Arístegui et al., 2009). Upwelling is highly seasonal and strongest in winter. In this sub- tropical region, the SPF assemblage is dominated by two sardinella species (round and flat sardinella (Sardinella maderensis)), stocks that are now considered to be over-exploited (FAO, 2018a). 3.2. The Canary/Iberian Current System phytoplankton and higher trophic levels at a variety of scales (Guti´errez et al., 2009; Ay´on et al., 2011; Salvatteci et al., 2018). Although tem perature was considered the main driver of the spatial dynamics of small pelagic fish in the HCS in the early 2000 s, the demonstration of plas ticity in the response of pelagic fish towards temperature (e.g., McFar lane et al., 2002; Bertrand et al., 2004; Espinoza and Bertrand, 2008; Guti´errez et al., 2008) suggested more complex relationships between SPF and vertical and horizontal water-mass characteristics, particularly in relation to the Oxygen Minimum Zones (OMZs) and prey distribution (Ay´on et al., 2008a, 2011; Swartzman et al., 2008; Ball´on et al., 2011; Bertrand et al., 2011; Salvatteti et al., 2019) at multiple, dynamic scales (Bertrand et al., 2014). These more complex ecosystem characteristics not only influenced bottom-up drivers but also the magnitude of top- down control from both predators and fisheries (Joo et al., 2015; Pas suni et al., 2016; Barbraud et al., 2018). Between the two time periods compared in the present literature review, many long-held beliefs changed. This included diet studies suggesting that Peruvian anchoveta and Pacific sardine do not mainly feed on phytoplankton but on zooplankton (Espinoza and Bertrand 2008, 2014; Espinoza et al., 2009, 2017) in agreement with observations in the Benguela system, (van der Lingen et al., 2006b). Moreover, anchovy and sardine in the HCS forage on larger prey items such as euphausiids to a greater degree than in other systems (Espinoza et al., 2009). An important shift in knowledge was the realization that the sediment record indicates that anchovy and sardine do not consistently alternate in biomass at decadal scales (e.g., Chavez et al., 2003) but that these species display inconsistent patterns of alternation across a range of timescales which are not correlated with the PDO and other remote climate drivers (Vald´es et al., 2008; Guti´errez et al., 2009; Salvatteci et al., 2018). The Canary/Iberian Current System (CanCS) is one of the four major EBUS, extending from the NW African coast (12◦N) through the Iberian Peninsula (to 43◦N). At the northern and southern limits, the strength and extent of upwelling varies seasonally (Arístegui et al., 2009; K¨ampf and Chapman, 2016) while between 10 and 20◦N upwelling becomes semi-continuous (Lathuili`ere et al., 2008). The system contains a broad diversity of marine habitats with three, distinct sub-regions: Iberian, Moroccan and West Saharan, and Mauritanian-Senegalese. 3.2. The Canary/Iberian Current System During the two periods reviewed here, research effort on the main species (Peruvian anchovy) focused on field studies, statistical time se ries analyses on population-level responses (distribution and produc tivity) as well as reproduction (Fig. 6). Only a few papers reported results from laboratory studies but a notable amount of field and labo ratory work reported on aspects of the early life-stage physiology. The coverage of abiotic drivers examined included not only temperature but also salinity with special emphasis on dissolved oxygen. Not only prey quantity but also prey quality was examined (Fig. 6). Fig. 6. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) on Peruvian anchovy in the Humboldt Current System. In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only re sponses are also shown (color and thickness of circles). Studies published on SPF in the CanCS represent about 9% of those included in this review (Fig. 5). Most studies focused on the European sardine (69%), followed by the anchovy (19%) and both Sardinellas (12%). The number of studies published in the two periods was similar but the majority of published research stems from the Iberian subregion. Relatively little research has been conducted on stocks of SPF in Mauritanian-Senegalese subregion with most historical research pub lished in regional journals or within technical reports (e.g., Boely et al., 1982; Fr´eon, 1983; John, 1986). Interestingly, some of the seminal research on recruitment mechanisms of SPF stem from this subregion such as Cury and Roy’s (1989) “optimal environmental window”. Fig. 6. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) on Peruvian anchovy in the Humboldt Current System. In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. 3.1. The Humboldt Current System Moreover, anchovy and sardine in the HCS forage on larger prey items such as euphausiids to a greater degree than in other systems (Espinoza et al., 2009). An important shift in knowledge was the realization that the sediment record indicates that anchovy and sardine do not consistently alternate in biomass at decadal scales (e.g., Chavez et al., 2003) but that these species display inconsistent patterns of alternation across a range of timescales which are not correlated with the PDO and other remote climate drivers (Vald´es et al., 2008; Guti´errez et al., 2009; Salvatteci et al., 2018). 3.1. The Humboldt Current System ringens; or (iv) the complex processes (from physics to seabirds and fisheries) impacting small pelagic fish (Bertrand et al., 2008a,c, 2014; Joo et al., 2014; Passuni et al., 2016; Barbraud et al., 2018). i The productivity of fish in the HCS is mostly controlled by climate- driven bottom-up processes impacting on the production of Fig. 5. The numbers of studies on small pelagic fish published during two, six-year time periods in 17 different biogeographic regions. The location of each circle often represents a much broader region (e.g., one or more large marine ecosystems). Some studies (statistical analyses / reviews) were counted in a multiple regions. Fig. 5. The numbers of studies on small pelagic fish published during two, six-year time periods in 17 different biogeographic regions. The location of each circle often represents a much broader region (e.g., one or more large marine ecosystems). Some studies (statistical analyses / reviews) were counted in a multiple regions. M.A. Peck et al. Progress in Oceanography 191 (2021) 102494 phytoplankton and higher trophic levels at a variety of scales (Guti´errez et al., 2009; Ay´on et al., 2011; Salvatteci et al., 2018). Although tem perature was considered the main driver of the spatial dynamics of small pelagic fish in the HCS in the early 2000 s, the demonstration of plas ticity in the response of pelagic fish towards temperature (e.g., McFar lane et al., 2002; Bertrand et al., 2004; Espinoza and Bertrand, 2008; Guti´errez et al., 2008) suggested more complex relationships between SPF and vertical and horizontal water-mass characteristics, particularly in relation to the Oxygen Minimum Zones (OMZs) and prey distribution (Ay´on et al., 2008a, 2011; Swartzman et al., 2008; Ball´on et al., 2011; Bertrand et al., 2011; Salvatteti et al., 2019) at multiple, dynamic scales (Bertrand et al., 2014). These more complex ecosystem characteristics not only influenced bottom-up drivers but also the magnitude of top- down control from both predators and fisheries (Joo et al., 2015; Pas suni et al., 2016; Barbraud et al., 2018). Between the two time periods compared in the present literature review, many long-held beliefs changed. This included diet studies suggesting that Peruvian anchoveta and Pacific sardine do not mainly feed on phytoplankton but on zooplankton (Espinoza and Bertrand 2008, 2014; Espinoza et al., 2009, 2017) in agreement with observations in the Benguela system, (van der Lingen et al., 2006b). 3.3. The Benguela Current System Economically and ecologically important SPF found in the BCS include European (Cape) anchovy, west coast (redeye) round herring (Etrumeus whiteheadi), Pacific sardine and the sardinellas Sardinella aurita and S. maderensis, all of which are primarily caught using purse- seines. Small catches of sardinellas are only taken in the NB and small catches of west coast round herring are only taken in the SB, and these species are not considered further here. The Lüderitz upwelling cell limits the exchange of epipelagic ichthyoplankton between the NB and SB due to the combination of a surface hydrodynamic and a subsurface thermal barrier (Lett et al., 2007), hence anchovy and sardine are considered to comprise separate sub-populations in the two sub-systems. Sardine was the initial target of purse-seiners in both sub-systems, and annual catches peaked during the 1960s before declining rapidly (van der Lingen et al., 2006c). Whilst sardine catches in the NB have not recovered to historical levels, those in the SB peaked again in the early 2000s but have again declined (de Moor et al., 2017). Anchovy were targeted in both sub-systems following the 1960s decline in sardine, but initial moderate catches in the NB declined to insignificant levels and there is almost no anchovy currently caught off Namibia (Roux et al., 2013). In contrast, anchovy (primarily juveniles) sustained the South African small pelagic fishery in years of low sardine catches and remains an important target species in the SB. Whereas both the NB and the SB were historically characterized by large populations and high catches of SPF, these species have virtually been removed from the NB, primarily by overexploitation (Boyer et al., 2001; Roux et al., 2013) but likely also by adverse environmental conditions during the mid-1990s (Jarre et al., 2015). Their removal, particularly of sardine, has had substantial and possibly irreversible impacts on the structure and functioning of the NB, and appears to have promoted the proliferation of jellyfish there (Roux et al., 2013). Field work has also contributed fundamental knowledge on the reproduction and feeding of SPF. For example, to apply the Egg Pro duction Method, new knowledge was generated on sardine egg mortality (Bernal et al., 2012), spawning seasonality and energy allocation (Nunes et al., 2011), and fine-scale spawning and larval distribution (Zwolinski et al., 2006). 3.3. The Benguela Current System Studies on SPF in the Benguela Current System (BCS) represented about 6% of the total number of papers included in this review with about 70% of the studies published in the first (2001–2006) period (Fig. 5). The BCS off southwestern Africa is bounded by the warm Angola Current in the north and the warm Agulhas Current in the south, and is divided into northern (NB) and southern (SB) sub-systems by the Lüderitz upwelling cell in Namibia (ca. 27◦S; Hutchings et al., 2009; Kirkman et al., 2016). The NB and the coast of South Africa west of 20◦E are wind-driven, coastal upwelling systems whereas the south coast of South Africa, also considered part of the SB, shows characteristics of both an upwelling and a temperate, shallow shelf system with seasonal coastal, shelf-edge and dynamic upwelling. Upwelling is perennial and strongest in the NB, seasonal and moderate off the South African west coast, and seasonal and weakest off the south coast (Lamont et al., 2018). Due to these differences in upwelling intensity, productivity is highest in the NB and lowest off the South African south coast. Fig. 7. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) on European sardine in the Canary Current and Iberian Systems. In total, 10 categories of drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only responses are also shown (color and thickness of circles). upwelling events impact on the dispersal of sardine larvae (Santos et al., 2005a,b), and how sea surface temperature, intense easterly winds, and discharges from the Guadalquivir River are related to early life-stage mortality of anchovy in the Gulf of Cadiz (e.g., Ruiz et al., 2006). Work in the latter region has been used to help forecast the strength of anchovy recruitment, potentially increasing the reliability of process- based stock assessment models (Rinc´on et al., 2016). 3.2. The Canary/Iberian Current System Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only re sponses are also shown (color and thickness of circles). During the two periods reviewed here, a good balance of laboratory, field, statistical analyses and spatially-explicit modelling studies was published (Fig. 7). In the Iberian sub-region, long-term data from Spanish and Portuguese scientific surveys (since the 1980 s) and fish eries catch statistics (since the 1940 s) have provided insight on how environment factors and processes regulate the productivity of SPF. Examples include how prevailing wind conditions affect sardine recruitment (Borges et al., 2003; Guisande et al., 2004), how winter 10 Progress in Oceanography 191 (2021) 102494 M.A. Peck et al. Fig. 7. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) on European sardine in the Canary Current and Iberian Systems. In total, 10 categories of drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only responses are also shown (color and thickness of circles). logbooks) on catches and field ecology of Sardinella spp (Braham et al., 2014; Ba et al., 2016). The increase in scientific contributions from this sub-region is expected to continue as the results of large international projects continue to be released (see Ba et al., 2017; Tiedemann et al., 2017; Balde et al., 2019). The number of relatively large-scale studies investigating stock structure or spawning dynamics of sardine or an chovy also doubled in the 2011–2016 time period (from 2 to 5 studies), especially those using genetic tools (e.g., Baibai et al., 2012; Silva et al., 2014). 2014). 3.3. The Benguela Current System Those studies included an assessment of how within-season variation in wind- driven upwelling impacted anchovy recruitment strength (Roy et al., 2001), attempts to predict anchovy recruitment using a deterministic expert system using classification trees (Miller and Field, 2002), and how anchovy recruitment was related to SST patterns (Richardson et al., 2003; Wilhelm et al., 2005). No such studies were published in the second 6-yr period reviewed here. Anchovy and sardine in the SB have both shown recent, eastward expansions of their distributions (van der Lingen et al., 2011) that appear to be linked to a cooling of inshore waters along the South Af rican southwest and south coasts since the 1980s (Blamey et al., 2015) and may be a result of climate change. Similarly, sardine in the NB has shifted its preferred spawning location southwards which has been attributed to a general warming of that system (Kreiner et al., 2011), also possibly driven by climate change. Another bottom-up impact, possibly arising from climate change, is the deleterious effect on SB sardine of spatially and temporally extensive harmful algal blooms (HABs) that have occurred off the South African south coast in recent years. van der Lingen et al. (2016) reported that sardine in HAB areas had particularly low condition factors compared to conspecifics outside of bloom areas, and that low condition was attributed to a cessation of feeding likely due to chemical irritation by the small (50 µm) di noflagellates being entrapped on the sardine’s gill rakers. Because of their coarser branchial baskets, neither anchovy nor round herring are able to entrap these small particles and hence were unaffected. y p Because the spawning and nursery areas of anchovy (and to a lesser extent sardine) in the SB are spatially separated, with most spawning occurring on the western Agulhas Bank and south coast and the major nursery ground located on the west coast, substantial research effort focused on the transport of eggs and early larvae from spawning to nursery grounds, and several biophysical IBMs were developed during the first review period. These were principally for Cape (European) anchovy in the SB (Mullon et al., 2002; Huggett et al., 2003; Mullon et al., 2003; Parada et al., 2003; Skogen et al., 2003) (see Fig. 8), but also for sardine (Miller et al., 2006) in the SB and the NB (Stenevik et al., 2003). 3.3. The Benguela Current System The IBMs developed for anchovy suggested a low probability of successful transport to the west coast nursery ground for eggs spawned east of Cape Agulhas, and that transport success of eggs spawned to the west of this cape was markedly seasonal and increased with increasing spawning depth. The latter is counter-intuitive given that most anchovy eggs occur in the upper 20 m (Dopolo et al., 2005). The IBM developed for SB sardine suggested the same results and identified Cape Agulhas as a break point between transport-based (west coast) and a retention- based (south coast) recruitment systems. Lett et al. (2006) reinforced these differences using a Lagrangian approach to simulate enrichment and retention, two processes fundamental to SPF recruitment (Bakun, 1996). Similar to anchovy, the successful recruitment of sardine to nursery areas increased in both the SB and NB with increasing depth of spawning which, again, was inconsistent with observations of near- surface sardine egg vertical distributions in both sub-systems (Stenevik et al., 2001; Dopolo et al., 2005). Only one biophysical IBM (Kone et al., 2013) and a review of previous IBMs (Lett et al., 2015) were published from the Benguela during the second 6-year period reviewed here. The decrease is attributed to a decrease in international (particularly French) collaborative programs (CvdL personal communication). p p Important publications examining bottom-up controls on SPF in the SB have been published outside of the two periods reviewed here. Roy et al. (2007) investigated potential environmental drivers of the abrupt change during the mid-1990s in the relative (i.e., proportion of total) distribution of European (Cape) anchovy spawners from being pre dominately located off the South African west coast to being predomi nantly located off the south coast. Those authors reported a significant positive correlation between the cross-shelf SST gradient on the south coast and the proportion of the anchovy spawner biomass there for the period 1984–2005, and suggested that coastal cooling in that region resulting from increased wind-induced upwelling off the south coast may have enhanced anchovy feeding conditions relative to those on the west coast. Subsequent inclusion of data up to 2011 corroborated this positive relationship and supported the hypothesis that the distribution change was environmentally mediated (Augustyn et al., 2018). 3.3. The Benguela Current System Field studies exploring the trophic ecology revealed the high efficiency of retention of microplankton prey by sardines (Garrido et al., 2007b) allowing adults of this species to consume phytoplankton, particularly during periods of low zooplankton abundance (Garrido et al., 2008a). Adult sardines were also shown to have high fish egg consumption (mainly from sardines and anchovies), similarly to Atlantic chub mackerel, which has high diet overlap with sardines (Garrido et al., 2015). Laboratory experiments conducted on sardine larvae focused on the influence of temperature and prey quantity on larval feeding (Caldeira et al., 2014), growth and survival (Garrido et al., 2016), and swimming capacity (Silva et al., 2014). The strong influence of temperature on the vital rates of sardine larvae observed in the laboratory agree well with results of field studies (Coombs et al., 2006; Stratoudakis et al., 2007). Field studies have also described larval diel vertical migration (Santos et al., 2006; Zwolinski et al., 2007). These laboratory and field results have generated hypotheses on the bottom-up mechanisms controlling sardine recruitment strength (e.g., Garrido, 2017). In the Canary and African sub-regions, more than 70% of research studies were field studies with half of those performed on early life stages (eggs and larvae) of European sardine and anchovy (e.g., Ettahiri et al., 2003; B´ecogn´ee et al., 2006; Moyano et al., 2014; Abdelouahab et al., 2016). In the most recent period, hydrodynamic modelling examined the transport dynamics of early life stages (Brochier et al., 2011). Twice as many research articles were published on SPF in the Mauritanian-Senegalese region in the most recent period (see Fig. 5) including attempts to compile unpublished, archived datasets (e.g., Several studies exploring environmental links to recruitment and abundance of anchovy and sardine in both the NB and the SB were published during 2001–2006. In the NB, studies on sardine included a synopsis of population fluctuations and possible environmental causes during the 1990s (Boyer et al., 2001), and relating recruitment to SST in the tropical Atlantic and coastal wind stress (Daskalov et al., 2003) and to sea surface height (Hardman-Mountford et al., 2003). Studies in the SB focused on anchovy recruitment, considered at the turn of this 11 M.A. Peck et al. Progress in Oceanography 191 (2021) 102494 century to be negatively correlated with southeasterly wind anomalies during the October-March spawning period because of advective loss of early life history stages (van der Lingen and Huggett, 2003). 3.3. The Benguela Current System Recent results from simulations using a regional oceanographic model of the south coast (Agulhas Bank) run with long-term (1948–2008) environ mental forcing corroborated the mid-1990s shift from warmer to colder temperatures and suggested that this was caused by a north–south migration in the large-scale wind belts rather than by changes in the Agulhas Current itself (Malan et al., 2019). van der Sleen et al. (2018) investigated the relationship between Cape anchovy recruitment and upwelling over the period 1985–2014 and found that recruitment was significantly and positively correlated with cumulative December- March (austral summer) upwelling. Those authors also reported that the slope of that positive, linear relationship increased when anchovy spawner biomass off the west coast in November/December of the preceding year was above a threshold of 0.74 MMT. By combining the two regressions for spawner biomass levels above and below that threshold, van der Sleen et al. (2018) accounted for 82% of the observed variability in anchovy recruitment. Sakamoto et al. (2020) examined otolith oxygen isotope ratios and microstructure of sardine from the SB and showed that nursery temperature affected early growth rates, with fish from cooler (by around 3 ◦C) waters in the west reaching 37–46 mm FL at 60 dph compared to those in warmer waters to the east that reached 43–52 mm. Those studies have provided strong evidence that bottom-up processes can have marked impacts on the distribution, abundance and vital rates of SPF in the SB. Fig. 8. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) on anchovy in the Benguela System. The species was reported as either European or Cape anchovy. In total, 10 categories of bottom-up drivers (below dotted line) were included: from left, 5 abiotic fac tors, 3 physical / mixing processes, and 2 aspects of prey. Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population- level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only responses are also shown (color and thickness of circles). Fig. 8. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) on anchovy in the Benguela System. 3.3. The Benguela Current System The species was reported as either European or Cape anchovy. In total, 10 categories of bottom-up drivers (below dotted line) were included: from left, 5 abiotic fac tors, 3 physical / mixing processes, and 2 aspects of prey. Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population- level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only responses are also shown (color and thickness of circles). 3.4. The California Current System The California Current System (CalCS) was among the regions with the most studies published on SPF in the two periods reviewed here (Fig. 5). The CaICS is broadly defined as the eastern limb of the North Pacific subtropical gyre and includes an equatorward flowing core that is generally offshore of the shelf break and more coastal, poleward flowing currents with seasonal variability in their depths and intensities. The CalCS extends from the eastern edge of the North Pacific Current 12 Progress in Oceanography 191 (2021) 102494 M.A. Peck et al. expanded its focus beyond its original goal of investigating reasons for variability in the sardine population to include sampling that is relevant to ocean responses to climate variability and change. The observations off California are now complimented by the Investigaciones Mexicanas de la Corriente de California (IMECOCAL) Program off Baja California which was initiated in 1997. near Vancouver Island, Canada at about 50◦N to Baja California Sur, Mexico near 25◦N where the surface flow turns westward to eventually merge with the North Equatorial Current. Wind-driven upwelling (both coastal upwelling and positive wind-stress curl) is a crucial element of the physical climate processes influencing biological productivity in the CalCS. Similar to other wind-driven EBUS, biological productivity in the CalCS is rich, but temporally variable across multiple time scales. Recent research on SPF in the CalCS has been shaped by a long history of studies in the region that began in the first two decades of the 20th century with the rapidly expanding industrial fishery. The species of focus for these research efforts has varied through time as the pop ulations of commercial interest have alternated; northern anchovy became the focus of most research in the 1960s, 1970s, and 1980s during periods of anomalously low biomass of Pacific sardine. Research originating from the region has contributed to about 10% of the total work assessed during the two periods considered in this manuscript (2001–2006 and 2011–2016) with a consistent number of studies pub lished during the two periods (n = 52 and 51, respectively). Roughly 75% of the publications during these periods focused on Pacific sardine, though several studies also considered species interactions. As noted above, much of the fundamental physiological and life-history work on SPF in the CalCS was conducted in the 20th century. 3.4. The California Current System Work published in recent decades has emphasized the consequences of changes in ocean conditions (e.g., physical conditions and planktonic prey fields) on the distribution and population productivity of SPF (Fig. 9). i p y p A number of noteworthy review articles describe the physical oceanography of the CalCS. Perhaps the most recent of these reviews includes description of the current understanding of the biological re sponses to physical variability (Checkley and Barth, 2009). The body of literature on processes in the CalCS describes relationships between physical processes and fisheries in the region, but also recognizes that these descriptions remain incomplete; observational and modelling constraints often limit understanding of processes that occur at the mesoscale and submesoscale, while the durations of observational time series limit abilities to describe processes occurring at multidecadal time scales. The seasonal cycles of upwelling favourable winds and biological productivity vary along a meridional gradient in the CalCS. Upwelling favourable winds are relatively weak but persistent year-round in the southern portion (south of Pt. Conception near 35◦N) of the system. The meridional direction and intensity of the winds exhibit increasing sea sonality from Pt. Conception northward, typically with intense winds from the north (upwelling favourable) in summer and winds from the south (downwelling favourable) in winter. Variability in ecosystem conditions has been attributed to changes in the intensity, persistence, spatial pattern, and seasonal timing of these upwelling winds (Barth et al., 2007; Bograd et al., 2009; Rykaczewski and Checkley, 2008). Additional sources of ecosystem variability stem from regional scale changes in characteristics of the subtropical gyre circulation and coastally trapped Kelvin waves (stimulated by El Ni˜no events in the tropics) that can modulate the depth of the nutricline (Jacox et al., 2018). Low-frequency variability (e.g., at decadal and multidecadal time scales) in the climate conditions in the region have been associated with changes in fish and invertebrate assemblages with implications for commercial fisheries (Chelton et al., 1982). i In the first period considered here (2001–2006), a notable amount of research was dedicated to investigating some of the physical processes that influence the distribution, composition, and survival of larval SPF and other members of the ichthyoplankton. These works were facilitated by the 5 decades of ichthyoplankton data collected by CalCOFI (e.g., Logerwell et al., 2001; Smith et al., 2001; Smith and Moser, 2003; Lynn, 2003). 3.4. The California Current System Additionally, changes in distribution associated with the 1997–1998 El Ni˜no event and the initiation of the IMECOCAL Program inspired investigation of the stock structure and distribution that span ned political boundaries (e.g., Funes-Rodrı́guez et al., 2001; Nev´arez- Martínez et al., 2001; Morales- Boj´orquez et al., 2003; Avendano-Ibarra et al., 2004; Felix-Uraga et al., 2004; S´anchez-Velasco et al., 2004; Morales-Boj´orquez and Nev´arez-Martínez, 2005). i Historically, commercial fisheries focused on massive populations of northern anchovy (Engraulis mordax) and Pacific sardine which made significant contributions to the regional economies in California and Baja California. The fishery for Pacific sardine grew to be the largest fishery in North America in the 1930s and 1940s and inspired pio neering research on SPF dynamics and the sustainability of their fish eries (Sette, 1943). The alarming collapse of the sardine population in the mid-1940s, however, had severe consequences for communities of central California (John Steinbeck: Cannery Row). Observation of this astonishing rearrangement of pelagic species in the mid-20th century was the initial motivation for 1949 commencement of the California Cooperative Oceanic Fisheries Investigations (CalCOFI), an oceano graphic survey program with a mission of understanding the human impacts and the influence of climate variability and climate change on living marine resources in the CalCS (McClatchie, 2013). Despite sub stantial investment in the collection of physical, chemical and biological data associated with the initial decades of the CalCOFI program, debate concerning the relative influence of commercial fishing and natural climate and population variability persisted (Clarke and Marr, 1955) and inspired alternative approaches to explore SPF variability. Investi gation of ocean sediments provided a perspective on population vari ability that precedes records of commercial landings (Soutar, 1967). This paleoceanographic approach stimulated a new field of SPF research and has been repeated in other ecosystems around the globe. Research during the second period (2011–2016) also included sig nificant contributions from scientists at Mexican institutions aimed at resolving patterns of ichthyoplankton distribution and the sensitivity of Fig. 9. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) on Pacific sardine in the California Current System. In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. 3.4. The California Current System During both time periods, this region produced a large amount of papers reporting the results of modelling studies exploring changes in the productivity and distribution (exampled for Pacific sardine shown in Fig. 9). A resurgence in investigation of vital rates was also evident during this more recent period (e.g., Zwolinski and Demer, 2013, 2014), perhaps motivated by early evidence that the northern stock of Pacific sardine was exhibiting successive recruitment failures. Although the population of northern anchovy was studied during both periods, a greater amount of anchovy-specific research was evident in the latter period (e.g., Takahashi et al., 2012; Fissel et al., 2011). Additionally, the decades of regular observations to quantify physical and biological conditions in the CalCS has facilitated one of the more unique aspects of research the region: application of theoretical numerical approaches to explore causal relationships in long time series of observational data (Sugihara et al., 2012; Deyle et al., 2013) (green arrows, Fig. 9). As data continue to accumulate over time, the value of oceanographic and fisheries observations to exploratory numerical analyses is certain to increase. along with fishery-dependent stock surveys (Ichinokawa et al., 2017) and complimentary acoustic surveys (Miyashita, 2018). In waters off South Korea, the distribution and abundance of SPF has been examined using trawls along with some acoustic data (Park et al., 2016). The population dynamics of SPF in the Kuroshio Current system are characterized by alternating cycles of abundance linked to basin- and multidecadal-scale climate variability. A prominent example is the shifts in dominance between anchovy and sardine which coincides with different phases of the Pacific Decadal Oscillation (PDO) index (Mantua and Hare 2002). When the PDO index was positive(negative), SST was lower(higher) in the NW Pacific and sardine(anchovy) was relatively abundant (Takasuka et al., 2007). In the early 1980 s, sardine was abundant while anchovy was relatively scarce and, by the end of that decade, the situation was reversed. Since 2010, sardine has started to increase in abundance while the SSB of anchovy has declined. This type of cycle has occurred twice during the last 100 years. 3.5. The northwest Pacific The oceanographic setting of the Northwest Pacific (NW Pacific) is characterized by dynamic fronts, meanders, and eddies formed during the interaction between cold- and warm-water currents (Nakata et al., 2000; Yasuda, 2003). The western boundary current system in the North Pacific is formed from the interaction between the warm Kuroshio and cold Oyashio Currents which converge off the coast of Japan and flow eastward forming the Kuroshio Extension and Kuroshio-Oyashio tran sition regions. In shelf areas to the north and east of the Kuroshio, a number of smaller currents interact in the vicinity of the Korean Peninsula including the warm Tsushima and East Korean Water Currents which intersect the cold Liman Current in the Sea of Japan. The dynamic mixing of these relatively nutrient-poor warm-water currents and nutrient-rich cold currents creates a highly productive environment supporting a variety of SPF such as Japanese sardine (Sardinops mela nostictus), Japanese anchovy (Engraulis japonicus), round herring (Etru meus teres), and Pacific herring. Studies published during the two time periods on these 3 species were the second most numerous of any region reviewed here (Fig. 5). i Research effort (in terms of the number of publications) was similar in the earlier (n = 60) and later (n = 53) periods reviewed here with most of the studies being field research. Unlike EBUS such as the HCS where anchovy often obtains the highest SSB, sardine obtained the highest biomass within the Kuroshio western boundary current system. Such a difference seems to be reflected in the study effort during the 1980s and 90s when most studies were focused on Japanese sardine. In the two more recent periods reviewed here, however, the majority of studies focused on anchovy (~70%) with fewer on sardine (~30%). Also, the number of studies examining both species has increased in the most recent period. Since the late 2000s, studies have attempted to advance a mechanistic understanding of the differences between Japa nese sardine and Japanese anchovy. These include inter-specific com parisons of the response of growth (Takasuka et al., 2007, 2009; Itoh et al., 2011), spawning (Oozeki et al., 2007; Takasuka et al., 2008a, 2008b), and transport (Itoh et al., 2009, 2011) to environmental factors. Laboratory studies have also examined energy allocation strategies for reproduction (Yoneda et al., 2014; Tanaka et al., 2016). 3.4. The California Current System The pattern of the species alternation in the NW Pacific was synchronous with that observed in eastern regions of the Pacific (California and Humboldt Current systems) despite the reversed temperature regimes between the opposite sides of the Pacific in the past, although the pattern observed in the California Current system has already been out of synchrony (Ka wasaki, 1983; Chavez et al., 2003; Takasuka et al., 2008b). Although “regimes” in productivity and abundance of different SPF have been recognized since Kawasaki (1983) compared sardine among various current systems, the physical-biological mechanisms linking climate variability to the population dynamics of SPF are still debated. Some hypotheses are based on changes in the winter mixed layer depth and spring phytoplankton bloom in the nursery grounds of sardine (Noto and Yasuda, 1999; Nishikawa and Yasuda, 2008; Nishikawa et al., 2011, 2013). For example, Noto and Yasuda (1999) found a close relationship between the mortality during the early life stages and winter SST in the nursery ground of sardine. Other hypotheses are based on changes in water temperature and species-specific differences in the optimal ther mal regimes for the early growth and spawning (Takasuka et al., 2007, 2008b). It is likely that an amalgam of factors are responsible for fluc tuations in the productivity of different SPF such as the interaction be tween temperature and food availability reported to control the growth rate of larval anchovy (Takahashi and Watanabe, 2005) or prey avail ability, temperature and intra-guild competition (Kawasaki and Omori, 1995; Kim et al., 2006) and/or fishing pressure (Suda et al., 2005; Katsukawa, 2007; Wan and Bian, 2012). 3.4. The California Current System Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only re sponses are also shown (color and thickness of circles). Fig. 9. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) on Pacific sardine in the California Current System. In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only re sponses are also shown (color and thickness of circles). Fig. 9. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) on Pacific sardine in the California Current System. In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only re sponses are also shown (color and thickness of circles). Although the magnitudes of the commercial SPF catches have declined substantially since the mid-20th century, the research meth odology developed in the region formed the foundation for many modern SPF surveys (e.g., the examination of batch fecundity by Hunter and Leong (1981) and the development of DEPM by Lasker (1985)). The CalCOFI Program has now continued for more than 70 years and 13 Progress in Oceanography 191 (2021) 102494 M.A. Peck et al. 3.4. The California Current System those patterns to climate conditions in the IMECOCAL region and the Gulf of California (e.g., Funes-Rodríguez et al., 2012; Vergara-Solana et al., 2013; Valencia-Gasti et al., 2015). Contributions that were focused on the Pacific sardine stock found along the US west coast included several efforts aimed at exploring the implications of climate variability for resource management, including end-to-end models (Fiechter et al., 2015; Rose et al., 2015) and stock forecasts (Kaplan et al., 2016); population dynamics (Lindegren et al., 2013; Asch et al., 2013; Song et al., 2012; Weber et al., 2015); and migration patterns (Demer et al., 2012; McDaniel et al., 2016; Zwolinski et al., 2011). During both time periods, this region produced a large amount of papers reporting the results of modelling studies exploring changes in the productivity and distribution (exampled for Pacific sardine shown in Fig. 9). A resurgence in investigation of vital rates was also evident during this more recent period (e.g., Zwolinski and Demer, 2013, 2014), perhaps motivated by early evidence that the northern stock of Pacific sardine was exhibiting successive recruitment failures. Although the population of northern anchovy was studied during both periods, a greater amount of anchovy-specific research was evident in the latter period (e.g., Takahashi et al., 2012; Fissel et al., 2011). Additionally, the decades of regular observations to quantify physical and biological conditions in the CalCS has facilitated one of the more unique aspects of research the region: application of theoretical numerical approaches to explore causal relationships in long time series of observational data (Sugihara et al., 2012; Deyle et al., 2013) (green arrows, Fig. 9). As data continue to accumulate over time, the value of oceanographic and fisheries observations to exploratory numerical analyses is certain to increase. those patterns to climate conditions in the IMECOCAL region and the Gulf of California (e.g., Funes-Rodríguez et al., 2012; Vergara-Solana et al., 2013; Valencia-Gasti et al., 2015). Contributions that were focused on the Pacific sardine stock found along the US west coast included several efforts aimed at exploring the implications of climate variability for resource management, including end-to-end models (Fiechter et al., 2015; Rose et al., 2015) and stock forecasts (Kaplan et al., 2016); population dynamics (Lindegren et al., 2013; Asch et al., 2013; Song et al., 2012; Weber et al., 2015); and migration patterns (Demer et al., 2012; McDaniel et al., 2016; Zwolinski et al., 2011). 3.5. The northwest Pacific The biological parameters obtained through those field and laboratory studies have been incorporated into bioenergetics sub-routines for Japanese sardine and saury (Cololabis saira) within end-to-end ecosystem models designed to test different hypotheses of biological mechanisms and to make pro jections of future population dynamics of these species (Ito et al., 2004; Kishi et al., 2007; Ito et al., 2010; Okunishi et al., 2012a, b). The number of modelling studies and review articles were higher in the more recent The SPF in the NW Pacific utilize water current systems for passive drift and as migration pathways to create spatially complex life cycle strategies. For example, Japanese sardine and anchovy utilize coastal spawning grounds in the Pacific and a major portion of their eggs and larvae are transported offshore by the Kuroshio Current. Juveniles migrate north in the Kuroshio Extension to nursery and feeding grounds in the Kuroshio–Oyashio transition region. As these fish grow and recruit to adult stocks, they return inshore and move south for spawning. The distribution and spawning areas of sardine and anchovy contract coastward or expand offshore during period of low and high SSB, respectively (Watanabe et al., 1996; Takasuka et al., 2008a). In contrast, round herring is a subtropical species mainly distributed and spawning in coastal waters whereas Pacific herring is a sub-Arctic species mainly distributed and spawning in the cold waters of the Oyashio and Liman Currents (Watanabe, 2007). The spawning patterns of SPF have been well documented from monthly ichthyoplankton and zooplankton sur veys conducted off the Pacific coast of Japan since 1978 (Oozeki, 2018) 14 Progress in Oceanography 191 (2021) 102494 M.A. Peck et al. nearshore spawning grounds for herring in the SW Baltic (e.g., Dodson et al., 2019). period (2011–2016). The research map for Japanese anchovy in the NW Pacific (Fig. 10) highlights the emphasis placed on field work exploring aspects of the vital rates at the individual level (growth, feeding, reproduction and survival) and the spatial distribution and productivity of the population. Reviewing the research conducted on sprat in the SW Baltic Sea, Voss et al. (2012) indicated that bottom-up processes (temperature, trans port, prey abundance) had a greater influence on year-class survival compared to top-down processes (predation mortality). 3.6. The Baltic Sea The Baltic Sea is the largest semi-enclosed, brackish water area in the world with an opening in its southwest corner through the straights of Denmark to the North Sea. The Baltic has a large catchment area and river runoff creates a permanent halocline between surface waters with low salinity and deeper, more saline waters (Fonselius, 1970). The Baltic Sea has a series of submarine sills and deep basins whose deep waters are hypoxic or anoxic (Fonselius, 1970). Atmospheric conditions cause episodic inflow events of dense marine water from the North Sea which renew and ventilate deep waters. In the most recent decades, these inflow events are often followed by long stagnation periods (Lehmann et al., 2002) and a decrease in the frequency of inflow events since the 1990s has been correlated to changes in zooplankton and fish pop ulations within deep basins including the two main SPF, European sprat and Atlantic herring (Alheit et al., 2005). Studies on SPF in the Baltic Sea formed a relatively high proportion of those reviewed here (within the top 4 of the 17 regions) (Fig. 5). During the two periods reviewed here, a considerable amount of research was conducted on various life stages of both Atlantic herring and sprat including reviews of how bottom-up processes may impact on the recruitment dynamics of sprat (K¨oster et al., 2003; Voss et al., 2012), herring (von Dorrien et al., 2013) or both species (M¨ollmann et al., 2005; Casini et al., 2006). These and other studies have exploited rich (long-term, often spatially-explicit) historical field data on the dominant zooplankton and fish species (e.g., Casini et al., 2014), such as re constructions of sprat spawning stock biomass back to the early 1930s (e.g., Eero, 2012). These time series continue to grow in length and be exploited to understand environmental drivers of recruitment success of SPF. An example is examining seasonal / thermal windows of larval survival using the Rügen Herring Larvae Survey, a survey started in the mid-1970s and continued consistenly since the mid 1990s, which in cludes weekly monitoring of larvae at one of the most important, There are a number of unique features of the Baltic Sea (from its topography and hydrography to its populations of SPF) driving specific research efforts. First, the Baltic Sea contains different sub-populations and genetically distinct populations of Atlantic herring which display different migration patterns and spawning seasons and locations. 3.5. The northwest Pacific For example, at high spawning stock biomass, warm water temperatures and wind- driven transport of sprat larvae from offshore spawning grounds to coastal areas was correlated with poor year-class success (Baumann et al., 2006). This was postulated to be due to density-dependent, top- down control of zooplankton resources in coastal areas leading to poor condition and starvation (Baumann et al., 2005). Post-larval sprat have high rates of growth and feeding and considerable laboratory work has been conducted to calibrate in situ proxies for these rates (Peck et al., 2012a, 2015; Günther et al., 2015). A second, critical period was considered to be the long over-winter period when relatively small, young-of-the-year (age-0 juvenile) sprat may not survive (Peck et al., 2012a; Voss et al., 2012). The work on Baltic sprat demonstrates how environmental controls (such as temperature thresholds) can affect various life stages to affect life cycle closure and population dynamics (Haslob et al., 2012b). Research in the Baltic Sea has also revealed how changes in the abundance of SPF can lead to density-dependent impacts on their growth and/or condition (Casini et al., 2006) highlighting how population dynamics of SPF are tightly coupled to the interaction be tween extrinsic (environmental) drivers and intrinsic factors. 3.6. The Baltic Sea Considerable work was conducted in the time periods reviewed here to resolve the spatial stock dynamics across the N. Atlantic basin (McPherson et al., 2004) and among co-existing herring stocks in the Baltic Sea (e.g., Jorgensen et al., 2005; Gr¨ohsler et al., 2013; Teacher et al., 2013). A second feature is the strong vertical and depth gradients in temperature, salinity and oxygen (water mass characteristics) struc turing and controlling the survival and development (Peck et al., 2012b, Moyano et al., 2016), feeding dynamics (Rajasilta et al., 2014), growth (Baumann et al., 2006), spatial distribution (Nielsen et al., 2001; Nis sling et al., 2003), and recruitment dynamics (Arula et al., 2016) of herring and sprat. Finally, given potential changes in prey availability and type driven by abiotic forcing, both laboratory and field studies have examined how changes in the quantity and quality (such as the contents of lipid including fatty acids or trophic upgrading) of zooplankton can influence larval survival and somatic growth of Atlantic herring (Illing et al., 2015; Paulsen et al., 2014, 2016) and sprat (Peck et al., 2015; Peters et al., 2015) (see Fig. 11). Fig. 10. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) on Pacific sardine in the California Current System. In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only re sponses are also shown (color and thickness of circles). 3.7. The Mediterranean Sea Responses (above dotted line) were separated into 9 categories including 7 at the indi vidual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only responses are also shown (color and thickness of circles). important, sub-regions supporting productive SPF populations (e.g., the Aegean Sea, the Gulf of Lions and nearby Catalan Coast, the Alboran Sea, the Straits of Sicily/Tunisian Coast, and the Adriatic Sea: Agostini and Bakun, 2002; Basilone et al., 2006; Zarrad and Missaoui, 2006; Tugores et al., 2011; Giannoulaki et al., 2013; Bonanno et al., 2014). From 2000 to 2013, four species contributed roughly 50% of the SPF landings including, in order of economic value, European anchovy, European sardine, round sardinella and European sprat (FAO, 2018b). The large economic and cultural importance of European anchovy (see Fig. 12 for research conducted on this species) and other SPF in Mediterranean countries has generated a wealth of research on bottom-up controls and this is reflected by this region having the most published research on SPF during the two time periods reviewed here (Fig. 5). This region had the highest number of publications in both of the two periods reviewed here including a considerable number of review arti cles. A thorough review of the research conducted on European anchovy across the Mediterranean was published in 1996 (Palomera and Rubíes, 1996) and the latest period has seen an upsurge in reviews that are both regional (Palomera et al., 2007; Sabat´es et al., 2007b, Van Beveren et al., 2016) and thematic-oriented (Peck et al., 2013; Stergiou et al., 2016). Despite this wealth of research, it is challenging to disentangle the effect of bottom-up processes controlling SPF when stocks experience heavy fishing pressure and this is the case in the Mediterranean Sea. Efforts to curtail the over-exploitation of SPF in this region are challenged due to the plethora of countries, shared stocks, abundant shelters/ports and high number of small fishing boats (e.g., Tsikliras et al., 2015). 3.7. The Mediterranean Sea The semi-enclosed, temperate Mediterranean Sea is rich in mesoscale activity resulting from its complex topography and strong and varied local winds (reviewed in Millot and Taupier-Letage, 2005). There are several unique features of the Mediterranean Sea which offer opportu nities to advance our understanding of bottom-up controls of SPF. First, both temperate and tropical (e.g., round sardinella) SPF co-occur in the region which offers a good test bed to examine historical (and projected) changes in climate will impact SPF (e.g., Sabat´es et al., 2006; Maynou et al., 2014, 2020; Stergiou et al., 2016). Second, although the whole region is classified as a Large Marine Ecoystem (McLeod et al., 2005), considerable east-to-west and north-to-south gradients exist in abiotic factors, biological productivity and patterns of biodiversity. The mean annual temperature and salinity, degree of oligotrophy and number of introduced invasive species all increase from the NW to the SE (Coll et al., 2010). Third, differences in topography, river discharge and amounts of upwelling and mesoscale activity have created several Fig. 10. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) on Pacific sardine in the California Current System. In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only re sponses are also shown (color and thickness of circles). 15 Progress in Oceanography 191 (2021) 102494 M.A. Peck et al. Fig. 11. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) on European sprat in the Baltic Sea. In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. Responses (above dotted line) were separated into 9 categories including 7 at the indi vidual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. 3.7. The Mediterranean Sea The color represents the type of study (not all types may be present). The number and type of studies considering only responses are also shown (color and thickness of circles). Short-term, bottom-up processes impacting the dynamics of SPF populations in the Mediterranean Sea include changes in riverine inputs (Lloret et al., 2004; Santojanni et al., 2006; Macías et al., 2014), advection and eddies (Sabat´es et al., 2007a, Catal´an et al., 2013; Ruiz et al., 2013, ´Alvarez et al., 2015; Ospina-Alvarez et al., 2015) and temperature (Maynou et al., 2014). Changes in the strength of bottom- up control are clearly associated with climate variability and climate change (e.g., Martín et al., 2011). For example, the mean temperature- at-catch has steadily increased during the last two decades (Tsikliras and Stergiou, 2014; Tzanatos et al., 2014) and Stergiou et al. (2016) reported strong relationships between warming and shifts in abundance of SPF, particularly after the 1990s. A northward expansion of round sardinella in the Mediterranean Sea tracks warming in the northern (Sabat´es et al., 2006; Maynou et al., 2014), central (Sinovcic et al., 2004) and eastern regions (Tsikliras, 2008) and has been linked to a marked decline in the abundance of European sardine. In the Gulf of Lions, de creases in somatic growth rate, size-at-age and somatic condition, and an increased age truncation of European anchovy and sardine have co- occurred with an order of magnitude increase in the abundance of Eu ropean sprat (Van Beveren et al., 2014; Brosset et al., 2016). Bottom-up processes impacting the community composition of zooplankton, together with reduced trophic niche partitioning are thought to be the drivers of these intra-guild changes (Van Beveren et al., 2014; Brosset et al., 2016). Shifts in seasonal spawning triggers have also occurred (Tsikliras et al., 2010; Palomera et al., 2007). Similar to other regions, low frequency teleconnections and periodic oscillations in local climate conditions have been linked to fluctuations in SPF stocks (Grbec et al., 2002; Lloret, 2000; Martín et al., 2011; Katara et al., 2011). Fig. 11. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) on European sprat in the Baltic Sea. In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. 3.7. The Mediterranean Sea More over, the region is experiencing rapid warming and accelerated species invasion (Libralato et al., 2015) disrupting the provision of natural ecosystem services such as healthy, historically dominant catches of SPF (Liquete et al., 2016; Stergiou et al., 2016). The intense exploitation and warming combined with accelerated ecosystem changes due to species invasions and other species changes (Stergiou et al., 2016; Brosset et al., 2016) offers many examples of potential situations that SPF may suffer elsewhere. Gaps exist, however, due to a scarcity of work employing laboratory experiments to study the ecophysiology of different life stages (reviewed in Peck et al., 2013). Also, similar to most systems, habitats and potential life-cycle bottlenecks related to the juvenile stage are poorly investigated. Increased research in southern Mediterranean areas fostered within programs of the United Nations Food and Agri cultural Organization (FAO) may partly ameliorate this situation. Fig. 12. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) on European anchovy in the Mediterranean Sea. In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only responses are also shown (color and thickness of circles). During the two periods reviewed here, research has been imbalanced with respect to species, stages, bottom-up drivers and geographic loca tions. For example, in both periods, ≥90% of the published studies stemmed from European as opposed to African countries. The majority of research has been conducted by Spain, Greece and Italy (on their local, SPF sub-components). There was, however, a five-fold increase in the number of studies published that stemmed from African countries (Algeria, Tunisia, Morocco) between 2001 and 06 and 2011–16. The increase in the number of publications in the second period was Mediterranean-wide with almost twice the number of papers published in the second compared to the first period. Most publications examined Fig. 12. 3.7. The Mediterranean Sea The potential diet overlap between anchovy and sardine has also been extensively studied (e.g., Chouvelon et al., 2015). Similar to other areas, most of the research conducted in the Medi terranean Sea was field research (79% in 2001–2006 and 63% in 2011–2016). In both periods, field research focused on relationships between environmental variables and distribution/abundance/struc ture, followed by studies on maturation or spawning and somatic growth. Our review found no published studies employing laboratory studies (e.g., Fig. 12 for Europen anchovy). During the second period, there was a marked increase in the number of studies examining diets across life stages (e.g., Nikolioudakis et al., 2012; Costalago et al., 2012; Costalago and Palomera, 2014; Rumolo et al., 2016) and exploring SPF using physical-biogeochemcal models and Lagrangian, particle tracking simulations conducted on anchovy (Catal´an et al., 2013; Ospina-´Alvarez et al., 2013; Schismenou et al., 2013; Palatella et al., 2014) or end-to-end models (Politikos et al., 2014; Coll et al., 2016). In both the North Sea and Bay of Biscay, spatially-explicit models have been extensively employed to advance understanding of various aspects of the ecology of SPF (Fig. 13a,b). Some of the earliest bio physical models for fish early life stages were championed in the NE Atlantic such as the drift modelling of herring early life stages by Bartsch et al. (1989) in the North Sea. When IBMs started to be used more frequently to examine SPF in the early 2000 s, an important advance was coupling a Lagrangian tracking model to otolith-based back-calculated growth trajectories of anchovy in the Bay of Biscay (Allain et al., 2003). Daewel et al. (2011) illustrated how an IBM for European sprat could be linked to a lower trophic level model to explore both direct (tempera ture, water currents) and indirect (prey field) impacts of climate vari ability on potential survival to the end of the larval stage. The rich spatiotemporal data series and laboratory work on herring larvae in the North Sea were employed to create physiological-based (foraging and growth) biophysical model to formulate and/or test hypotheses on bottom-up drivers of recruitment (Hufnagl and Peck, 2011; Huebert and Peck, 2014; Hufnagl et al., 2015). In the Bay of Biscay, juvenile and adult life stages were included in spatially-explicit, bioenergetics models of growth and movement in anchovy (Politikos et al., 2015). 3.7. The Mediterranean Sea Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) on European anchovy in the Mediterranean Sea. In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only responses are also shown (color and thickness of circles). 16 Progress in Oceanography 191 (2021) 102494 M.A. Peck et al. European anchovy (58% and 73% in consecutive periods) followed by European sardine (33% and 21%) and Round sardinella (8% and 1%). A second notable trend is the increase in basin-wide research from the first to second periods on essential habitats of European sardine and an chovy, including juveniles (Tugores et al., 2011; Giannoulaki et al., 2013). A remarkable upsurge in the amount of research on sprat and round sardinella track the increase in abundance of these SPF species. Fernandez et al., 2015) and to thoroughly document long-term changes in the intensity of site-specific spawning (Harma et al., 2012) and productivity (Corten, 2013). A primary impetus for exploring these herring data was to understand the collapse (in the early 1970 s) and recovery (by the mid 1980 s) of the autumn-spawning North Sea herring stock. In the Bay of Biscay, considerable research has been conducted on many life history facets of European anchovy, particularly because this stock collapsed and has now been rebuilt after a 5-yr closure of the fishery (2005 to 2010). The fishery closure provided opportunities to study natural mortality rates which appear very high for two-year olds, suggesting senescence is an important, intrinsic limitation impacting on population dynamics (Uriate et al., 2016). Retrospective analyses of the seasonal dynamics of growth and/or distribution (Ibaibarriaga et al., 2013; Petitgas et al., 2014; Boyra et al., 2016) have made use of annual, broad-scale surveys conducted since the early 1990s during the anchovy spawning season in spring. Separate in-shore and offshore spawning populations have been identified (Montes et al., 2016). 3.7. The Mediterranean Sea This IBM approach was linked to a Dynamic Energy Budget (DEB) model including energy allocation rules (Gatti et al., 2013) giving rise to one of only a handful of end-to-end models linking physics, lower trophic levels (zooplankton), and life cycle ecophysiology of SPF. 3.8. The northeast Atlantic This region includes the European continental shelf area from the Bay of Biscay in the south, the North Sea and waters surrounding the British Isles, a spatial extent that includes a transition zone between warmer-water (anchovy, sardine) and colder-water (sprat, herring) SPF. The hydrography of these shelf systems is dynamic with an array of tidal-mixing, river plume and shelf break fronts fuelling an extremely productive and complex food web. That many of these mesoscale fea tures are strongly influenced by changes in physical forcing and many SPF occur near their (high or low) latitudinal limit, are reasons why strong changes in the distribution and/or abundance of SPF populations have been correlated to climate variability in this region (Beare et al., 2004; Petitgas et al., 2012; Alheit et al., 2012). i A variety of field studies published during the second 6-year period (2011–2016) sought to understand habitat and resource partitioning by a “novel” co-occurrence of SPF in the southern North Sea (Raab et al., 2012; Bils et al., 2012). In, 2006, the productivity of a relict (coastal) population of European anchovy in the southern North Sea dramatically increased due to more favourable temperature and feeding conditions and adult anchovy started to be identified in offshore trawl surveys (Petitgas et al., 2012; Raab et al., 2013). During the same time period, ichthyoplankton surveys found mixtures of European sardine and sprat in the southern North Sea (Kanstinger et al., 2009). In late spring (May/ June), early life stages of European sardine, sprat and anchovy in offshore frontal areas (Kanstinger et al., 2009) indicated the close proximity and timing of spawning by the three species although Euro pean anchovy were more abundant at shallower stations closer to the coast. 3.9. The northwest Atlantic The NW Atlantic includes regions supporting warmer and cooler water species of clupeoid fish such as bay anchovy (Anchoa mitchilli) in the Chesapeake Bay and other coastal areas at lower latitudes, Atlantic herring using spawning grounds on Georges Bank and in the Gulf of Maine., as well as Atlantic menhaden (Brevoortia tyrannus) overlapping in some habitats with both of the former species. Atlantic menhaden supports one of the oldest and largest fisheries (by volume) in the NW Atlantic (for fishing communities in Chesapeake Bay and other areas of the mid-Atlantic bight of the east coast of the United States). That stock declined dramatically in the 1990s and remained low for nearly a decade but has subsequently increased under lower levels of fishing pressure despite low recruitment indices. A number of research groups have consistently used laboratory studies to explore aspects of the ecophysiology of North Sea Atlantic herring such as how intrinsic and/or extrinsic factors influence funda mental aspects of growth physiology. Examples include parental effects on traits of early life stages (Bang et al., 2006) and how environmental factors affect otolith ring formation (Fox et al., 2003), embryonic growth (Geffen, 2002), muscle development (Temple et al., 2001; Temple et al., 2001) or schooling behaviour (Domenici et al., 2002). This laboratory work is complimented with large field datasets such as the dedicated autumn and winter surveys for herring larvae that have been conducted in the North and Irish Seas since 1959. Indices of abundance of larvae from different spawning sites are available since 1972. Exploration of these long-term survey data have helped researchers understand envi ronmental drivers of recruitment such as winter water temperature and /or zooplankton (Payne et al., 2009; Lusseau et al., 2014; Alvarez- In this region in the 1970s and 1980s, seminal laboratory studies were conducted on Atlantic menhaden that provided a rare, mechanistic understanding of the relationship between swimming, feeding (and diet) and somatic growth (e.g., Durbin and Durbin, 1975; Durbin et al., 1980). 3.9. The northwest Atlantic The number and type of studies considering only responses are also shown (color and thickness of circles). Chesapeake Bay for menhaden populations (Luo et al., 2001) and another linking primary production to growth (Annis et al., 2011). A specific life history trait of menhaden includes offshore spawning with transport and ingress of larvae to coastal estuarine nursery areas and several studies examined the sources and characteristics of cohorts entering nursery grounds (Warlen et al., 2002; Light and Able, 2003; Lozano et al., 2012). Although fundamental studies on topics such as ontogenetic changes in diets (Friedland et al., 2006) continued in both periods, long-term data from menhaden sampling programs was mined in the second period to explore temperature-growth characteristics (Humphrey et al., 2014), mortality rates of larvae (Simpson et al., 2016) and links between recruitment climate indices such as the Atlantic Multi-decadal Oscillation (Buchheister et al., 2016). The impetus for most of these studies has been to understand the decreased recruitment strength of menhaden in this region since the 1990 s. Fig. 14. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) in the northwest Atlantic on bay anchovy. In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only responses are also shown (color and thickness of circles). Bay anchovy represents another species that was studied intensively in the laboratory and mesocosms in the 1980s to gain fundamental data on bioenergetics rates leading to seminal modelling studies of its role (as predator and prey) within Chesapeake Bay (Lou and Brandt, 1993). Within both periods reviewed here, our search string found 11 papers that explored a variety of processes from larval transport (North and Houde, 2004), recruitment dynamics (Jung and Houde, 2004) and the growth dynamics of a population close to the high latitudinal limit of the species (Lapolla et al., 2001a,b). 3.9. The northwest Atlantic An emphasis of several of these studies was to test hypotheses by applying spatial or ecosystem (size-based) models to field data. This was continued in the only study published on Bay anchovy in the second period by Adamack et al. (2014) who explored how hypoxia (driven by scenarios of nutrient loading and rainfall) would impact on the mortality of larvae (see research map, Fig. 14). That work followed on from field measurements indicating that Bay anchovy recruitment was inversely related to dissolved oxygen concentrations in different regions of the estuary (Jung and Houde, 2004). The marked decline in studies published from the first to the second period suggests a waning interest in conducting studies on bottom-up processes and factors affecting Bay anchovy. Fig. 14. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) in the northwest Atlantic on bay anchovy. In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only responses are also shown (color and thickness of circles). subsequent larval rentential areas a few months were examined by Stephenson et al. (2015) showing the complex behavioural and bio physical (advective) processes influencing the metapopulation structure of herring in the Bay of Fundy. 3.9. The northwest Atlantic During the first of the two periods reviewed here, studies on menhaden utilized those ecophysiological results to create various types of growth models such as one providing 3-D estimates of the carrying capacity of In this region in the 1970s and 1980s, seminal laboratory studies were conducted on Atlantic menhaden that provided a rare, mechanistic understanding of the relationship between swimming, feeding (and diet) and somatic growth (e.g., Durbin and Durbin, 1975; Durbin et al., 1980). During the first of the two periods reviewed here, studies on menhaden utilized those ecophysiological results to create various types of growth models such as one providing 3-D estimates of the carrying capacity of 17 M.A. Peck et al. Progress in Oceanography 191 (2021) 102494 M.A. Peck et al. Fig. 13. Research emphasis in papers pub lished during two time periods (2001–2006, and 2011–2016) in the northeast Atlantic including Atlantic herring in the North Sea (Panel A) and European anchovy in the Bay of Biscay (Panel B). In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. Re sponses (above dotted line) were separated into 9 categories including 7 at the individ ual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population- level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only responses are also shown (color and thickness of circles). Fig. 13. Research emphasis in papers pub lished during two time periods (2001–2006, and 2011–2016) in the northeast Atlantic including Atlantic herring in the North Sea (Panel A) and European anchovy in the Bay of Biscay (Panel B). In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. Re sponses (above dotted line) were separated into 9 categories including 7 at the individ ual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population- level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). 3.10. Sub-polar regions (Gulf of Alaska and Bering, Barents and Norwegian Seas) g., Holst et al., 2002), relative ease of laboratory culture, and intense long-term changes including stock collapses and recoveries. In the sub- polar NE Atlantic, large stocks of plantivorous fish co-occur and may compete for resources, which has been a catalyst for studying diets (e.g., Godiksen et al., 2006; ´Oskarsson et al., 2015) and distributions (Utne et al., 2012b) and for the development of individual-based models of movements and habitat utilization (Utne and Huse, 2012). i Long-term field data exist for herring in sub-polar waters and, not unexpectedly, a number of retrospective studies have been published. In the NE Atlantic, the Norwegian Spring Spawning (NSS) herring stock collapsed to very low biomass levels in the 1960s and was rebuilt in the 1980s. Reproductive characteristics differed between high and low (collapsed) stock biomass levels with fish maturing at larger body sizes and at decreased ages during the collapse period (Engelhard and Heino, 2004) which could suggest a release from density-dependent growth regulation. For NSS herring in the Barents Sea, the relationship between recruitment and temperature was relatively weak (e.g., Fiksen and Slotte, 2002) which agrees with the results of a longer (1913 to 2007) time series analysis in which only a weak (albeit significant) positive relationship was observed, where recruitment variability was not correlated with temperature, and recruitment variability was lower prior to the stock collapse (Bogstad et al., 2013). In Pacific herring, density dependence was suggested in a time series of stock size and temperature during certain regimes identified using fuzzy logic (Chen, 2001). In the analysis of a shorter (10-year) but highly seasonally resolved (e.g., weekly to monthly sampling) time series, Reum et al. (2013) found no evidence for density-dependent growth of larvae (but growth was related to temperature) but significant density-dependent growth during the early juvenile phase highlighting how the dominant mechanisms controlling growth can be stage-specific. p p p ( ) g ( g , , ) Studies have examined behavioural-mediated movements of herring at a variety of spatial scales from the sub-micro (thin layers and diurnal vertical migration (DVM) of larvae) to meso (feeding migrations of adult). For example, field sampling by Ferreira et al. (2012) found that Atlantic herring larvae perform type I DVM (shallow during day, deep at night) likely resulting mainly from foraging tactics. 3.10. Sub-polar regions (Gulf of Alaska and Bering, Barents and Norwegian Seas) Laboratory work on Pacific herring demonstrated how the presence of thin layers but not necessarily prey patches can attract larvae and impact their vertical distribution (Clay et al., 2004). In adult Atlantic herring in the Barents Sea, foraging tactics were also used to explain the large-scale feeding migrations. In that case, the seasonal movements of schools appeared to be linked to regional differences in the timing of the ascent into shallow waters of a key prey species, the copepod Calanus finmarchicus (Broms et al., 2012). Earlier work by Kvamme et al. (2003) documented changes in the movement of herring schools towards locations with higher concentrations of prey. Changes in depth distribution of adult Atlantic herring across 12 years were suggested to be due to inter-annual dif ferences in the strength of intra- and inter-specific competition for prey, indicating density-dependent changes in habitat utilization (Huse et al., 2012). In another example, Langård et al. (2014) documented consis tent, pre-spawning schooling dynamics of herring in a coastal Norwe gian fjord and reported on the influence of variation in fish size and spawning phase on school tightness. Finally, work on first-time migrants and changes in wintering grounds demonstrated the importance of so cial learning in shaping the large-scale migration patterns of NSS herring (Huse et al., 2010). i i There were a considerable number of studies published on the spatial dynamics of populations of both sister species of herring (Fig. 15). In sub-polar waters of both the Atlantic and Pacific, complex herring stock structures exist and a variety of studies have employed genetics to explore the relatedness between and within herring species (Jørstad, 2004). For instance, comparisons of allozyme loci and vertebral numbers suggested large differences between NSS herring captured simultaneously with individuals more closely related to Pacific herring The ability to artificially spawn and rear herring in the laboratory has promoted laboratory research on various aspects of early life stages such as the effects of environmental stressors on survival (Lefebvre et al., 2005; Froehlich et al., 2015), feeding (Utne-Palm, 2004; Ingvarsd´ottir Fig. 15. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) in sub-polar systems in the northern hemisphere including Pacific herring in the northeast Pacific (Panel A) and Atlantic herring in the Barents and North Sea (Panel B). 3.10. Sub-polar regions (Gulf of Alaska and Bering, Barents and Norwegian Seas) Two sister species of herring dominate the SPF assemblage in sub- polar waters in the northern hemisphere (e.g., between 50 and 70◦N). Atlantic herring dominates in the Nordic, and Irminger and Labrador Seas while Pacific herring dominates in the Gulf of Alaska and Berring Sea. Both Pacific and Atlantic herring are extremely valuable fisheries resources not only to commercial fisheries but also to indigenous peo ples in sub-polar regions. Atlantic and Pacific herring are, arguably, among the most thoroughly studied fish species in the world for which seminal research has established fundamental paradigms of factors controlling fish productivity and year-class success (e.g., Hj¨ort, 1914). This review, mostly focusing on a 12-year period in the new millenium, ignores a long history of examination fuelled then and now by the unique life history attributes and traits of herring. These traits include a Long-term bottom trawl survey data allowed an exploration of how habitat occupancy contracted under stock collapse and expanded again after the recover of Atlantic herring across three key sub-regions of the NW Atlantic as well as within a specific sub-region (Overholtz, 2002; Overholtz and Friedland, 2002). Another study explored small-scale swimming movements of adult spawners using ultrasonic tags revealing, in some cases, selective tidal stream transport (Lacoste et al., 2001). Work on movement patterns of adult spawners was continued in earnest in the second period with studies using spatial and temporal data derived from hydroacoustic survey revealing seasonal movements including spawning aggregations (Jech and Stroman, 2012; Wurtzell et al., 2016). The locations of adult spawner aggregations and 18 Progress in Oceanography 191 (2021) 102494 M.A. Peck et al. sampled in the Japan Sea and the Gulf of Alaska ((Jørstad et al., 2004). Work in the early 2000s developed more powerful techniques to distinguish both intra- and inter-stock genetic variation (e.g., kinship analysis) using microsatellites (Olsen et al., 2002). Inter-breeding be tween NSS and a coastal (fjord) stock explained the loss of differences in larval growth rate and vertebral numbers over several decades sug gesting that metapopulation structure in herring is dynamic (Johanne sen et al., 2014). Studies of introgression between these Pacific and Atlantic sister species have generated hypotheses regarding post-glacial population separation and (re-)mixing (e.g., Laakkonen et al., 2015). complex stock structure, recurrent migration patterns (feeding and reproductive migrations including homing to specific spawning sites, e. 4.1. Projecting future productivity of SPF For example, based on future changes in water temperature projected by climate climate models, the center of the distribution of Peruvian anchoveta was expected to shift to higher latitudes at rates between 13 and 33 km / decade depending on the severity of global warming (Jones and Cheung, 2015; Jones et al., 2015). These shifts in distribution will impact accessibility of stocks and landings with downstream, global impacts on fish meal and fish oil prices. Finally, a mechanistic understanding of how physical and biogeochemical processes impact SPF populations is required for advancing short-term forecasts (months to years) that could be valuable for management of SPF stocks. Within the four EBUS (Humboldt, California, Benguela and Canary), understanding how upwelling strength is linked to phytoplankton and zooplankton production and how upwelling may change will be key to making robust projections of how climate change impacts. In the northern Humboldt, recent trends in SST are in accordance with the hypothesis that increasing greenhouse gas concentrations will force intensification of upwelling-favourable winds in EBUS (Bakun, 1990). Although this hypothesis was challenged by projections from global climate models (Vecchi and Soden, 2007; Lu et al., 2007), these models poorly represent the regional wind forcing and upwelling (Gent et al., 2010). Overall, a consensus is emerging that the intensity and duration of upwelling-favourable winds will increase in the southern Humboldt Current system, off Chile and will (moderately) decrease off Peru (Garreaud and Falvey, 2009; Falvey and Garreaud, 2009; Goubanova et al., 2011; Echevin et al., 2012; Belmadani et al., 2014; Garcia-Reyes, 2015; Wang, 2015). Decreased upwelling would decrease the produc tivity of lower trophic levels and warming-induced increases in strati fication would ultimately increase the area of the OMZ. Such changes are in agreement with those constructed from the paleo record in sedi ments (Salvatteci et al., 2019). In central Chile, upwelling is predicted to occur earlier, end later and be of greater intensity, especially in summer (Garreaud and Falvey, 2009; Belmadani et al., 2014; Rykaczewski et al., Differences among the types of studies across regions can be visu alized by comparing the ‘research maps’ (Fig. 16). In regions at rela tively high latitudes, herrings and sprat are amenable to laboratory and /or mesocosm research, allowing a wider array of responses (including physiology and behaviour) to be examined. 4.1. Projecting future productivity of SPF Although we have attempted to highlight key research exploring bottom-up controls of SPF which was published prior to (≤2000), be tween (2007–2010) and after (≥2017) the two, 6-year time periods exhaustively reviewed here. It should be noted that our global maps and figures would look considerably different if these other time periods were also included in a rigorous, systematic literature review. For example, considerable field research on SPF was conducted in the 1980s and 1990s in California as part of the long-running CalCOFI program. The types (and amounts) of studies performed in various regions are also influenced by the types of large-scale (global) initiatives such as GLOBEC (regional and national programs from 1990 to 2009) or specific regional programs such as BENEFIT (1998–2007) in the Benguela (Hampton and Sweijd, 2008), or the Humboldt Conference in 2006 (Bertrand et al., 2008b) and the DISCOH (Dynamics of the Humboldt Current System, 2010–2019) International Joint Laboratory in the Humboldt. These programs are often conducted in response to large- scale changes (collapses or increases) of stocks. For example, the Cal COFI program was promoted because of serious concerns over the decline of sardine (https://calcofi.org/about-calcofi/history.html). The type (and amount) of publications on SPF in specific regions can also be influenced by the research interest (and productivity) of principle in vestigators who have ‘championed’ SPF research. For example, the late 1970s through the 1990s was a very productive period at the Ches apeake Biological Laboratory for research conducted on bay anchovy). Finally, the history of research conducted in specific regions likely influenced the types of studies that were published in this millennium. For example, studies including robust conceptual and process models are, arguably, only possible in areas where long-term field data were available. Although it is beyond the scope of the present study to exhaustively review projections of climate change impacts on SPF populations, un derstanding how bottom-up factors such as temperature and prey availability regulate SPF populations is critical to making these pro jections. Moreover, making robust projections of changes in bottom-up forcing and SPF are critical because of the economic (and ecological and cultural) importance of SPF, particularly for providing fish meal and fish oil required by the growing aquaculture industry. 3.10. Sub-polar regions (Gulf of Alaska and Bering, Barents and Norwegian Seas) In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only responses are also shown (color and thickness of circles). Fig. 15. Research emphasis in papers published during two time periods (2001–2006, and 2011–2016) in sub-polar systems in the northern hemisphere including Pacific herring in the northeast Pacific (Panel A) and Atlantic herring in the Barents and North Sea (Panel B). In total, 10 categories of bottom-up drivers (below dotted line) were used including 5 abiotic factors, 3 physical / mixing processes, and 2 aspects of prey. Responses (above dotted line) were separated into 9 categories including 7 at the individual level (from left, 3 energetic costs or losses and 4 vital rates) and 2 at the population-level (see Fig. 4 for legend). The thickness of lines represents the number of studies. The color represents the type of study (not all types may be present). The number and type of studies considering only responses are also shown (color and thickness of circles). 19 Progress in Oceanography 191 (2021) 102494 M.A. Peck et al. biophysical modelling in some (e.g., California Current, NE Atlantic, Mediterranean Sea) but not all (e.g., NW Pacific, Humboldt System) of those regions. In terms of vital rates, relatively few studies were pub lished on reproduction compared to growth and feeding. Disease and parasites were rarely studied nor were aspects of the physiology of SPF. In terms of physical factors, turbidity was least examined followed by water density, pH and dissolved oxygen (albiet regional differences exist). Turbulence was the physical process least often studied. We speculate that at least two reasons might explain this lack of research effort on turbulence. First, seminal work in the 1980s and 1990s (e.g., see review by Dower et al., 1997) produced results on the effects of turbulence (microscale, on early life stages) that researchers may perceive are applicable across species and regions. 3.10. Sub-polar regions (Gulf of Alaska and Bering, Barents and Norwegian Seas) Similarly, the ability to capture and maintain large juvenile and adult herring in small-scale in situ enclosures has led to studies examining adaptive schooling behaviour such as changes in schooling in reponse to sounds made by predators or ships (Wilson et al., 2002; Handegard et al., 2015) or simulated attacks by predators (Rieucau et al., 2016). It should be noted that, across the 10 years of published research reviewed here, herring was the only SPF studied using mesocosms / in situ enclosures. In sub-polar waters, there is a rich history of using spatially-explicit models to develop and test hypotheses on mechanisms behind observed patterns in the growth and/or distribution of herring in both the Pacific (Snauffer et al., 2014; Ito et al., 2015) and Atlantic (Huse et al., 2002; Sætre et al., 2002; Vikebø et al., 2010) as well as the potential top-down control of Calanus finmarchicus (Utne et al., 2012a). As previously mentioned, these efforts stem from development of high-resolution physical and/or biogeochemical models in the 1990s in both regions. Since 2008 in the Barents and Norwegian Seas, operational oceano graphic tools have been forecasting the drift and distribution of larvae and young juveniles of Atlantic herring to, among other things, aid in survey design and implementation (Vikebø et al., 2011). 3.10. Sub-polar regions (Gulf of Alaska and Bering, Barents and Norwegian Seas) Second, from a methodological point of view, turbulence is not straightforward to study under controlled laboratory conditions. Another clear gap in knowledge is that relatively few studies were published that examined the effect of prey quality (e.g., changes in lipid or fatty acid content of phyto- and zooplankton) on SPF populations. Although only selected species are shown for each region, these species were dominant in terms of the amount of research conducted during the two 6-yr periods reviewed here. et al., 2012) and behaviour (Maneja et al., 2015) of embryos and larvae (Fig. 15). Research on herring larvae includes seminal studies doc umenting ontogenetic changes in digestive capacity (e.g., Rojas-Garcia et al., 2016) and nutritional requirements (e.g., Conceiç˜aoa et al., 2002). Similarly, the ability to capture and maintain large juvenile and adult herring in small-scale in situ enclosures has led to studies examining adaptive schooling behaviour such as changes in schooling in reponse to sounds made by predators or ships (Wilson et al., 2002; Handegard et al., 2015) or simulated attacks by predators (Rieucau et al., 2016). It should be noted that, across the 10 years of published research reviewed here, herring was the only SPF studied using mesocosms / in situ enclosures. et al., 2012) and behaviour (Maneja et al., 2015) of embryos and larvae (Fig. 15). Research on herring larvae includes seminal studies doc umenting ontogenetic changes in digestive capacity (e.g., Rojas-Garcia et al., 2016) and nutritional requirements (e.g., Conceiç˜aoa et al., 2002). Similarly, the ability to capture and maintain large juvenile and adult herring in small-scale in situ enclosures has led to studies examining adaptive schooling behaviour such as changes in schooling in reponse to sounds made by predators or ships (Wilson et al., 2002; Handegard et al., 2015) or simulated attacks by predators (Rieucau et al., 2016). It should be noted that, across the 10 years of published research reviewed here, herring was the only SPF studied using mesocosms / in situ enclosures. In sub-polar waters, there is a rich history of using spatially-explicit models to develop and test hypotheses on mechanisms behind observed patterns in the growth and/or distribution of herring in both the Pacific (Snauffer et al., 2014; Ito et al., 2015) and Atlantic (Huse et al., 2002; Sætre et al., 2002; Vikebø et al., 2010) as well as the potential top-down control of Calanus finmarchicus (Utne et al., 2012a). 3.10. Sub-polar regions (Gulf of Alaska and Bering, Barents and Norwegian Seas) As previously mentioned, these efforts stem from development of high-resolution physical and/or biogeochemical models in the 1990s in both regions. Since 2008 in the Barents and Norwegian Seas, operational oceano graphic tools have been forecasting the drift and distribution of larvae and young juveniles of Atlantic herring to, among other things, aid in survey design and implementation (Vikebø et al., 2011). et al., 2012) and behaviour (Maneja et al., 2015) of embryos and larvae (Fig. 15). Research on herring larvae includes seminal studies doc umenting ontogenetic changes in digestive capacity (e.g., Rojas-Garcia et al., 2016) and nutritional requirements (e.g., Conceiç˜aoa et al., 2002). Similarly, the ability to capture and maintain large juvenile and adult herring in small-scale in situ enclosures has led to studies examining adaptive schooling behaviour such as changes in schooling in reponse to sounds made by predators or ships (Wilson et al., 2002; Handegard et al., 2015) or simulated attacks by predators (Rieucau et al., 2016). It should be noted that, across the 10 years of published research reviewed here, herring was the only SPF studied using mesocosms / in situ enclosures. In sub-polar waters, there is a rich history of using spatially-explicit models to develop and test hypotheses on mechanisms behind observed patterns in the growth and/or distribution of herring in both the Pacific (Snauffer et al., 2014; Ito et al., 2015) and Atlantic (Huse et al., 2002; Sætre et al., 2002; Vikebø et al., 2010) as well as the potential top-down control of Calanus finmarchicus (Utne et al., 2012a). As previously mentioned, these efforts stem from development of high-resolution physical and/or biogeochemical models in the 1990s in both regions. Since 2008 in the Barents and Norwegian Seas, operational oceano graphic tools have been forecasting the drift and distribution of larvae and young juveniles of Atlantic herring to, among other things, aid in survey design and implementation (Vikebø et al., 2011). et al., 2012) and behaviour (Maneja et al., 2015) of embryos and larvae (Fig. 15). Research on herring larvae includes seminal studies doc umenting ontogenetic changes in digestive capacity (e.g., Rojas-Garcia et al., 2016) and nutritional requirements (e.g., Conceiç˜aoa et al., 2002). 4.1. Projecting future productivity of SPF These research maps also indicate that a considerable amount of field work has been published documenting the spatial distribution of SPF but that many of those studies did not explicitly examine any potential bottom-up driver(s) influencing those patterns (e.g., thick outline around spatial distribution symbols in 7 of the 12 regions) (Fig. 16). The availability of field data is likely a reason for the greater emphasis placed on spatially-explicit 20 Progress in Oceanography 191 (2021) 102494 M.A. Peck et al. Progress in Oceanography 191 (2021 eck et al. 6. Research maps of studies exploring fundamental aspects of small pelagic fishes focusing on bottom-up factors and processes. The number of f lines), laboratory experiments (red lines), spatially-explicit modelling (blue lines), statistical time series analyses (green lines) and mesocosm stud conducted on key small pelagic fish species in 12 different regions is shown. This is based on the pooled studies published across 12 years (2 2016). Line thickness denotes the number of studies (also for symbol outlines indicating the number of studies that examined only a response an m-up forcing factor or process). The legend for the symbols (see insert) are more easily seen in Fig. 4. (For interpretation of the references to color in d, the reader is referred to the web version of this article.) Fig. 16. Research maps of studies exploring fundamental aspects of small pelagic fishes focusing on bottom-up factors and processes. The number of field studies (grey lines), laboratory experiments (red lines), spatially-explicit modelling (blue lines), statistical time series analyses (green lines) and mesocosm studies (yellow lines) conducted on key small pelagic fish species in 12 different regions is shown. This is based on the pooled studies published across 12 years (2001–2006, 2011–2016). Line thickness denotes the number of studies (also for symbol outlines indicating the number of studies that examined only a response and not any bottom-up forcing factor or process). The legend for the symbols (see insert) are more easily seen in Fig. 4. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 16. Research maps of studies exploring fundamental aspects of small pelagic fishes focusing on bottom-up factors and processes. 4.1. Projecting future productivity of SPF or physical changes within ecosystems such as the shoaling of OMZ waters and habitat compression tending to increase overlap with pred ators. These top-down processes are not included in this review. As discussed in the Benguela section, other indirect (bottom-up) effects related to climate change appear highly relevant such as the increase in HABs associated with decreased somatic condition of sardine (van der Lingen et al., 2016). Outside of the four, major EBUS regions, projecting how climate change may alter bottom-up processes impacting the productivity of SPF is also challenging for a few reasons. First, in oligotrophic areas such as the Mediterranean Sea, the results of different biogeochemical models (e.g., depictions of the dynamics of the deep chlorophyll maximum) agree poorly in some regions (e.g., Ramírez-Romero et al., 2020) and making robust projections of the impacts of climate change on SPF will require a better representation of i) mesoscale ocean physics in topo graphically complex areas, ii) nutrient loading by rivers fueling local production, iii) conditions causing local upwelling events. Projecting the effects of climate change will not only require better representations of these physical processes but also continuing to collect time-series data as well as new physiological data from laboratory experiments testing climate change-related drivers across life stages. • Oxygen minimum zones and seasonal extents of hypoxia are expanding. More research is needed on the acute (survival) and chronic (reductions in growth, reproduction) impacts of hypoxia on SPF. At the same time, explorations of how low dissolved oxygen and other drivers of SPF populations (either via laboratory experiments or statistical analyses of field data) need to recognize the complexity and context-specific effects of multiple direct and indirect factors (e. i g., Bertrand et al., 2011). Taking this holistic view will help better build and parameterize end-to-end models that attempt to depict how expanding OMZs and other processes affect complex ecosystem dynamics. • The use of otoliths to infer exact ages and somatic growth rates of early life stages is challenging in some species of SPF. Continued work is needed to verify and corroborate daily increment formation in the otoliths of SPF, particularly when environmental conditions cause relatively low rates of somatic growth (cold temperatures, low prey abundance). 4.1. Projecting future productivity of SPF Quantifying and reducing the error associated larval growth trajectories (first weeks of life) from the otoliths of much older juveniles is needed to apply a “characteristics of survi vors” approach (Plaza et al., 2019). The application of advanced chemical analyses such as δ18O (Javor 2013; Darnaude et al., 2014; Sakamoto et al., 2020) could yield important information on popu lation structure, spatial dynamics and movement patterns. Climate change may cause no-analog combinations of abiotic and biotic conditions, at least in terms of modern-day field observations in various ecosystems. Reconstructing paleorecords, such as time periods when productivity was lower and OMZs were larger than at present in the HUC (Salvatteci et al., 2019) or changes in winter sea surface tem perature in the NW Pacific (Kuwae et al., 2017) may provide valuable analogs to situations that could be faced in the future (in the absence of fishing). 4.1. Projecting future productivity of SPF The number of field studies (grey lines), laboratory experiments (red lines), spatially-explicit modelling (blue lines), statistical time series analyses (green lines) and mesocosm studies (yellow lines) conducted on key small pelagic fish species in 12 different regions is shown. This is based on the pooled studies published across 12 years (2001–2006, 2011–2016). Line thickness denotes the number of studies (also for symbol outlines indicating the number of studies that examined only a response and not any bottom-up forcing factor or process). The legend for the symbols (see insert) are more easily seen in Fig. 4. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 21 21 M.A. Peck et al. Progress in Oceanography 191 (2021) 102494 2015; Wang et al., 2015). Furthermore, climate change may signifi cantly reduce the spawning success of small pelagic fish in the Humboldt Current (Brochier et al., 2013). In this EBUS, therefore, changes in bottom-up factors (temperature, prey and dissolved oxygen) are ex pected to cause relatively dramatic shifts in the productivity and dis tribution of SPF with implications for the world’s largest, single-species fishery. • Understanding bottom-up drivers requires a close marriage between laboratory experiments, field surveys and biophysical modelling. This tripartite approach has been fully realized in studies conducted on Atlantic herring, a species relatively easy to grow in the labora tory, but not for most other SPF (clupeid) species. Establishment of broodstocks, captive rearing and programs studying the ecophysi ology of various life stages will allow species-specific parameteriza tions of mechanistic (physiological-based) models. i i Climate-induced shifts in bottom-up factors are more complicated and less distinct in other EBUS. For example, recent trends in upwelling intensity in the Canary EBUS remain unclear (Sousa et al., 2017), especially when compared to other EBUS (Sydeman et al., 2014; Wang et al., 2015). During the recent period of global warming (1967–2007), no increase in upwelling intensity in the Iberian and NW African sectors was observed and sea surface temperature only marginally increased at a rate greater than 0.01 ◦C yr−1 (Barton et al., 2013). Zonal (sub-region specific) and seasonal shifts in bottom-up processes, however are ex pected in the future. 4.1. Projecting future productivity of SPF Recent projections suggest an overall increase in upwelling intensity in the W Iberian Peninsula and decrease in NW Af rica (Wang et al., 2015; Sousa et al., 2017), and total cumulative up welling has increased off the South African south coast in the southern Benguela but not in the northern Benguela (Lamont et al., 2018). Similarly, the magnitude and impact of ongoing ocean acidification is expected to be region-specific and highly dependent on changes in up welling intensity (Gonz´alez-D´avila et al., 2009). It is important to note that predation pressure on SPF has shifted and may also markedly shift in the future due novel mixtures of species resulting from climate-driven warming and latitudinal shifts of populations (e.g., Garrido et al., 2015) • Quantitative field sampling of young-of-the-year (YOY) juveniles needs to be developed. The body size of young juveniles reduces their catchability in gears often used in surveys. Young-of-the-year (YOY) juveniles can avoid plankton nets used to catch eggs and larvae and can be too small to be reliably retained by purse seines and trawl nets used to capture adults in stock assessment surveys. Due to their large numbers and high mass-specific rates of feeding, YOY juveniles can have very large trophodynamic impacts on prey and suffer food limitation, reducing rates of growth and survival. Where young ju veniles have been studied (e.g., Voss et al., 2012), it is clear that the dynamics of this life stage can have large impacts on the strength of recruitment. • More emphasis is needed on understanding the role of density- dependence in population dynamics of SPF (e.g., Takasuka et al., 2019) to examine the magnitude of top-down regulation of prey resources as well as intra-guild dynamics such as competition and predation. More emphasis on intra-guild processes may help unravel mechanisms controlling fluctuations in SPF stocks. l l • Continued study of the influence of climate variability on SPF and their ecosystems is needed including investigations of the impacts of ocean–atmosphere interactions and climate modes and current sys tems (Alheit et al., 2019a). These explorations should not only be correlative (detecting patterns in field time series data) but also advance knowledge on the mechanisms and processes in ocean ecosystems represented by changes in these atmospheric / oceanic indices of climate variability that affect the growth and survival of SPF. 5. Recommendations • Mesocosm studies contributed less than 1% of the research published on SPF in the time periods (2001–2006 and 2011 to 2016) reviewed here. In the 1980s and 1990s, mesocosm studies provided useful data on the growth, feeding and survival of SPF in semi-natural settings (Fuiman and Gamble, 1988; Cowan and Houde, 1990). The use of large mesocosms is, arguably, the best way to advance understanding on how both direct (e.g., temperature, pH) and indirect (prey field) factors influence the growth and survival of SPF (Sswat et al., 2018). Based on our global review of the studies published on SPF, we are able to make a number of recommendations for future research needed to advance our understanding of how bottom-up factors and processes control the dynamics of these ecologically and economically important fishes. These recommendations are not independent from one another. 22 Progress in Oceanography 191 (2021) 102494 M.A. Peck et al. Research programs utilizing mesocosms may, thus, provide much- needed information on how environmental factors interact to affect SPF. Research programs utilizing mesocosms may, thus, provide much- needed information on how environmental factors interact to affect SPF. Alheit, J., 1987. Egg cannibalism versus egg predation: their significance in anchovies. In The Benguela and Comparable Ecosystems. Payne, A.I.L., Gulland, J.A., Brink, K.H. (Eds). South African Journal of Marine Science 5, 467–470. Alheit, J., 1993. Use of the daily egg production method for estimating biomass of clupeoid fishes: a review and evaluation. Bulletin of Marine Science 53, 750–767. • Due to their global distribution, their economic and ecological importance and the similarity in their biology, research on SPF has been traditionally characterized by large international cooperation. Some of the shifts in the amount of peer-reviewed publications (and their research emphasis) between the two, 6-year periods reviewed here, can be attributed to changes in the magnitude of international collaboration. There is a successful history of multinational projects and meetings on SPF (Sharp and Csirke, 1983; Csirke and Sharp, 1984; Alheit and Bakun, 2010). For example, the regional GLOBEC program SPACC (Small Pelagic Fish and Climate Change), started in 1994, lasted 15 years and culminated in the publication of the SPACC book in 2009 (Checkley et al., 2009). Although recent symposia have provided important platforms for knowledge exchange (Peck et al., 2014; Alheit et. 5. Recommendations al., 2019b, Alheit and Peck, 2019), a coordinated, global platform with sufficient funding for participation of scientists from developing countries is needed to foster not only knowledge exchange but also comparative studies. A first, new step towards formal, global collaboration was recently taken by establishing a working group on small pelagic fish supported by two international marine science organizations (ICES – International Council for the Exploration of the Seas - WGSPF; and PICES – North Pacific Science Organization - WG43). The present review represents the first product stemming from this international collaborative effort. • Due to their global distribution, their economic and ecological importance and the similarity in their biology, research on SPF has been traditionally characterized by large international cooperation. Some of the shifts in the amount of peer-reviewed publications (and their research emphasis) between the two, 6-year periods reviewed here, can be attributed to changes in the magnitude of international collaboration. There is a successful history of multinational projects and meetings on SPF (Sharp and Csirke, 1983; Csirke and Sharp, 1984; Alheit and Bakun, 2010). For example, the regional GLOBEC program SPACC (Small Pelagic Fish and Climate Change), started in 1994, lasted 15 years and culminated in the publication of the SPACC book in 2009 (Checkley et al., 2009). Although recent symposia have provided important platforms for knowledge exchange (Peck et al., 2014; Alheit et. al., 2019b, Alheit and Peck, 2019), a coordinated, global platform with sufficient funding for participation of scientists from developing countries is needed to foster not only knowledge exchange but also comparative studies. A first, new step towards formal, global collaboration was recently taken by establishing a working group on small pelagic fish supported by two international marine science organizations (ICES – International Council for the Exploration of the Seas - WGSPF; and PICES – North Pacific Science Organization - WG43). The present review represents the first product stemming from this international collaborative effort. Alheit, J., Bakun, A., 2010. Population synchronies within and between ocean basins: Apparent teleconnections and implications as to physical–biological linkage mechanisms. Journal of Marine Systems 79, 267–285. Alheit, J., Hagen, E., 1997. 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https://openalex.org/W4225311238	https://tc.copernicus.org/articles/16/2629/2022/tc-16-2629-2022.pdf	English	null	Reply to RCs	null	2,022	cc-by	9,515	The Cryosphere, 16, 2629–2642, 2022 https://doi.org/10.5194/tc-16-2629-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. The Cryosphere, 16, 2629–2642, 2022 https://doi.org/10.5194/tc-16-2629-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Correspondence: Tom Chudley (chudley.1@osu.edu) Correspondence: Tom Chudley (chudley.1@osu.edu) Received: 6 February 2022 – Discussion started: 3 March 2022 Revised: 9 May 2022 – Accepted: 8 June 2022 – Published: 1 July 2022 Received: 6 February 2022 – Discussion started: 3 March 2022 Revised: 9 May 2022 – Accepted: 8 June 2022 – Published: 1 July 2022 Abstract. By utilising imagery from overlapping orbits, the Sentinel-2 programme offers high-frequency observations of high-latitude environments well in excess of its 5 d re- peat rate, which is valuable for obtaining large-scale records of rapid environmental change. However, the production of glacier velocity datasets from optical feature tracking of Sentinel-2 imagery is limited by the orthorectiﬁcation error in ESA products, which introduces signiﬁcant systematic er- rors (on the order of tens of metres) into displacement ﬁelds produced from cross-track image pairs. As a result, most standard processing chains ignore cross-track pairs, which limits the opportunity to fully beneﬁt from Sentinel-2’s high- frequency observations during periods of intermittent cover- age or for rapid dynamic events. Here, we use temporally complete glacier velocity datasets to empirically reconstruct systematic error, allowing for the corrected velocity datasets to be produced for four key fast-ﬂowing marine-terminating outlets across the Greenland Ice Sheet between 2017–2021. We show that corrected data agree well with comparison velocity datasets derived from optical (Landsat 8) and syn- thetic aperture radar (Sentinel-1) data. The density of avail- able velocity pairs produces a noisier dataset than for these comparative records, but a best-ﬁt velocity reconstructed by time-series modelling can identify periods of rapid change (e.g. summer slowdowns), even where gaps exist in other datasets. We use the empirical error maps to identify that the commercial DEM used to orthorectify Sentinel-2 scenes over Greenland between 2017–2021 likely shares data sources with freely available public DEMs, opening avenues for the analytical correction of Sentinel-2 glacier velocity ﬁelds in the future. 1 Introduction Continuous glacier velocity datasets derived from medium- resolution satellite programmes have become increasingly available in recent years, forming a key part of investiga- tions into ice discharge (King et al., 2018; Gardner et al., 2018; Mankoff et al., 2019), glacier dynamics (Poinar and Andrews, 2021; Dehecq et al., 2019), and characterisation of seasonal glacier behaviour (Vijay et al., 2021; Moon et al., 2014). Globally comprehensive scene-pair velocity ﬁelds from medium-resolution satellite data are available from both optical feature-tracking and SAR speckle-tracking tech- niques: e.g. for Landsat 8 optical data, the ITS_LIVE pro- gramme (Gardner et al., 2018, 2022), and for Sentinel-1 SAR data, the MEaSUREs and PROMIICE programmes for Greenland (Joughin, 2021a; Solgaard et al., 2021) and the RETREAT programme for glaciers and ice caps (Friedl et al., 2021). The Sentinel-2 mission holds further promise in deriving glacier velocities from optical imagery compared to Landsat 8, offering an improved repeat time of 5 d (with both Sentinel-2A and Sentinel-2B) compared to 16 d (now 8 d from 2022 onwards with the addition of Landsat 9) and a resolution of 10 m in the visible and near-infrared portion of the electromagnetic spectrum compared to 30 m (15 m panchromatic) for Landsat 8. This dense temporal coverage increases the chances of ﬁnding cloud-free image pairs, par- ticularly when making use of cross-track imagery at high lati- tudes. However, as of yet, the use of Sentinel-2 velocity ﬁelds – particularly in the form of large-scale public datasets – are limited. A particular problem for Sentinel-2 feature tracking is the presence of systematic orthorectiﬁcation errors. These are Empirical correction of systematic orthorectiﬁcation error in Sentinel-2 velocity ﬁelds for Greenlandic outlet glaciers Thomas R. Chudley1, Ian M. Howat1,2, Bidhyananda Yadav1, and Myoung-Jong Noh1 1Byrd Polar and Climate Research Center, Ohio State University, Columbus, OH, USA 2School of Earth Sciences, Ohio State University, Columbus, OH, USA Correspondence: Tom Chudley (chudley.1@osu.edu) Received: 6 February 2022 – Discussion started: 3 March 2022 Revised: 9 May 2022 – Accepted: 8 June 2022 – Published: 1 July 2022 T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error (2016) suggest that high-latitude source datasets are shared with the Viewﬁnder Panoramas 3′′ DEM (de Ferranti, 2014), which uses, among other sources, 20th century topographic maps to reconstruct high-latitude ice topography (Jonathan de Ferranti, personal communica- tion, 2021). This decades-old source data could explain the large vertical DEM errors (tens to hundreds of metres) com- pared to Landsat 8, which, in Collection-2 processing, uses more recent region-speciﬁc elevation models at high latitudes (Franks et al., 2020), such as the ArcticDEM, Greenland Ice Mapping Project (GrIMP) DEM, and Alaskan National Ele- vation Dataset. As a result, orthorectiﬁcation errors remain a signiﬁcant issue for producing consistent Sentinel-2 glacier velocity ﬁelds. Kääb et al. (2016) recommend that feature tracking using cross-track image pairs should be performed only for ice displacements that are at least 1 order of magni- tude larger than the expected orthorectiﬁcation error, whilst Nagy et al. (2019) recommend not using cross-track pairs at all. This limits the beneﬁts of Sentinel-2’s dense data cov- erage, reducing the number of available image pairs to only p g g A range of solutions have been implemented to account for orthorectiﬁcation errors in medium-resolution optical satel- lite imagery. Rosenau et al. (2015) provide their own im- proved orthorectiﬁcation for Landsat imagery by orthorecti- fying L1G data to the ∼1′′ ASTER Global Digital Elevation Model (GDEM) V2, rather than the ∼30′′ Global 30 Arc Second Elevation (GLOTOPO30) dataset that was standard for L1C products at the time. However, the non-orthorectiﬁed product (L1B) for Sentinel-2 is not made available, meaning that this approach is not viable. With access to the Planet- DEM 90, Ressl and Pfeifer (2018) were able to generate a predicted offset ﬁeld for Sentinel-2 relative orbits over Aus- tria by using the PlanetDEM, the known orbits of Sentinel- 2, and a reference DEM (the AustriaDEM) as ground truth. Here, rays were projected from the satellite orbital path to the reference DEM and intersected with the PlanetDEM to derive the off-nadir offset. However, this method not only requires access to the PlanetDEM (which is a commercial product) but also a reference elevation model that is accu- rate at the time of image acquisition, which is challenging for glaciated regions where, in places, surface elevations are changing signiﬁcantly on an interannual timescale. T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error 2630 those with temporal baselines of 5 (10, 15, etc.) days. Being able to remove or account for the off-nadir orthorectiﬁcation error is highly desirable to unlock the full potential of dense Sentinel-2 temporal coverage at high latitudes. lateral off-nadir offsets in orthorectiﬁed satellite imagery re- sulting from vertical differences between the digital elevation model (DEM) surface used to orthorectify the imagery and the true surface at the time of acquisition. Over solid bedrock, these offsets occur due to DEM errors, but the issue is exac- erbated in glacial environments, where signiﬁcant (tens of metres or more) real elevation change may occur between the DEM and image acquisition times due to changes in ice surface elevation (hereafter 1h) resulting from, mainly, sus- tained ﬂow acceleration, increased surface melt, and, subse- quently, rapid ice thinning (e.g. King et al., 2020). When tracking displacement between two optical scenes from the same orbital path (in Sentinel mission terminology, the “rel- ative orbit”), orthorectiﬁcation errors will be the same across the two images and will be eliminated in the ﬁnal displace- ment map. However, in scene pairs from different orbits, a systematic error will be present as the vector sum of the two orthorectiﬁcation errors. Sentinel-2 is particularly vul- nerable to orthorectiﬁcation error, suffering from an order- of-magnitude greater terrain bias than Landsat 8 (Altena and Kääb, 2017). This is in part due to the wide viewing angle of Sentinel-2 compared to Landsat 8: with Sentinel-2’s swath width of 290 km, a vertical DEM error of 1h can result in a worst-case offset of ∼1h/5.4 at the maximum off-nadir dis- tances, whilst for Landsat 8’s swath width of 185 km this is only ∼1h/7.8 (Kääb et al., 2016). However, in the L1C and L2A data provided by ESA, the large errors are also related to the DEM chosen to orthorectify Sentinel-2 imagery. Un- til 23 August 2021 (30 March 2021 for Europe and Africa), the commercial PlanetDEM 90 m global elevation model (https://planetobserver.com/global-elevation-data/, last ac- cess: 29 February 2022) was used to orthorectify Sentinel- 2 data. Little public information exists as to which data sources were used to construct the PlanetDEM outside of the Shuttle Radar Topography Mission (SRTM) acquisition zone. However, Kääb et al. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error subsetting Sentinel-2 tiles for each glacier AOI, reproject- ing the raster to a common WGS 84/National Snow and Ice Data Centre (NSIDC) Sea Ice Polar Stereographic North (EPSG:3413) projected coordinate system, mosaicking the adjacent overlapping tiles, and ﬁnally clipping the mosaicked raster to the glacier AOI. We only considered scenes un- til 23 August 2021, when the L1C orthorectiﬁcation pro- cess switched to a new geolocation procedure and underlying DEM (Sect. 4.3). dense velocity ﬁelds taking advantage of the entire Sentinel- 2 record, it would be desirable to develop a method that, like Altena and Kääb (2017), remains simple and computation- ally efﬁcient and does not require any prior knowledge of 1h but is also able to produce a geographically complete record of velocity that is not spatially limited by the relationship be- tween satellite geometry and ﬂow direction. Here, we take advantage of 5 years of Sentinel-2 imagery to generate empirical corrections for systematic orthorectiﬁ- cation error in ice surface velocity ﬁelds at four key marine- terminating outlet glaciers around the Greenland Ice Sheet. We describe the process by which we produce dense and con- tinuous velocity datasets from 2017–2021, before validating our results by comparing them to publicly available velocity datasets at four key outlet glaciers. Velocity ﬁelds at 100 m resolution were produced us- ing feature tracking methods, performed using a direc- tional weighted ﬁltering (DWF) algorithm based on the Sur- face Extraction from TIN-based Search-space Minimization (SETSM) approach (Noh and Howat, 2019). The method was originally developed for precisely estimating the sur- face displacement map (SDM) by compensating for relative sensor model biases (minimising co-registration errors) and removing orthorectiﬁcation errors caused by height changes through true DEMs. For the purpose of this research, we modiﬁed the algorithm to use orthorectiﬁed images directly, and we applied the relative sensor model bias compensation module as co-registrations. For co-registration, we deﬁne the off-ice region using the GrIMP ice mask (Howat et al., 2014). The SDM processing is fully automated except in using an a priori, or seed, velocity ﬁeld to specify maximum displace- ments for determining the initial resolution in the coarse-to- ﬁne processing scheme (Noh and Howat, 2019). Here, we used InSAR-derived velocity ﬁelds between 2016 and 2017 (Joughin, 2021b) as the seed. 2.1.2 Estimating orbit-pair offsets Velocity ﬁelds are grouped by the relative orbits of their re- spective source image pairs. Pairs of images acquired from the same relative orbit are hereafter referred to as repeat-track pairs, and those from different relative orbits as cross-track pairs. For any given outlet glacier, certain combinations of orbit pairs may have anywhere from a few to > 100 velocity ﬁelds. A reference ﬂow ﬁeld is constructed using the median U and V velocity values (in x and y EPSG:3413 Polar Stereo- graphic North grid directions) from all (2017–2021) repeat- track velocity ﬁelds. Before processing, uncorrected velocity ﬁelds are ﬁltered using a 3 × 3 median ﬁlter to reduce noise. For individual velocity ﬁelds, the expected displacement is calculated from these reference ﬂow ﬁelds and the temporal baseline of the scene pair. The offset between the uncorrected displacement and the expected displacement is then calcu- lated. Empirical orbit-pair offset ﬁelds are generated as the median offset for each orbit pair. Where these offset ﬁelds are constructed from fewer than ﬁve velocity ﬁelds, their quality is notably degraded. Hence, where a particular orbital pair has fewer the ﬁve observations, an empirical offset ﬁeld is not constructed, and the velocity ﬁelds are not processed fur- ther. 2 Methods We produce and present velocity data for four major marine- terminating glaciers: Sermeq Kujalleq (Store Glacier), Ser- meq Kujalleq (Jakobshavn Isbræ), Helheim Glacier, and Kangerlussuaq Glacier (Fig. 1). As two of these glaciers share a Greenlandic name, we hereafter refer to them by their alternative names used in scientiﬁc literature (Store Glacier and Jakobshavn Isbræ). As orthorectiﬁcation error is a function of satellite geome- try, it should result in a consistent offset (in units of absolute displacement rather than velocity) across all velocity ﬁelds generated from the same relative orbit pairs. We assume that other errors in the velocity ﬁeld (e.g. image matching error, coregistration error) are (a) random and (b) do not correlate with speciﬁc relative orbit pairings. Hence, by drawing from a large base dataset, we can infer the average orthorectiﬁca- tion error over the study period for speciﬁc relative orbit pairs by measuring the average offset between (i) the ice displace- ment measured from Sentinel-2 scenes and (ii) the expected displacement from a reference velocity ﬁeld. We refer to this difference as the orbit-pair offset ﬁeld and subtract it from measured ﬁelds to generate ﬁnal corrected ﬁelds (Fig. 2). T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error g g g y Finally, recent work has begun to develop frameworks that harmonise orbit and elevation offsets in Sentinel-2 glacier velocity datasets that do not require any prior knowledge of 1h (Altena and Kääb, 2017; Altena et al., 2019). How- ever, the complexity of these methods necessitates simpliﬁed pipelines for operational use and bulk processing (Altena and Kääb, 2017). The simpliﬁed method presented by Altena and Kääb (2017) takes advantage of the fact that, if glacier ﬂow is known a priori, it is possible to map the offset onto this ﬂow direction as the offset vector always occurs perpendic- ular to the ﬂight path (or, for dual orbits, along the epipolar line between the two satellite locations). This is an elegant solution that can be implemented in normal image matching pipelines without requiring elevation data, but it comes with two primary limitations. The ﬁrst is the assumption that ﬂow direction is stable over time, which is justiﬁed on sub-decadal timescales for ice streams and glaciers that are not undergo- ing signiﬁcant changes in their geometries, such as occurs during surges. However, the second limitation of this method is that comprehensive coverage is restricted by two criteria: (i) when the ﬂow direction bearing is in the same direction as the epipolar line, the displacement will be mapped to in- ﬁnity, and (ii) when displacement is within the measurement error, the same effect can occur. As a result, the authors ﬁlter velocities where the ﬂow direction is within 20◦of the epipo- lar line and where displacement is > 2.5 times the matching accuracy. Hence, the ﬁnal corrected velocity ﬁeld is discon- tinuous, depending on the relationship between satellite ge- ometry and surface topography. To produce continuous and https://doi.org/10.5194/tc-16-2629-2022 The Cryosphere, 16, 2629–2642, 2022 2631 The Cryosphere, 16, 2629–2642, 2022 2.1.1 Velocity ﬁeld production Data are produced at four marine-terminating glaciers, matching the spatial extent of the areas of interest (AOIs) in the MEaSUREs Selected Glacier Site Velocity Maps from Optical Images collection (Howat, 2020). We downloaded all bottom-of-atmosphere corrected Level-2A (L2A) Sentinel- 2 data with a cloud cover < 50% from the Amazon Web Services (AWS) Registry of Open Data using the sat-search STAC API (https://github.com/sat-utils/sat-search, last ac- cess: 29 February 2022). Sentinel-2 data are staged using a pre-deﬁned tiling scheme based on the Military Grid Refer- ence System (MGRS). The pre-processing pipeline includes https://doi.org/10.5194/tc-16-2629-2022 The Cryosphere, 16, 2629–2642, 2022 The Cryosphere, 16, 2629–2642, 2022 2.2.2 Gaussian process regression The output from our velocity correction process produces a dense time series of varying error estimates. Hence, we use Gaussian process (GP) regression (Rasmussen and Williams, 2006; Hugonnet et al., 2021) to estimate a continuous uncertainty-bounded time-series velocity from our sampled time-series observations. Two particular properties of GP re- gression make it useful for the current application: (i) the Bayesian nature of the method accommodates the incomplete velocity record, producing a smooth, nonlinear, interpolated output, and (ii) the probabilistic model can incorporate un- certainty estimates and provides an empirical conﬁdence in- terval to predictions. 2.1.4 Error assessment A ﬁrst-order estimate of error is taken as the root mean square error (RMSE) of the absolute velocity of the bedrock area (as deﬁned by the GrIMP bedrock mask). RMSEs tended to be low, with the median RMSE consistently beneath < 0.5 m d−1 for the study glaciers discussed in this paper (Fig. S1 in the Supplement). Additionally, the mean and stan- dard deviation of the U and V velocity ﬁelds of the bedrock area are also recorded, in order to assess systematic error within individual ﬂow ﬁelds due to poor co-registration for example. Where the mean of the U or V velocity is greater than 1 standard deviation away from zero, the ﬁeld is consid- ered to have a systematic error and is not included for pre- sentation in this study (Sect. 2.2). T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error Figure 2. Workﬂow for deriving corrected and ﬁltered glacier ve- locity ﬁelds from Sentinel-2 Level-2A data. the ice sheet boundaries. Due to changing ice boundaries at marine-terminating locations, areas within the GrIMP ocean mask are not ﬁltered or removed, but ice velocities beyond the extent of the GrIMP ice mask should not be considered reliable. To remove erroneous velocity measurements, areas within the GrIMP ice mask are ﬁltered where ﬂow directions are > 20◦offset from the reference ﬂow ﬁeld. If, after ﬁltering, no data remain (< 1% of the ice area has valid velocity mea- surements), the ﬁeld is discarded and no output data are gen- erated. 2.2.1 Sampling To present time series of glacier surface velocity, we sample four sites of increasing distance from the calving front at each of our sample glaciers (Fig. 1; Table S1 in the Supplement). We sample across a 1 km ×1 km area, calculating the median velocity across this sample region. We ﬁlter out data points where < 70% of the sample region contains data, or where the RMSE error estimate is > 5 m d−1. We further ﬁlter ﬁelds where the temporal baseline is only 2 d, where errors were signiﬁcantly greater than any other baselines (Fig. S3). Figure 2. Workﬂow for deriving corrected and ﬁltered glacier ve- locity ﬁelds from Sentinel-2 Level-2A data. between ∼160 and ∼400 m. Over the 4-year study period, this results in a potential time-dependent error in 1h of be- tween 0.3 % and 2.2 % at our study glaciers. Applying these uncertainties to typical maximum offsets (directly correlated with 1h) of between 40 and 60 m shows that, even using worst-case assumptions, offset vector errors range between ±0.2 and ±1.3 m, values which are subsumed by other error terms (e.g. miscorrelation and coregistration). 2.1.3 Velocity correction and ﬁltering Once orbit-pair offsets have been constructed, uncorrected velocity ﬁelds are converted to absolute displacement, cor- rected using the appropriate orbit-pair displacement offset ﬁeld, and converted back to velocity. Displacements are cor- rected only over ice as deﬁned in the GrIMP mask, as our correction scheme is designed to correct for surface eleva- tion change, which we assume is largely negligible outside of T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error 2632 gure 1. Reference velocity ﬁelds (median velocity of repeat-orbit pairs; see Sect. 2.1.2) for the four marine-terminating glaciers presented this paper, ordered anti-clockwise from north: (a) Store Glacier, (b) Jakobshavn Isbræ, (c) Helheim Glacier, and (d) Kangerlussuaq acier. The 1 km ×1 km sample sites a–d are marked for each glacier – see Table S2 for precise coordinates. Backgrounds are GrIMP DEM lshades (Howat et al., 2017); coordinates in NSIDC Sea Ice Polar Stereographic North (EPSG:3413). Inset: location in Greenland of all tlet glacier AOIs (red). Figure 1. Reference velocity ﬁelds (median velocity of repeat-orbit pairs; see Sect. 2.1.2) for the four marine-terminating glaciers presented in this paper, ordered anti-clockwise from north: (a) Store Glacier, (b) Jakobshavn Isbræ, (c) Helheim Glacier, and (d) Kangerlussuaq Glacier. The 1 km ×1 km sample sites a–d are marked for each glacier – see Table S2 for precise coordinates. Backgrounds are GrIMP DEM hillshades (Howat et al., 2017); coordinates in NSIDC Sea Ice Polar Stereographic North (EPSG:3413). Inset: location in Greenland of all outlet glacier AOIs (red). We note that over the course of the study period ongoing glacier surface elevation change will continue to change 1h, and hence the orthorectiﬁcation error will not be constant. However, our method of estimating orthorectiﬁcation error across the entire 2017–2021 study period implicitly assumes a constant 1h. We could improve this assumption by assess- ing offsets on shorter timescales – such as annually – but shorter timescales (smaller sample sizes) result in a notable reduction in the number of cross-track pairs available for cor- rection (i.e. satisfying our threshold of ﬁve available velocity ﬁelds) and a lower quality of offset ﬁelds even for cross-track pairs where sufﬁcient data are available. However, given the large initial 1h values, surface elevation change over the study period likely has a negligible impact on the orthorec- tiﬁcation error. Using annual surface elevation change rates between 2003–2019 from Smith et al. (2020) as approxima- tions, maximum surface elevation change rates at our study sites range between −0.3 (Store Glacier) and −3.6 m a−1 (Kangerlussuaq Glacier). Maximum estimated \|1h\| values (Sect. 3.1), which roughly correlate with these values, range https://doi.org/10.5194/tc-16-2629-2022 The Cryosphere, 16, 2629–2642, 2022 T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error 2633 https://doi.org/10.5194/tc-16-2629-2022 https://doi.org/10.5194/tc-16-2629-2022 The Cryosphere, 16, 2629–2642, 2022 2634 T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error Figure 3. Median offset between measured displacement and expected displacement (from reference velocity ﬁeld) for different orbital pairs at Helheim Glacier. Magnitude is shown in colour; vectors are shown as white arrows. The orbital geometry of the constituent relative orbits is visualised in Fig. S2. Figure 3. Median offset between measured displacement and expected displacement (from reference velocity ﬁeld) for different orbital pairs at Helheim Glacier. Magnitude is shown in colour; vectors are shown as white arrows. The orbital geometry of the constituent relative orbits is visualised in Fig. S2. Figure 4. Comparison between DEM vertical differences and velocity offset for Helheim Glacier. (a) DEM difference between the GrIMP DEM and the Viewﬁnder DEM. (b) Absolute vertical difference between the Viewﬁnder DEM and GrIMP DEM. (c) Median offset between the measured displacement and expected displacement for velocity ﬁelds taken from relative orbits 053 and 139 (identical to Fig. 3c). (d) Hexbin density plot comparing the absolute vertical differences between the Viewﬁnd DEM and GrIMP DEM with the empirically determined orbital displacement. Figure 4. Comparison between DEM vertical differences and velocity offset for Helheim Glacier. (a) DEM difference between the GrIMP DEM and the Viewﬁnder DEM. (b) Absolute vertical difference between the Viewﬁnder DEM and GrIMP DEM. (c) Median offset between the measured displacement and expected displacement for velocity ﬁelds taken from relative orbits 053 and 139 (identical to Fig. 3c). (d) Hexbin density plot comparing the absolute vertical differences between the Viewﬁnd DEM and GrIMP DEM with the empirically determined orbital displacement. https://doi.org/10.5194/tc-16-2629-2022 The Cryosphere, 16, 2629–2642, 2022 T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error 2635 Figure 5. Velocity difference maps at Jakobshavn over a period of summer slowdown in 2019 (see Figs. 6 and 7). (a) Velocity ﬁeld pre- slowdown, from uncorrected Sentinel-2 feature tracking. (b) Velocity ﬁeld post-slowdown, from uncorrected Sentinel-2 feature tracking. (c) Difference between pre- and post-slowdown from uncorrected Sentinel-2 feature tracking. (d–f) Same as (a–c), but for corrected Sentinel- 2 feature tracking. (g–i) Same as (a–c), but for contemporaneous Sentinel-1 speckle tracking. Note log scale used on difference maps. Backgrounds are GrIMP DEM hillshades (Howat et al., 2017). Figure 5. https://doi.org/10.5194/tc-16-2629-2022 Velocity difference maps at Jakobshavn over a period of summer slowdown in 2019 (see Figs. 6 and 7). (a) Velocity ﬁeld pre- slowdown, from uncorrected Sentinel-2 feature tracking. (b) Velocity ﬁeld post-slowdown, from uncorrected Sentinel-2 feature tracking. (c) Difference between pre- and post-slowdown from uncorrected Sentinel-2 feature tracking. (d–f) Same as (a–c), but for corrected Sentinel- 2 feature tracking. (g–i) Same as (a–c), but for contemporaneous Sentinel-1 speckle tracking. Note log scale used on difference maps. Backgrounds are GrIMP DEM hillshades (Howat et al., 2017). We performed GP regression using the GaussianProcess- Regressor() implementation in the Python scikit-learn li- brary. We model ice velocity as the sum of two ker- nels (covariance functions) representing seasonal and short- term variability. Our implementation follows Rasmussen and Williams (2006), ignoring long-term variability over the 5- year period we assess. Seasonal variability is incorporated as the product of an exponential sine squared kernel (ExpSi- neSquared()) and a radial basis function kernel (RBF()). The exponential sine squared kernel has a ﬁxed periodicity of 1 year and the length scale as a free parameter. The radial basis function kernel has a free length scale. We implement short- medium term variability as a rational quadratic kernel (Ratio- nalQuadratic()), with free length scale and alpha (controlling the diffusivity of the length scale) parameters. Finally, we incorporate velocity error estimates (Sect. 2.1.3) into mod- elling directly via the alpha parameter of the GaussianPro- cessRegressor(). DEM from GeoEye and WorldView Imagery (Version 1) (Howat et al., 2014, 2017; hereafter the “GrIMP DEM”), which was produced from imagery between 2009–2015. We compare our generated velocity ﬁelds to two other public datasets. To compare with medium-resolution SAR velocity ﬁelds, we make use of MEaSUREs Greenland 6 and 12 d Ice Sheet Velocity Mosaics from SAR (Version 1) ve- locity ﬁelds from speckle-tracked Sentinel-1 data (Joughin et al., 2018; Joughin, 2021a). These were downloaded from the NSIDC data portal. To compare with optical velocity ﬁelds derived from Landsat 8 imagery, we make use of the ITS_LIVE dataset (Gardner et al., 2018, 2021). These were downloaded using the ITS_LIVE API, and, to match our Sentinel-2 data thresholds, ﬁltered to velocity ﬁelds where (i) the maximum interval between data pairs was 30 d and (ii) at least 1 % of the data contained valid pixels. For both Sentinel-1 and Landsat 8 ﬁelds, we extracted time-series data in the same way as for Sentinel-2 data: i.e. 2.3 Supplementary data As orthorectiﬁcation errors scale with 1h, we compare gen- erated offset maps to DEM difference as a proxy for 1h. The ﬁrst DEM is the Viewﬁnder Panoramas 3′′ DEM for Green- land (Version 1) (de Ferranti, 2014; hereafter the “Viewﬁnder DEM”). Kääb et al. (2016) inferred that the Viewﬁnder DEMs likely shared source data with the PlanetDEM for a test site in northern Norway. The second DEM is the GrIMP https://doi.org/10.5194/tc-16-2629-2022 ﬁltering to data that cover at least 70 % of the 1 km ×1 km sample region and < 5 m d−1 error. The Cryosphere, 16, 2629–2642, 2022 T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error The lowest level of agreement between the two datasets occurs at Helheim Glacier point b, where only 52.3 % of Sentinel-1 velocity values lie within error of the Sentinel-2 GP ﬁt, and the Sentinel-1 record is, on average, 0.47 m d−1 slower than that of the GP ﬁt. If the measured offsets are due to orthorectiﬁcation error, they should directly correlate to the 1h between the times of DEM and image acquisition. We can thus validate that our offsets are meaningful by comparing them to the DEM difference between the Viewﬁnder DEM, which we use as a proxy for the PlanetDEM (Sect. 2.3), and version 2 of the GrIMP DEM, which acts as our reference elevation, or at least a closer approximation to the surface elevation at the time of image acquisition (Fig. 4). The spatial pattern of \|1h\| (Fig. 4b) matches the magnitude of the orbital off- set (Fig. 4c), with a strong, positive correlation (R2 = 0.67, p < 0.01) between the two variables (Fig. 4d). The direction of the offset (white vectors in Fig. 4c) is also predicted by the direction of 1h (Fig. 4a): where 1h values are negative, off- set error occurs in the ESE direction (towards relative orbit 053), whereas positive 1h values occur where offset errors are in the WNW direction (towards relative orbit 139). This high level of agreement also occurs in comparison with the ITS_LIVE dataset derived from optical Landsat 8 imagery (orange triangles in Fig. 7). Across all sites, the median difference between the ITS_LIVE record and the GP ﬁt is 0.12 m d−1, 68 % (95 %) of values lie within 0.29 (0.63) m d−1 of one another, and 87.0 % of values lie within error of each other (Table S3). The increased density of the Sentinel-2 record relative to the Landsat 8 record allows for a higher temporal precision in identifying rapid drainage events. For instance, the summer slowdown at sites a and b at Store Glacier are not as not well captured in the Land- sat 8 or Sentinel-1 records, where a sparse record means that the timing of the slowdown often appears to take more than a month, whilst the dense Sentinel-2 dataset and the GP ﬁt show the slowdown to occur on a timescale of ∼2 weeks (Fig. S5). T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error 2636 land. Comparing offset ﬁelds constructed for various orbit pairs at Helheim Glacier (Fig. 3) shows that the behaviour of empirical offset ﬁelds are consistent with theory. Offset ﬁelds for repeat orbit pairs (Fig. 3a, e, i) have negligible off- set, whilst the offset is largest for the cross-track pairs from relative orbits 053 and 139 (Fig. 3c, g), which have the great- est distance between their respective orbital paths (Fig. S2). Empirical offset vectors are, to within reasonable error, uni- formly parallel and occur along the epipolar line of the satel- lite pair viewing geometry (Figs. 3, cf. S2), consistent with the hypothesis that these systematic errors occur due to or- thorectiﬁcation error in the off-nadir direction. short-baseline pairs are increasingly offset from the reference dataset value, even where orbit pairs are identical (Fig. S4). This is because orthorectiﬁcation error is an absolute dis- placement offset and as such becomes a higher relative er- ror component of short-baseline velocities. In all cases, the corrected velocity data converge near the reference Sentinel- 1 time series. The correction is greatest for observations at Jakobshavn Isbræ and Kangerlussuaq Glacier, where 1h val- ues are the largest. short-baseline pairs are increasingly offset from the reference dataset value, even where orbit pairs are identical (Fig. S4). This is because orthorectiﬁcation error is an absolute dis- placement offset and as such becomes a higher relative er- ror component of short-baseline velocities. In all cases, the corrected velocity data converge near the reference Sentinel- 1 time series. The correction is greatest for observations at Jakobshavn Isbræ and Kangerlussuaq Glacier, where 1h val- ues are the largest. The high density of data points reveals a noisy dataset compared to the SAR-derived data. The use of GP regres- sion highlights an effective way of interpolating a continuous time series from this dataset (blue line in Fig. 7), whilst ac- counting for sample-speciﬁc error values and time-variable data densities. Across all sites, the median difference be- tween the Sentinel-1 record and the GP ﬁt is 0.08 m d−1, 68 % (95 %) of values lie within 0.41 (0.73) m d−1 of one another, and 90.5 % of Sentinel-1 values lie within the er- ror of the Sentinel-2 GP ﬁt (Table S2). These differences, on the order of decimetres, align with our error estimates from off-ice displacement of between 0.3 and 0.5 m d−1 on average (Fig. S1). T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error However, the Landsat 8 record displays a higher precision at lower velocity values (sub-4 m d−1, e.g. site d at Store Glacier), likely because any errors in the empirically derived displacement ﬁeld have greater relative inﬂuence at low ﬂow velocities. The efﬁcacy of the orbital correction ﬁelds is visualised for an example case at Jakobshavn Isbræ (Fig. 5), which shows the relative ability of corrected and uncorrected glacier ve- locity ﬁelds to properly capture the magnitude and extent of a summer slowdown occurring in late July 2019 (see also Figs. 6 and 7 for time series of this event). In the uncor- rected velocity ﬁelds (Fig. 5a and b), orthorectiﬁcation er- ror in the cross-track velocity ﬁelds (relative orbits 025/111 and 068/025 respectively) introduces an apparent difference in the ﬁnal velocity ﬁeld in excess of 10 m d−1 at the calving front and 2 m d−1 even tens of kilometres inland, a rate of change that is unphysical. After correction (Fig. 5d and e), this change is reduced to ∼2–3 m d−1 at the front and negli- gible amounts inland (Fig. 5f), an observation that is in line with contemporaneous Sentinel-1 observations (Fig. 5g–i). 3.2 Ice velocity time series For the sampled 1 km ×1 km sectors of our four study glaciers, we compare our corrected Sentinel-2 data against other data sources. Comparing our corrected velocity data to the uncorrected dataset shows the improvement that our empirical correction has (Fig. 6). Uncorrected data show a characteristic error distribution, with points offset from the Sentinel-1 -derived velocities based on their constituent or- bit pairs and their temporal baseline. Prior to correction, 3.1 Orbital offset generation Empirically determined systematic offsets can reach values on the order of tens of metres at outlet glaciers around Green- https://doi.org/10.5194/tc-16-2629-2022 The Cryosphere, 16, 2629–2642, 2022 The Cryosphere, 16, 2629–2642, 2022 Figure 6. Figure 6. (Figs. 5, 6). As orthorectiﬁcation error is linearly propor- tional to 1h between the orthorectiﬁcation DEM and the true glacier surface (Fig. 4), errors are greatest at glaciers where ice surface elevation has changed the most. This is exempliﬁed by Store Glacier, which is notable for its long- term stability induced by a sill at the glacier terminus (e.g. Morlighem et al., 2016) and has relatively low orthorectiﬁ- cation errors prior to correction (Fig. 6). here differ from Sentinel-1 observations by, on average, only 0.08 m d−1, and 90.5 % of Sentinel-1 values lie within the GP ﬁt error (Rable S2). Unlike monthly averaging, the use of GP regression retains the ability to capture short-term variation at approximately weekly timescales and provides a time-varying conﬁdence interval based on training data un- certainty and coverage. The data display high agreement with comparative datasets sourced from Sentinel-1 and Landsat 8 (Fig. 7). Al- though Sentinel-1 has been presented as a “true” reference velocity in this study, it is of note which two will vary when only two of the three records presented agree with each other: contrast, for instance, Helheim site b (Sentinel-2 and Landsat 8 agree), Jakobshavn site a (Sentinel-2 and Sentinel-1 agree), and Kangerlussuaq site a (Sentinel-1 and Landsat 8 agree). All three methods are orthorectiﬁed using different DEMs whose ages, accuracies, and resultant 1h values are vari- able. As such, it is likely that systematic biases are present in all three datasets and are spatially variable on a kilo- metre scale. However, the characteristics of the cross-track Sentinel-2 dataset will make it advantageous over Landsat 8- derived datasets in scenarios where a high density of obser- vations is required, as the increased observation frequency will allow for a greater chance of successful image pairs over critical dynamic periods, such as the early melt season ac- celeration and late summer slowdown. In contrast, individ- The empirical corrections described in this paper demon- strably reduce the inﬂuence of systematic orthorectiﬁcation error on Sentinel-2-derived glacier velocity ﬁelds, reducing the variance from the order of metres to decimetres (Figs. 6, 7). This allows for the incorporation of cross-track pairs into the glacier velocity time series, increasing the tempo- ral density of observations, in particular for pairs with short (< 10 d) baselines, by as much as a factor of 7 (Fig. S4b). 4.1 Data quality In the absence of empirical correction, orthorectiﬁcation errors can result in errors in excess of 10 m d−1 in short-baseline velocity ﬁelds at Greenlandic outlet glaciers https://doi.org/10.5194/tc-16-2629-2022 The Cryosphere, 16, 2629–2642, 2022 T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error 2637 T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error Figure 6. T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error 2638 Figure 6. Corrected (blue) and uncorrected (red) velocity time series for four sample glaciers in 2019. Top to bottom are points (a–d) in Fig. 1. The black line and diamonds mark the MEaSUREs Sentinel-1 velocity dataset for comparison. Figure 6. Corrected (blue) and uncorrected (red) velocity time series for four sample glaciers in 2019. Top to bottom are points (a–d) in Fig. 1. The black line and diamonds mark the MEaSUREs Sentinel-1 velocity dataset for comparison. ual Landsat 8 velocity ﬁelds appear to be more precise and may be preferable when the accuracy of individual velocity ﬁelds are necessary (e.g. comparing early and late season ve- locities). Sentinel-2 also provides a temporal advantage over Sentinel-1 datasets, which are limited to a ﬁxed 6 d repeat cy- cle, and may also provide a valuable alternative for applying to small and steep glaciers, such as in high mountain regions, where the use of synthetic aperture radar for velocity extrac- tion can be challenging (e.g. Paul et al., 2022). DEM opens new avenues for deriving high-latitude Sentinel- 2 orthorectiﬁcation error from analytical methods (Ressl and Pfeifer, 2018). To do so would require not only the Viewﬁnder DEM but also a “true” glacier surface elevation DEM contemporaneous to image acquisition, which is difﬁ- cult to acquire in rapidly changing sectors of the cryosphere. Here, we highlight the utility of time-evolving DEMs and the potential of ArcticDEM strips (Porter et al., 2018) and monthly/annual composites thereof to address this challenge in the future. https://doi.org/10.5194/tc-16-2629-2022 Figure 6. The temporal density of observations means that short-term variations in speed, such as the slowdown at the terminus of Store Glacier, can be well resolved (Fig. S5). The noise of the dataset highlights the utility of effective ﬁltering and time series modelling, such as GP regression (Fig. 7), in extracting a continuous velocity estimate – following other studies that have made use of, for example, Kalman ﬁlter- ing (King et al., 2018) to similar effect – and it provides the advantage of a greater accuracy through the synthesis of multiple estimates. GP ﬁts over the four glaciers assessed https://doi.org/10.5194/tc-16-2629-2022 The Cryosphere, 16, 2629–2642, 2022 Figure 7. for example, Landsat Collection 2), so this discontinuity be- tween pre- and post-2021 data will remain into the future. As such, the method outlined here remains applicable for the correction of Sentinel-2-derived glacier velocity across the wider cryosphere for Sentinel-2 L1C and L2A data be- tween 2015–2021. The more recent dataset will reduce the value of 1h and as such the orthorectiﬁcation error in the new datasets – although as the DEM used for orthorectiﬁca- tion consists of pre-2015 data, some orthorectiﬁcation error will remain. As such, offset ﬁelds such as those calculated in this study will need to be calculated separately for the pre- and post-2021 data. Additionally, the lower 1h value will likely invalidate the assumption we make in this study that further elevation change will have a negligible impact on our assumption of stable geometry. However, GLO-90 is avail- able for public download (European Space Agency, 2021), allowing for correction via analytical methods. In combina- tion with the opportunities we highlight in Sect. 4.2, this provides a framework for the production of a continuous Sentinel-2 velocity dataset from 2016–present with cross- track velocities corrected analytically according to their un- derlying DEM. for example, Landsat Collection 2), so this discontinuity be- tween pre- and post-2021 data will remain into the future. As such, the method outlined here remains applicable for the correction of Sentinel-2-derived glacier velocity across the wider cryosphere for Sentinel-2 L1C and L2A data be- tween 2015–2021. The more recent dataset will reduce the value of 1h and as such the orthorectiﬁcation error in the new datasets – although as the DEM used for orthorectiﬁca- tion consists of pre-2015 data, some orthorectiﬁcation error will remain. As such, offset ﬁelds such as those calculated in this study will need to be calculated separately for the pre- and post-2021 data. Additionally, the lower 1h value will likely invalidate the assumption we make in this study that further elevation change will have a negligible impact on our assumption of stable geometry. However, GLO-90 is avail- able for public download (European Space Agency, 2021), allowing for correction via analytical methods. In combina- tion with the opportunities we highlight in Sect. 4.2, this provides a framework for the production of a continuous Sentinel-2 velocity dataset from 2016–present with cross- track velocities corrected analytically according to their un- derlying DEM. 4.3 Future datasets The differencing of orbital pair offsets from a reference ﬂow ﬁeld has been shown to be an effective method of recon- structing systematic orthorectiﬁcation errors when the un- derlying DEM is not available. Furthermore, we show that orthorectiﬁcation errors over Helheim Glacier are consistent with Sentinel-2 data being orthorectiﬁed using data sources present in the Viewﬁnder Panoramas 3′′ DEM (de Ferranti, 2014). This ﬁnding agrees with that of Kääb et al. (2016), who found similar agreement between a 1h estimated from the Viewﬁnder DEM and Sentinel-2 offsets for a test site in northern Norway. Further testing is required to establish if the Viewﬁnder DEM and PlanetDEM data sources are con- sistent across broader regions of Greenland and the high lat- itudes, but if they are then the freely available Viewﬁnder The empirical offsets derived in this study are only applica- ble up to 23 August 2021. On this date (or 30 March 2021 for coverage of Europe and Africa), L1C/L2A Sentinel-2 products switched to an improved geometric reﬁnement with two major changes: (i) co-registration of scenes to a Global Reference Image (GRI) and (ii) the use of a new DEM, the Copernicus DEM at 90 m resolution (“GLO-90”), for topo- graphic correction. This new DEM is based on 2011–2015 radar satellite data acquired during the TanDEM-X mission, meaning there will be a discontinuity in 1h between the pre- and post-August 2021 data. There is no currently announced reprocessing of the Sentinel-2 archive with the new geometric reﬁnements (see, The Cryosphere, 16, 2629–2642, 2022 https://doi.org/10.5194/tc-16-2629-2022 T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error Figure 7. for example, Landsat Collection 2), so this discontinuity be- tween pre- and post-2021 data will remain into the future. As such, the method outlined here remains applicable for the correction of Sentinel-2-derived glacier velocity across the wider cryosphere for Sentinel-2 L1C and L2A data be- tween 2015–2021. The more recent dataset will reduce the value of 1h and as such the orthorectiﬁcation error in the new datasets – although as the DEM used for orthorectiﬁca- tion consists of pre-2015 data, some orthorectiﬁcation error will remain. As such, offset ﬁelds such as those calculated in this study will need to be calculated separately for the pre- and post-2021 data. 4.3 Future datasets Additionally, the lower 1h value will likely invalidate the assumption we make in this study that further elevation change will have a negligible impact on our assumption of stable geometry. However, GLO-90 is avail- able for public download (European Space Agency, 2021), allowing for correction via analytical methods. In combina- tion with the opportunities we highlight in Sect. 4.2, this provides a framework for the production of a continuous Sentinel-2 velocity dataset from 2016–present with cross- track velocities corrected analytically according to their un- derlying DEM. 5 Conclusions By taking advantage of the complete Sentinel-2 reco tween 2017–2021, we demonstrate an empirical met correcting for systematic orthorectiﬁcation error in velocity ﬁelds derived from cross-track image pairs in scale datasets. The method is simple and computationa demanding, whilst allowing for spatially continuous velocity ﬁelds to be constructed without limitations duced by satellite and ﬂow geometry. This method i to produce a complete dataset of glacier velocity for fo marine-terminating glaciers across the Greenland Ice Comparison with alternative datasets highlights the k vantages of the high temporal frequency of Sentinel agery, but this density of data also necessitates the use tistical techniques to account for noise and uncertainty dataset. Furthermore, we use this method to identify a publicly available data source for the DEM data used thorectify Sentinel-2 scenes over Greenland between 2021, which will be useful for future studies concerne orthorectiﬁcation error in the region. The transition to geometric reﬁnement method for Sentinel-2 scenes b T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error 2639 T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error Figure 7. Figure 7. T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error T. R. Chudley et al.: Empirical correction of systematic orthorectiﬁcation error 2640 Figure 7. Corrected Sentinel-2 (blue dots), ITS_LIVE Landsat-8 (orange triangles), and MEaSUREs Sentinel-1 (black diamonds) velocity time series for four sample glaciers in 2019. Top to bottom are points (a–d) in Fig. 1. The blue line marks the output of the Gaussian process regression, with the blue shading marking the 2σ uncertainty bound. Figure 7. Corrected Sentinel-2 (blue dots), ITS_LIVE Landsat-8 (orange triangles), and MEaSUREs Sentinel-1 (black diamonds) velocity time series for four sample glaciers in 2019. Top to bottom are points (a–d) in Fig. 1. The blue line marks the output of the Gaussian process regression, with the blue shading marking the 2σ uncertainty bound. provides the opportunity for this to occur through analytical rather than empirical methods. Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/tc-16-2629-2022-supplement. 5 Conclusions By taking advantage of the complete Sentinel-2 record be- tween 2017–2021, we demonstrate an empirical method of correcting for systematic orthorectiﬁcation error in glacier velocity ﬁelds derived from cross-track image pairs in large- scale datasets. The method is simple and computationally un- demanding, whilst allowing for spatially continuous glacier velocity ﬁelds to be constructed without limitations intro- duced by satellite and ﬂow geometry. This method is used to produce a complete dataset of glacier velocity for four key marine-terminating glaciers across the Greenland Ice Sheet. Comparison with alternative datasets highlights the key ad- vantages of the high temporal frequency of Sentinel-2 im- agery, but this density of data also necessitates the use of sta- tistical techniques to account for noise and uncertainty in the dataset. Furthermore, we use this method to identify a likely publicly available data source for the DEM data used to or- thorectify Sentinel-2 scenes over Greenland between 2015– 2021, which will be useful for future studies concerned with orthorectiﬁcation error in the region. The transition to a new geometric reﬁnement method for Sentinel-2 scenes beyond August 2021 will require new correction offsets to be gen- erated, but the transition to a publicly available DEM (and the identiﬁcation of 2015–2021 data sources in this study) https://doi.org/10.5194/tc-16-2629-2022 The Cryosphere, 16, 2629–2642, 2022 References Altena, B. and Kääb, A.: Elevation Change and Improved Velocity Retrieval Using Orthorectiﬁed Optical Satel- lite Data from Different Orbits, Remote Sens., 9, 300, https://doi.org/10.3390/rs9030300, 2017. 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Y., van den Broeke, M. provides the opportunity for this to occur through analytical rather than empirical methods. Data availability. The velocity datasets produced for the glaciers in this study are available from Zenodo (https://doi.org/10.5281/zenodo.6676196, Chudley et al., 2022), and an expanded record covering Greenlandic marine- terminating glaciers will be made available as part of the MEaSUREs project through the NSIDC. The NSIDC also hosts access to the supplementary data used in this work: ITS_LIVE data (https://doi.org/10.5067/IMR9D3PEI28U; Gardner et al., 2022), MEaSUREs Sentinel-1 velocity data (https://doi.org/10.5067/6JKYGMOZQFYJ; Joughin, 2021a), GrIMP DEM (https://doi.org/10.5067/H0KUYVF53Q8M; Howat et al., 2017), and the GrIMP mask (https://doi.org/10.5067/B8X58MQBFUPA; Howat, 2017). Version 1 of the 3′′ Viewﬁnder Panoramas DEM for Greenland can be found at http://viewﬁnderpanoramas.org/GL-ReadMe.html (de Ferranti, 2014). Sentinel-2 scenes were accessed via the AWS Registry of Open Data (https://registry.opendata.aws/sentinel-2, last access: 29 February 2022). Velocity processing was performed using SETSM, available at https://github.com/setsmdeveloper/SETSM (Noh and Howat, 2019). Author contributions. TRC and IMH conceptualised and designed the study. TRC, BY, and MJN processed the data. TRC performed the analysis. TRC wrote the manuscript with contributions from all authors. Competing interests. The contact author has declared that neither they nor their co-authors have any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Acknowledgements. The velocity ﬁelds contain modiﬁed Coperni- cus Sentinel data (2017–2021), processed by ESA. 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https://openalex.org/W4393060728	https://journal.amikveteran.ac.id/index.php/kreatif/article/download/2101/1687	Indonesian	null	Pelatihan Pembuatan Origami Untuk Meningkatkan Kreativitas Anak Bekebutuhan Khusus Di Panti Asuhan Bhakti Luhur Banjarmasin	Kreatif	2,023	cc-by-sa	2,867	KREATIF: Jurnal Pengabdian Masyarakat Nusantara Vol. 3, No. 1 Maret 2023 e-ISSN: 2962-3839; p-ISSN: 2962-4436 , Hal 211-216 KREATIF: Jurnal Pengabdian Masyarakat Nusantara Vol. 3, No. 1 Maret 2023 e-ISSN: 2962-3839; p-ISSN: 2962-4436 , Hal 211-216 Pelatihan Pembuatan Origami Untuk Meningkatkan Kreativitas Anak Bekebutuhan Khusus Di Panti Asuhan Bhakti Luhur Banjarmasin Training Of Making The Origami To Improve The Creativity Of Children With Special Needs At The Bhakti Luhur Orphanage, Banjarmasin Getrudis Tutpai1, Ermeisi Er Unja2, Lucia Andi Chrismilasari3, Aulia Rachman4 1,2,3,4Sekolah Tinggi Ilmu Kesehatan Suaka Insan, Banjarmasin meisiunja10@gmail.com2 Article History: Received: 30 Januari 2023 Revised: 12 Februari 2023 Accepted: 22 Maret 2023 Abstract: Children with needs require special treatment to help develop their fine motor skills. Through this community service activity it is hoped that it can develop fine motor skills in children with special needs. This activity is carried out using paper media packaged in the art of paper folding or origami. This paper folding art training activity is given to children with special needs with the method of lectures, questions and answers and demonstrations in the form of paper folding training in various forms. The results of this community service show that there has been an increase in knowledge in groups of children with needs where after the activities the children know about the art of paper folding and can practice how to fold paper in various shapes of animals, flowers and stars. It is hoped that accompanying teachers at the Bhakti Luhur Orphanage can do paper folding or other types of art more often to be able to further develop the abilities of children with special needs. Because fun activities like this are more in demand by children. Keywords: ABK, Motor Ability, Origami. Abstrak Anak berkebutuhan membutuhkan perlakuan khusus dalam membantu mengembangkan kemampuan motoric halusnya. Melalui kegiatan pengabdian masyarakat ini diharapkan dapat mengembangkan keterampilan motorik halus pada anak-anak berkebutuhan khusus. Kegiatan ini dilaksanakan dengan menggunakan media kertas yang dikemas dalam seni melipat kertas atau origami. Kegiatan pelatihan seni melipat kertas inidiberikan kepada anak-anak berkebutuhan khusus dengan metode ceramah, tanya jawab dan demonstrasi dalam bentuk pelatihan melipat kertas dalam berbagai bentuk. Hasil pengabdian masyarakat ini menunjukkan bahwa terjadi i k t t h d k l k k k b k b t h di t l h dil k k Abstract: Children with needs require special treatment to help develop their fine motor skills. Through this community service activity it is hoped that it can develop fine motor skills in children with special needs. KREATIF: Jurnal Pengabdian Masyarakat Nusantara Vol. 3, No. 1 Maret 2023 e-ISSN: 2962-3839; p-ISSN: 2962-4436 , Hal 211-216 This activity is carried out using paper media packaged in the art of paper folding or origami. This paper folding art training activity is given to children with special needs with the method of lectures, questions and answers and demonstrations in the form of paper folding training in various forms. The results of this community service show that there has been an increase in knowledge in groups of children with needs where after the activities the children know about the art of paper folding and can practice how to fold paper in various shapes of animals, flowers and stars. It is hoped that accompanying teachers at the Bhakti Luhur Orphanage can do paper folding or other types of art more often to be able to further develop the abilities of children with special needs. Because fun activities like this are more in demand by children. Article History: Received: 30 Januari 2023 Revised: 12 Februari 2023 Accepted: 22 Maret 2023 Keywords: ABK, Motor Ability, Origami. Abstrak Anak berkebutuhan membutuhkan perlakuan khusus dalam membantu mengembangkan kemampuan motoric halusnya. Melalui kegiatan pengabdian masyarakat ini diharapkan dapat mengembangkan keterampilan motorik halus pada anak-anak berkebutuhan khusus. Kegiatan ini dilaksanakan dengan menggunakan media kertas yang dikemas dalam seni melipat kertas atau origami. Kegiatan pelatihan seni melipat kertas inidiberikan kepada anak-anak berkebutuhan khusus dengan metode ceramah, tanya jawab dan demonstrasi dalam bentuk pelatihan melipat kertas dalam berbagai bentuk. Hasil pengabdian masyarakat ini menunjukkan bahwa terjadi peningkatan pengetahuan pada kelompok anak-anak berkebutuhan dimana setelah dilakukan kegiatan anak-anak mengetahui mengenai seni melipat kertas dan dapat mempraktekan bagaimana cara melipat kertas dalam beberapa bentuk binatang, bunga maupun bintang. Bagi para guru pendamping di Panti Asuhan Bhakti Luhur diharapkan dapat lebih sering melakukan seni melipat kertas atau jenis seni lainnya untuk dapat lebih mengembangkan kemampuan anak-anak berkebutuhan khusus. Karena kegiatan yang menyenangkan seperti ini lebih diminati oleh anak- anak. https://journal.amikveteran.ac.id/index.php/kreatif 211 https://journal.amikveteran.ac.id/index.php/kreatif 211 PENDAHULUAN Melipat kertas adalah sesuatu hal yang sangat menyenangkan bagi anak-anak karena mereka dapat mebiat apa saja, dari melipat sesuatu yang sederhana seperti bentuk segi tiga, segi empat, sampai dengan bentuk yang agak sulit. Melipat tidak hanya untuk anak-anak, namun juga orang dewasa. Sebab dengan seni origami ini dapat dilakukan dengan bersama-sama sehingga akan meningkatkan interaksi dan komunikasi serta pendekatan antar guru dan anak. Ketertarikan anak terhadap seni origami, terletak pada keunikan dari origami tersebut yang merupakan karya seni yang menyenangkan, anak-anak sangat berminat pada pembelajaran seni origami yang sangat menarik itu. Hal ini dapat terlihat dari kecerian anak, ketika sehelai kertas yang dipegang dan kemudian dilipat beberapa kali dan pada detik yang berikutnya berubah menjadi karya seni tiga dimensi yang tidak terbayangkan anak sebelumnya. Kegiatan melipat kertas atau origami adalah sebuah cara yang dapat mengembangkan kemampuan motorik halus anak. Origami memiliki peran penting sebagai media komunikasi yang menyenangkan antara guru, anak, juga orangtua. Jika dilatih secara konsisten dan diaplikasikan dengan metode yang tepat, maka bisa meningkatkan daya konsentrasi anak. Keterampilan origami adalah suatu kegiatan yang sangat baik untuk merangsang kreativitas anak, serta membangun daya ingat anak, membangun daya imajinasi anak, dapat menumbuhkan rasa penasaran anak dan membangun sosial yang baik bersama orang-orang yang ada disampingnya. g y g g g y g p g y Di negara Jepang, kertas origami ini dipakai saat mengajar anak-anak di TK, termaksud anak yang tidak bisa diam di kelas. Begitu pula pada anak-anak berkebutuhan khusus, metode ini juga dapat dilakukan. Secara khusus kegiatan melipat bertujuan untuk melatih daya ingat, pengamatan, keterampilan tangan, mengembangkan daya fantasi, kreasi, ketelitian, kerapian,dan perasaan keindahan. Melalui kegiatan melipat kertas juga dapat mengembangkan keterampilan motorik halus anak, seperti melatih gerak otototot tangan sehingga anak memiliki kemampuan untuk memegang pensil, meremas kertas, ataupun membentuk benda dari adonan atau bahan lain. Melipat kertas (origami) merupakan salah satu pengembangan motorik halus yang membutuhkan keterampilan, ketelitian, dan bimbingan. Damayanti (2012) mengatakan manfaat melipat kertas (origami) yaitu sebagai berikut: anak berlajar tentang konsep, sebagai alat komunikasi, dapat meningkatkan keterampilan motorik halus anak, anak belajar mengenai ukuran dan bentuk, anak belajar mengikuti instruksi yang runtun, anak belajar berkreativitas, anak belajar membuat mainan sendiri, anak belajar berimajinasi, anak dilatih konsentrasi.Hal-hal ini yang dianggap dapat membantu anak-anak yang berkebutuhan khusus untuk dapat mengembangkan kemampuan motoriknya. Anak berkebutuhan khusus adalah anak yang mempunyai karakteristik khusus dibanding dengan anak normal pada umumnya. Kata Kunci: ABK, Kemampuan Motorik, Origami. Kata Kunci: ABK, Kemampuan Motorik, Origami. https://journal.amikveteran.ac.id/index.php/kreatif 211 212 KREATIF Vol. 3, No. 1 Maret 2023 KREATIF Vol. 3, No. 1 Maret 2023 https://journal.amikveteran.ac.id/index.php/kreatif PENDAHULUAN Terkait pengertian anak berkebutuhan khusus diungkapkan oleh Desiningrum, 2016 yang mengatakan secara umum, ABK memiliki karakteristik khusus yang berbeda dengan anak pada umumnya, namun tidak selalu menunjukkan ketidakmampuan mental. Milyartini (2012) juga mengemukakan anak berkebutuhan khusus adalah anak dengan karakteristik khusus yang berbeda dengan anak pada umumnya, baik berbeda karena memiliki keterbatasan/ketidakmampuan (fisik, mental dan sosial emosi), maupun memiliki kelebihan atau keistimewaan (gifted and tallented). Anak berkebutuhan khusus biasanya tidak mampu beradaptasi dengan lingkungan sehingga mereka memerlukan layanan, perawatan, pengawasan dan dukungan serta pembelajaran yang https://journal.amikveteran.ac.id/index.php/kreatif 213 KREATIF: Jurnal Pengabdian Masyarakat Nusantara Vol. 3, No. 1 Maret 2023 e-ISSN: 2962-3839; p-ISSN: 2962-4436 , Hal 211-216 213 sesuai secara terus menerus. Namun, keterbatasan/ketidakmampuan yang dimiliki seorang ABK tidak mengartikan bahwa mereka tidak berhak mendapatkan pendidikan. sesuai secara terus menerus. Namun, keterbatasan/ketidakmampuan yang dimiliki seorang ABK tidak mengartikan bahwa mereka tidak berhak mendapatkan pendidikan. Banyak hal yang perlu diperhatikan bagi anak-anak yang berkebutuhan khusus diantaranya adalah bagaimana mengembangkan kemampuan motorik halus anak dengan sebuah pembelajaran yang menarik dan menyenangkan. Salah satunya adalah dengan menggunakan seni origami atau sering disebut juga dengan seni melipat kertas. Hirai ( 2010 :8) seni melipat kertas atau origami adalah suatu seni yang berasal dari cina yang diperkenalkan oleh seseorang yang bernama Ts’ai Lun yang awal mulanya terbuat dari kertas yang berasal dari hancuran tumbuhan dan kain yang sudah tidak terpakai. Namun Salsabila (2011:2), mengatakan bahwa origami adalah seni melipat kertas yang berasal dari negeri jepang dan dikembangkan ke berbagai negara lain sebagai pelengkap kegiatan keterampilan atau sekedar mengisi waktu luang. Kegiatan di dalam seni origami terdapat macam-macam aktivitas tangan meliputi memegang, membalik, memutar, mengambil, dan melipat kertas yang dapat meningkatkan kemampuan motoric pada anak. g , p y g p g p p Anak berkebutuhan khusus (ABK) adalah anak yang memiliki kelainan pada dirinya baik secara fisik maupun mental dan membutuhkan perlakuan khusus. Melalui kegiatan melipat kertas diharapkan dapat mengembangkan keterampilan motorik halus terlebih pada anak-anak berkebutuhan khusus. Metode ini diketahui dapat melatih gerak otot-otot tangan sehingga anak memiliki kemampuan untuk memegang pensil, meremas kertas, ataupun membentuk benda dari adonan atau bahan lain. Melipat kertas (origami) merupakan salah satu pengembangan motorik halus yang membutuhkan keterampilan, ketelitian, dan bimbingan. pada anak-anak berkebutuhan khusus, metode ini juga dapat dilakukan. Secara khusus kegiatan melipat bertujuan untuk melatih daya ingat, pengamatan, keterampilan tangan, mengembangkan daya fantasi, kreasi, ketelitian, kerapian,dan perasaan keindahan. PENDAHULUAN Dengan dilakukannnya kegiatan ini, diharapkan anak-anak berkebutuhan khusus bisa lebih mandiri seperti melipat baju maupun melipat apa saja yang perlu dilipat. METODE Sasaran program PKM dalam bentuk penyuluhan dan pelatihan ini adalah anak-anak berkebutuhan khusus di Panti asuhan Bhakti Luhur, Banjarmasin sebagai mitra PKM. Dalam kegiatan ini proses meningkatkan motorik halus anak-anak berkebutuhan khusus dilakukan dengan menggunakan media kertas yang dikemas dalam seni melipat kertas atau origami. Kegiatan pelatihan seni melipat kertas diberikan kepada anak-anak berkebutuhan khusus dengan metode ceramah, tanya jawab dan demonstrasi dalam bentuk pelatihan. Adapun tahapan-tahapan dalam pelaksanaan kegiatan pengabdian sebagai berikut: a. Ceramah dan tanya jawab interaktif bersama anak-anak tentang manfaat,fungsi dan pentingnya seni melipat kertas yang dapat dimanfaatkan sebagai media alternatif untuk meningkatkan motorik halus anak usia dini. b. Menonton video mengenai seni melipat atau origami c. Demonstrasi digunakan untuk mempraktekkan cara melipat kertas yang sesuai dengananak usia dini https://journal.amikveteran.ac.id/index.php/kreatif https://journal.amikveteran.ac.id/index.php/kreatif 214 KREATIF Vol. 3, No. 1 Maret 2023 KREATIF Vol. 3, No. 1 Maret 2023 DISKUSI Melalui kegiatan ini diharapkan mampu membantu anak-anak yang berkebutuhan khusus untuk lebih mengembangkan cara berpikir mereka. Keterbatasan/ketidakmampuan yang dimiliki seorang Anak berkebutuhan khusus tidak mengartikan bahwa mereka tidak berhak mendapatkan pendidikan. Anak-anak tersebut juga berhak mendapatkan cara pendidikan yang sama seperti anak- anak lainnya, agar kemampuan motorik halus anak juga dapat berkembang. Saputra & Rudyanto (2005) Motorik Halus adalah kemampuan anak beraktivitas dengan menggunakan otot-otot halus (kecil) seperti melipat, menulis, meremas, menggenggam, menyusun balok, dan memasukkan kelereng. Untuk itu dalam kegiatan ini menggunakan seni yang menyenangkan seperti seni melipat kertas atau origami. Kegiatan origami secara khusus memiliki manfaat dalam melatih daya ingat, pengamatan, keterampilan tangan atau motoric halus, mengembangkan daya fantasi, kreasi, ketelitian, kerapian,dan perasaan keindahan. Sementara dampaknya bagi anak yaitu bisa belajar tentang gerakan tangan, latihan konsentrasi dan kreativitas. Hal tersebut sejalan dengan pendapat Damayanti (2012) mengatakan manfaat melipat kertas (origami) yaitu sebagai berikut: anak belajar tentang konsep, sebagai alat komunikasi, dapat meningkatkan keterampilan motorik halus anak, anak belajar mengenai ukuran dan bentuk, anak belajar mengikuti instruksi yang runtun. Keterampilan origami juga sangat baik untuk merangsang kreativitas anak, serta membangun daya ingat anak, membangun daya imajinasi anak, dapat menumbuhkan rasa penasaran anak dan membangun sosial yang baik bersama orang-orang yang ada disampingnya (Herlina, 2021). Origami memberikan manfaat yang baik bagi anak-anak berkebutuhan khusus agar lebih kreatif dengan menggunakan barang yang mudah didapat, sederhana dan biaya yang mudah dijangkau. Kegiatan PkM tentang pelatihan origami atau seni melipat kertas untuk meningkatkan motorik halus anak-anak berkebutuhan khusus berlangsung dengan baik dan lancar sesuai dengan rencana dan jadwal yang telah disepakati bersama antara tim PkM dengan pihak panti asuhan Bhakti Luhur. Hasil kegiatan menunjukkan bahwa peserta dapat melipat kertas, memahami cara dan seni melipat kertas dengan berbagai bentuk. Anak-anak sangat terlihat senang dan ceria setelah kegiatan selesai. Hal ini dapat dilihat dari kemampuan motorik halus anak sebelum menggunakan origami dimana masih banyak anak yang masih kesulitan dalam menggambar, belum mampu melipat dengan lipatan 5-8 lipatan, tetapi setelah dilakukan origami ada peningkatan kemampuan motorik halus anak dimana anak sudah dapat melipat dengan berbagai kreasi. KESIMPULAN Hasil pengabdian masyarakat ini menunjukkan bahwa terjadi peningkatan pengetahuan pada kelompok anak-anak berkebutuhan dimana setelah dilakukan kegiatan anak-anak mengetahui mengenai seni melipat kertas dan dapat mempraktekan bagaimana cara melipat kertas dalam beberapa bentuk binatang, bunga maupun bintang. Bagi para guru pendamping di Panti Asuhan Bhakti Luhur diharapkan dapat lebih sering melakukan seni melipat kertas atau jenis seni lainnya untuk dapat lebih mengembangkan kemampuan anak-anak berkebutuhan khusus. Karena kegiatan yang menyenangkan seperti ini lebih diminati oleh anak-anak. https://journal.amikveteran.ac.id/index.php/kreatif 215 215 KREATIF: Jurnal Pengabdian Masyarakat Nusantara Vol. 3, No. 1 Maret 2023 e-ISSN: 2962-3839; p-ISSN: 2962-4436 , Hal 211-216 HASIL Kegiatan pengabdian masyarakat ini laksanakan pada diikuti oleh 30 orang anak berkebutuhan khusus di Panti Asuhan Bhakti Luhur, Banjarmasin. Pada kegiatan ini diberikan materi mengenai origami atau seni melipat kertas dan cara seni melipat kertas kepada anak-anak. Pada tahap pertama, pemateri memberikan video mengenai origami serta jenis-jenis origami yang sering dilakukan oleh anak-anak usis dini. Kemudian setelah selesai menonton video dan melakukan tanya jawab, Anak- anak diminta mencoba untuk melipat kertas namun hampir Sebagian besar anak-anak hanya melipat kertas menjadi 2 bagian atau 4 bagian tanpa membentuk pola apapun. Hal ini menunjukkan mereka belum pernah mengenal seni melipat kertas atau origami sebelumnya. Selanjutnya pemateri dan beberapa orang mahasiswa memberikan contoh cara melipat kertas bermacam bentuk kepada anak-anak. Kegiatan ini dimulai dengan menyiapkan alat dan bahan yang diperlukan untuk melipat kertas. Alat dan bahan yang diperlukan yaitu kertas manila, kertas origami, bekas kardus, lem, gunting, kaleng, isolotif, double tip,pensil warna dan spidol. Setelah selesai menyiapkan alat dan bahan, tahap selanjutnya anak-anak dibagi dalam beberapa kelompok yang dipimpin oleh seorang dosen dan mahasiswa. Pembagian dalam kelompok tersebut bertujuan agar kegiatan dapat berjalan dengan lancar. j g g p j g Setelah anak-anak berkumpul dalam kelompok-kelopoknya, tahapan selanjutnya adalah melipat kertas. Proses pembuatan media ini, dilakukan dengan cara mendemonstrasikan langkah- langkah melipat kertas kegiatan melipat kertas berlangsung dengan lancar. Peserta sangat semangat dan antusias mengikuti pelatihan. Hal tersebut terlihat dari hasil melipat kertas yang dibuat oleh peserta. Proses melipat kertas yang dibuat oleh peserta sangat menarik, bervariasi dan selesai tepat waktu. Anak-anak yang sebelumnya tidak mengetahui dan memahami cara melipat kertas, setelah dilakukan pembimbingan anak-anak dapat melipat kertas dengan bermacaram-macam bentuk. Ada yang melipat membentuk Binatang ikan dan burung, bunga, serta bintang. Semuanya terlihat sangan senang dengan kegiatan ini. Setelah anak-anak berkumpul dalam kelompok-kelopoknya, tahapan selanjutnya adalah melipat kertas. Proses pembuatan media ini, dilakukan dengan cara mendemonstrasikan langkah- langkah melipat kertas kegiatan melipat kertas berlangsung dengan lancar. Peserta sangat semangat dan antusias mengikuti pelatihan. Hal tersebut terlihat dari hasil melipat kertas yang dibuat oleh peserta. Proses melipat kertas yang dibuat oleh peserta sangat menarik, bervariasi dan selesai tepat waktu. Anak-anak yang sebelumnya tidak mengetahui dan memahami cara melipat kertas, setelah dilakukan pembimbingan anak-anak dapat melipat kertas dengan bermacaram-macam bentuk. Ada yang melipat membentuk Binatang ikan dan burung, bunga, serta bintang. Semuanya terlihat sangan senang dengan kegiatan ini. https://journal.amikveteran.ac.id/index.php/kreatif https://journal.amikveteran.ac.id/index.php/kreatif 215 PENGAKUAN/ACKNOWLEDGEMENTS Terimakasih diberikan kepada STIKES Suaka Insan atas dukungan biaya yang diberikan pada kegiatan pengabdian kepada masyarakat ini sehingga kegiatan ini dapat berjalan dengan baik. Kepada pihak Panti Asuhan Bhakti Luhur sebagai mitra pelaksana yang turut terlibat secara langsung, juga kami ucapakan terimakasih yang sebanyak-banyaknya. https://journal.amikveteran.ac.id/index.php/kreatif 216 KREATIF Vol. 3, No. 1 Maret 2023 DAFTAR REFERENSI Ardhiyanti, Yulrina, dkk. (2014). Panduan Lengkap Keterampilan Dasar Kebidanan I. Ed 1, Cet1.Yogyakarta: Deepublish. 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https://openalex.org/W2771688344	https://discovery.dundee.ac.uk/ws/files/19742612/embj.201798099.full.pdf	English	null	Rab29 activation of the Parkinson's disease‐associated LRRK2 kinase	EMBO journal	2,017	cc-by	20,043	University of Dundee Rab29 activation of the Parkinson's disease-associated LRRK2 kinase Purlyte, Elena; Dhekne, Herschel S. ; Sarhan, Adil R.; Gomez, Rachel ; Lis, Pawel; Wightman, Melanie Published in: EMBO Journal DOI: 10.15252/embj.201798099 Publication date: 2018 Licence: CC BY Document Version Publisher's PDF, also known as Version of record Link to publication in Discovery Research Portal Citation for published version (APA): Purlyte, E., Dhekne, H. S., Sarhan, A. R., Gomez, R., Lis, P., Wightman, M., Martinez, T. N., Tonelli, F., Pfeffer, S. R., & Alessi, D. R. (2018). Rab29 activation of the Parkinson's disease-associated LRRK2 kinase. EMBO Journal, 37(1), 1-18. https://doi.org/10.15252/embj.201798099 Rab29 activation of the Parkinson's disease-associated LRRK2 kinase Link to publication in Discovery Research Portal Citation for published version (APA): Purlyte, E., Dhekne, H. S., Sarhan, A. R., Gomez, R., Lis, P., Wightman, M., Martinez, T. N., Tonelli, F., Pfeffer, S. R., & Alessi, D. R. (2018). Rab29 activation of the Parkinson's disease-associated LRRK2 kinase. EMBO Journal, 37(1), 1-18. https://doi.org/10.15252/embj.201798099 General rights Copyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Citation for published version (APA): Purlyte, E., Dhekne, H. S., Sarhan, A. R., Gomez, R., Lis, P., Wightman, M., Martinez, T. N., Tonelli, F., Pfeffer, S. R., & Alessi, D. R. (2018). Rab29 activation of the Parkinson's disease-associated LRRK2 kinase. EMBO Journal, 37(1), 1-18. https://doi.org/10.15252/embj.201798099 Abstract ~1% of sporadic Parkinson’s patients, making LRRK2 one of the most commonly mutated genes linked to Parkinson’s disease (Simon-Sanchez et al, 2009). LRRK2 is a large, multi-domain protein kinase consisting of an armadillo repeat domain (residues 150–510), an ankyrin domain (residues 690–860), leucine-rich repeats (resi- dues 984–1278), a ROC-type GTPase domain (residues 1335–1510) that closely resembles a Rab GTPase and is associated with a COR domain (C-terminal of Roc, residues 1511–1878), a serine/threonine protein kinase domain (residues 1879–2138), and a WD40 repeat- containing domain (residues 2142–2496). The most common patho- genic mutation lies within the catalytic domain (G2019S) and increases kinase activity, suggesting that LRRK2 inhibitors might offer therapeutic benefit for Parkinson’s disease (Greggio et al, 2006; Ozelius et al, 2006; Smith et al, 2006; Jaleel et al, 2007; Hatcher et al, 2017). Parkinson’s disease predisposing LRRK2 kinase phosphorylates a group of Rab GTPase proteins including Rab29, within the effector- binding switch II motif. Previous work indicated that Rab29, located within the PARK16 locus mutated in Parkinson’s patients, operates in a common pathway with LRRK2. Here, we show that Rab29 recruits LRRK2 to the trans-Golgi network and greatly stim- ulates its kinase activity. Pathogenic LRRK2 R1441G/C and Y1699C mutants that promote GTP binding are more readily recruited to the Golgi and activated by Rab29 than wild-type LRRK2. We iden- tify conserved residues within the LRRK2 ankyrin domain that are required for Rab29-mediated Golgi recruitment and kinase activa- tion. Consistent with these findings, knockout of Rab29 in A549 cells reduces endogenous LRRK2-mediated phosphorylation of Rab10. We show that mutations that prevent LRRK2 from interact- ing with either Rab29 or GTP strikingly inhibit phosphorylation of a cluster of highly studied biomarker phosphorylation sites (Ser910, Ser935, Ser955 and Ser973). Our data reveal that Rab29 is a master regulator of LRRK2, controlling its activation, localization, and potentially biomarker phosphorylation. Members of the Rab GTPase family, including Rab8A, Rab10, and Rab29 (also known as RAB7L1), are substrates for LRRK2 (Steger et al, 2016). Recent work has defined a subset of 14 Rab proteins (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35, and Rab43) that are potential direct substrates for LRRK2 (Steger et al, 2017). The LRRK2 phosphorylation site (Thr72 for Rab8A and Thr73 for Rab10) for all of these Rab proteins lies within the effector-binding, switch II motif (Pfeffer, 2001; Cherfils & Zeghouf, 2013). 1 MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK 2 Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA 3 The Michael J. Fox Foundation for Parkinson’s Research, New York, NY, USA Corresponding author. Tel: +44 1382 385602; E-mail: f.tonelli@dundee.ac.uk Corresponding author. Tel: +1 650 725 5130; E-mail: pfeffer@stanford.edu Corresponding author. Tel: +44 1382 385602; E-mail: d.r.alessi@dundee.ac.uk †Equal Experimental Contribution Abstract LRRK2 phosphorylation of Rab8A and Rab10 proteins blocks binding to Rab GDP-dissociation inhibitor (GDI) that is required for Rab protein membrane delivery and recycling; phos- phorylation also inhibits binding of Rab8A to Rabin-8, its cognate guanine nucleotide exchange factor (GEF) (Steger et al, 2016, 2017). Pathogenic mutations located within the GTPase (R1441G/C) and COR (Y1699C) domains do not directly stimulate LRRK2 kinase activity in vitro (Jaleel et al, 2007; Nichols et al, 2010); neverthe- less, they markedly enhance phosphorylation of Rab isoforms to an even greater extent than the G2019S mutation in vivo (Ito et al, 2016; Steger et al, 2016). These mutations promote GTP binding to the LRRK2 Roc domain (Guo et al, 2007; Lewis et al, 2007; Li et al, 2007; Daniels et al, 2011; Webber et al, 2011; Liao et al, 2014). One Keywords Golgi; GTPase; PARK genes; phosphorylation; Rab10 Subject Categories Membrane & Intracellular Transport; Molecular Biology of Disease; Post-translational Modifications, Proteolysis & Proteomics DOI 10.15252/embj.201798099 \| Received 27 August 2017 \| Revised 13 November 2017 \| Accepted 16 November 2017 Keywords Golgi; GTPase; PARK genes; phosphorylation; Rab10 Subject Categories Membrane & Intracellular Transport; Molecular Biology of Disease; Post-translational Modifications, Proteolysis & Proteomics DOI 10.15252/embj.201798099 \| Received 27 August 2017 \| Revised 13 November 2017 \| Accepted 16 November 2017 General rights i h d Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Published online: December 6, 2017 ª 2017 The Authors. Published under the terms of the CC BY 4.0 license Activation of LRRK2 by Rab29 Elena Purlyte et al Activation of LRRK2 by Rab29 Elena Purlyte et al In this study, we demonstrate that Rab29 functions as a critical upstream regulator of LRRK2 by stimulating kinase activity, inferred by assessing autophosphorylation of Ser1292 as well as phosphory- lation of LRRK2 substrates such as Rab10. We find that the patho- genic LRRK2 mutants that bind GTP with higher affinity are activated by Rab29 to a much greater extent than wild-type LRRK2, suggesting a mechanism by which such mutations stimulate LRRK2 activity in vivo. Our studies suggest that LRRK2 interacts with Rab29 via its N-terminal ankyrin domain, and, strikingly, mutations that disrupt regulation by Rab29 prevent phosphorylation of a clus- ter of highly studied biomarker phosphorylation sites (Ser910, Ser935, Ser955 and Ser973) (Dzamko et al, 2010; Doggett et al, 2011). Our findings suggest that Rab29 plays a major role in regulat- ing LRRK2 Golgi localization and kinase activity as well as poten- tially triggering phosphorylation of biomarker sites. explanation for why the R1441G/C and Y1699C LRRK2 mutants are more active in cells is that enhanced GTP binding induces a confor- mational change, rendering LRRK2 more susceptible to activation by an as yet unknown, upstream activator. explanation for why the R1441G/C and Y1699C LRRK2 mutants are more active in cells is that enhanced GTP binding induces a confor- mational change, rendering LRRK2 more susceptible to activation by an as yet unknown, upstream activator. Rab29 is one of five genes contained within the PARK16 locus linked to Parkinson’s disease (Simon-Sanchez et al, 2009; Tucci et al, 2010). It is most closely related to Rab32 and Rab38 GTPases that are needed for lysosome-related organelle biogenesis (Bultema & Di Pietro, 2013; Wang et al, 2014). In contrast to Rab29, Rab32 and Rab38 do not possess a LRRK2 phosphorylation site within their Switch II effector-binding motifs. There is significant variability within the PARK16 locus and Parkinson’s association patterns across populations, and it is currently unclear how mutations within the PARK16 locus are linked to Parkinson’s disease. Transcriptome analysis has suggested that the PARK16 locus enhances expression of Rab29 (Beilina et al, 2014). Other genetic studies of Parkinson’s patient cohorts have found common variants within the LRRK2 and Rab29 genes that function coordinately to increase Parkinson’s risk, as human genetic variants at these loci impact Parkinson’s risk non- additively (MacLeod et al, 2013; Pihlstrom et al, 2015). Rab29 activates LRRK2 To explore whether Rab29 influences LRRK2 kinase activity, we co- expressed Rab29 with wild-type and pathogenic mutants of LRRK2 in HEK293 cells, and assessed LRRK2 autophosphorylation of Ser1292 (Sheng et al, 2012) as well as phosphorylation of endoge- nous Rab10 (Thr73) and Rab29 (Thr71), employing well-character- ized phospho-specific antibodies (Fig EV1). Overexpression of Rab29 significantly enhanced both LRRK2 Ser1292 and Rab10 phos- phorylation (Fig 1A). Rab29 stimulated activity of the “enhanced GTP-binding mutants” (R1441C, R1441G, R1441H, Y1699C) to a much greater extent than wild-type LRRK2 (Fig 1A). Rab29 was also phosphorylated by the R1441G/C and Y1699C mutants to a much greater extent than wild-type LRRK2, consistent with the higher acti- vation state of these pathogenic mutants (Fig 1A). Due to its higher basal activity, the G2019S mutant displayed elevated kinase activity in the absence of Rab29 overexpression, which was further enhanced upon Rab29 overexpression. The I2020T, T2031S, and G2385R pathogenic mutants behaved more like wild-type LRRK2 and were activated by Rab29 overexpression to a lesser extent than the enhanced GTP-binding mutants (Fig 1A). In general, the amount of Rab10 phosphorylation correlates with the extent of LRRK2 acti- vation; however, some variation correlating with the level of Rab29 expression is observed. Furthermore, there are 14 Rab proteins that are phosphorylated by LRRK2 (Steger et al, 2017) and conceivably, LRRK2 mutants may have slightly different localization or prefer- ences for diverse Rab proteins, which could also account for varia- tion between Ser1292 phosphorylation and Rab10 phosphorylation observed (Fig 1A). We also found that stimulation of Ser1292 as well as Rab10 phosphorylation induced by overexpression of Rab29 was abolished by introducing a kinase-inactivating D2017A muta- tion (Fig 1A, right panels), confirming that Rab29 was enhancing phosphorylation by stimulating LRRK2 kinase activity. Genetic investigations in Caenorhabditis elegans neurons revealed that the RAB29 (GLO-1) orthologue acts upstream of LRRK2 (LRK-1) in a signaling pathway controlling axon termination (Kuwahara et al, 2016). It was also reported that Rab29 and LRRK2 double-knockout mice exhibit an enlarged kidney phenotype that was non-additive, rela- tive to single Rab29 or LRRK2 knockout, further implying that these genes act in a common pathway (Kuwahara et al, 2016). Activation of LRRK2 by Rab29 Elena Purlyte et al A haplotype located near the 50 region of RAB29 is associated with Parkinson’s and epistasis between Rab29 and LRRK2 gene variants has been demonstrated (Pihlstrom et al, 2015). ª 2017 The Authors Introduction Autosomal dominant missense mutations within the leucine-rich repeat protein kinase 2 (LRRK2) gene predispose to Parkinson’s disease (Paisan-Ruiz et al, 2004; Zimprich et al, 2004). Mutations in LRRK2 account for ~5% of familial Parkinson’s, and are observed in 1 The EMBO Journal ª 2017 The Authors. Published under the terms of the CC BY 4.0 license The EMBO Journal Published online: December 6, 2017 Activation of LRRK2 by Rab29 Elena Purlyte et al Rab29 selectively activates LRRK2 We next evaluated the effect that 11 Rab proteins including Rab32 and Rab38 (that are highly related to Rab29) have on Ser1292 phos- phorylation of wild-type LRRK2 (Fig EV2A) and LRRK2[R1441G] (Fig EV2B). We found that for wild-type LRRK2, Rab29 markedly stimulated Ser1292 phosphorylation, but with exception of Rab12, which induced a modest ~twofold increase, none of the other Rab proteins including Rab32 and Rab38 activated LRRK2 significantly (Fig EV2A). For the LRRK2[R1441G] mutant, Rab29 also markedly increased Ser1292 phosphorylation more than any of the other Rab proteins (Fig EV2B), but Rab8A and Rab38 also stimulated Ser1292 phosphorylation two- to threefold (Fig EV2B). Rab29 is localized to the Golgi complex (Wang et al, 2014) where it overlaps with p115 protein (Fig 3A). Like LRRK2 R1441G (Fig 2), LRRK2 G2019S also displays a distributed, punctate pattern when expressed on its own (Fig 3E). A small amount of perinuclear LRRK2 may co-localize with endogenous Rab29, but available anti- bodies were unable to label endogenous Rab29 protein. However, when LRRK2 G2019S was co-expressed in cells with Rab29, the proteins showed significant co-localization over an extended, Rab29 activates LRRK2 WT is wild-type and D2017A corresponds to the kinase-inactive LRRK2 mutant. Similar results were obtained in two separate experiments. Right: As in left panel except that kinase-inactivating D2017A LRRK2 mutation was inserted into the indicated LRRK2 pathogenic mutant. Similar results were obtained in two independent experiments. B As in (A) except that LRRK2[R1441G] pathogenic variant was co-transfected with wild-type and indicated mutants of Rab29. EE indicates Rab29[T71E,S72E] mutation and AA indicates Rab29[T71A,S72A] mutation. Similar results were obtained in two separate experiments, each performed in duplicate. A Left: HEK293 cells were transfected with the indicated wild-type and human full-length pathogenic LRRK2 variants with either HA-empty vector () or HA-tagged Rab29 (+). 24 h post-transfection, cells were lysed and analyzed by immunoblotting with the indicated antibodies. WT is wild-type and D2017A corresponds to the kinase-inactive LRRK2 mutant. Similar results were obtained in two separate experiments. Right: As in left panel except that kinase-inactivating D2017A LRRK2 mutation was inserted into the indicated LRRK2 pathogenic mutant. Similar results were obtained in two independent experiments. B As in (A) except that LRRK2[R1441G] pathogenic variant was co-transfected with wild-type and indicated mutants of Rab29. EE indicates Rab29[T71E,S72E] mutation and AA indicates Rab29[T71A,S72A] mutation. Similar results were obtained in two separate experiments, each performed in duplicate. of cytoplasmic protein obscures the localization of the membrane- associated pool in fixed cells. To overcome this challenge, and to avoid the possibility of spurious precipitation of cytosolic LRRK2 protein onto cellular structures during fixation, we employed an established liquid nitrogen coverslip freeze–thaw protocol (Seaman, 2004) to deplete cytosolic proteins and reveal the localization of membrane-associated LRRK2 protein. Figure 2 shows the localiza- tion of R1441G LRRK2 in HeLa cells upon transient transfection. The protein is localized predominantly in the perinuclear region but distinct, peripheral punctae are also detected, and 60% of these also contain Rab10 protein (Fig 2A and C). In the perinuclear region, more than 40% of the LRRK2 punctae co-localized with Rab8 protein (Fig 2B and D). These findings are consistent with the fact that Rab10 and Rab8 are significant LRRK2 substrates (Steger et al, 2016). Rab29 by LRRK2, we mutated the phosphorylation sites to Glu and observed that the Rab29[T71E,S72E] mutant failed to activate LRRK2 (Fig 1B). This suggests that Rab29 phosphorylation may decrease its ability to activate LRRK2. Rab29 activates LRRK2 In an attempt to mimic phosphorylation of 2 The EMBO Journal ª 2017 The Authors Elena Purlyte et al Activation of LRRK2 by Rab29 Published online: December 6, 2017 Published online: December 6, 2017 Rab29 (HA) WT D2017A R1441C R1441G R1441H Y1699C R1728H G2019S I2020T T2031S G2385R Rab29-pT71 Rab10-pT73 (endogenous) Rab10-total (endogenous) LRRK2-total LRRK2-pS1292 R1441G R1441G D2017A Y1699C Y1699C D2017A G2019S G2019S D2017A 25kDa 25kDa 25kDa 25kDa 250kD 250kD A B enhanced GTP-binding LRRK2 Rab29 D2017A = kinase inactive Rab29 LRRK2 R1441G 25kDa 250kD wt T71E S72E EE AA 250kD Rab29 (HA) Rab10-pT73 (endogenous) Rab10-total (endogenous) LRRK2 LRRK2-pS1292 25kDa 25kDa Figure 1. Rab29 activates LRRK2. A Left: HEK293 cells were transfected with the indicated wild-type and human full-length pathogenic LRRK2 variants with either HA-empty vector () or HA-tagged Rab29 (+). 24 h post-transfection, cells were lysed and analyzed by immunoblotting with the indicated antibodies. WT is wild-type and D2017A corresponds to the kinase-inactive LRRK2 mutant. Similar results were obtained in two separate experiments. Right: As in left panel except that kinase-inactivating D2017A LRRK2 mutation was inserted into the indicated LRRK2 pathogenic mutant. Similar results were obtained in two independent experiments. B As in (A) except that LRRK2[R1441G] pathogenic variant was co-transfected with wild-type and indicated mutants of Rab29. EE indicates Rab29[T71E,S72E] mutation and AA indicates Rab29[T71A,S72A] mutation. Similar results were obtained in two separate experiments, each performed in duplicate. Elena Purlyte et al Activation of LRRK2 by Rab29 The EMBO Journal Published online: December 6, 2017 The EMBO Journal Rab29 (HA) WT D2017A R1441C R1441G R1441H Y1699C R1728H G2019S I2020T T2031S G2385R Rab29-pT71 Rab10-pT73 (endogenous) Rab10-total (endogenous) LRRK2-total LRRK2-pS1292 R1441G R1441G D2017A Y1699C Y1699C D2017A G2019S G2019S D2017A 25kDa 25kDa 25kDa 25kDa 250kD 250kD A enhanced GTP-binding LRRK2 Rab29 D2017A = kinase inactive A D2017A = kinase inactive B Rab29 LRRK2 R1441G 25kDa 250kD wt T71E S72E EE AA 250kD Rab29 (HA) Rab10-pT73 (endogenous) Rab10-total (endogenous) LRRK2 LRRK2-pS1292 25kDa 25kDa B B Rab29 LRRK2 R1441G 25kDa 250kD wt T71E S72E EE AA 250kD Rab29 (HA) Rab10-pT73 (endogenous) Rab10-total (endogenous) LRRK2 LRRK2-pS1292 25kDa 25kDa Figure 1. Rab29 activates LRRK2. Figure 1. Rab29 activates LRRK2. g A Left: HEK293 cells were transfected with the indicated wild-type and human full-length pathogenic LRRK2 variants with either HA-empty vector () or HA-tagged Rab29 (+). 24 h post-transfection, cells were lysed and analyzed by immunoblotting with the indicated antibodies. Rab29 activates LRRK2 Others have suggested that LRRK2 and various Rab proteins including Rab29 inter- act, largely based on co-immunoprecipitation analysis, but binding domains have not yet been pinpointed (Dodson et al, 2012; MacLeod et al, 2013; Beilina et al, 2014; Waschbusch et al, 2014; Zhang et al, 2015). It has also been reported that Rab29 recruits LRRK2 to the Golgi apparatus (MacLeod et al, 2013; Beilina et al, 2014). LRRK2 is constitutively phosphorylated at a cluster of Ser residues lying between the ankyrin domain and leucine-rich repeat region (Ser910, Ser935, Ser955 and Ser973) that plays a role in regulating 14-3-3 binding and cytosolic localization (Nichols et al, 2010; Doggett et al, 2011). These sites have received a lot of attention as they are controlled by LRRK2 kinase activity, and therefore become rapidly dephosphorylated in response to diverse LRRK2 inhibitors (Dzamko et al, 2010; Doggett et al, 2011). Monitoring the dephosphorylation of these residues, especially Ser935, has become the principal biomarker strategy to assess the in vivo efficacy of LRRK2 inhibitors (Hatcher et al, 2017). Despite a lot of research, it is currently unclear how LRRK2 kinase activity influences phosphorylation of these biomarker sites. Autophosphorylation would be the simplest model to account for the data obtained to date. However, autophosphorylation of the biomarker residues has not been observed in in vitro studies under- taken thus far, perhaps indicating a missing factor is required to stim- ulate autophosphorylation of these sites (Dzamko et al, 2010). Other kinases not known to be regulated by LRRK2 including CK1 (Chia et al, 2014) and PKA (Muda et al, 2014) have also been reported to phosphorylate these sites. In macrophages, the IkappaB kinase family phosphorylates Ser910 and Ser935 sites independently from LRRK2 kinase activity (Dzamko et al, 2012). To test whether phosphorylation of Rab29 at Thr71 and Ser72 by LRRK2 was required for activation of LRRK2, we mutated these sites both to Ala. We found that the Rab29[T71A,S72A] mutant still acti- vated LRRK2[R1441G] to the same extent as wild-type Rab29, indi- cating that phosphorylation of Rab29 is not required for its ability to activate LRRK2 (Fig 1B). ª 2017 The Authors The EMBO Journal Rab29 activates LRRK2 on Golgi membranes At steady state, LRRK2 is primarily cytosolic; approximately 10% associates with membranes upon cell fractionation. The large pool 3 The EMBO Journal The EMBO Journal ª 2017 The Authors The EMBO Journal Published online: December 6, 2017 Activation of LRRK2 by Rab29 Elena Purlyte et al Rab8 Rab10 Rab8 0 20 40 60 Co-localized puncta near nucleus (percent) D Rab10 R1441G LRRK2 Rab8 R1441G LRRK2 **** the periphery and Rab8 near the nucleus. G-LRRK2 (red) and either GFP-Rab10 (A) or GFP-Rab8 (B) in green. Scale bar, 10 lm. The second row in bove. Scale bar, 2 lm. ndicated Rab in the indicated cell regions was determined from a Mander’s coefficient after automatic P < 0.0001; P = 0.0076 by Student’s unpaired, two-tailed t-test (n = 13 from two experiments). For were generated (see A) that included the plasma membrane and excluded the nucleus. For perinuclear perinuclear, reticular structure (Fig 3B). This structure is di d l i l i i b Co-localization of LRRK2 G2019S (Fig 3B) and R1441G proteins (see i b l ) i h b d l i hi h b Rab10 Rab8 0 20 40 60 80 Co-localized puncta at cell periphery (percent) Rab10 Rab8 0 20 40 60 Co-localized puncta near nucleus (percent) A D C B Rab10 R1441G LRRK2 Rab8 R1441G LRRK2 * ** R1441G-LRRK2 co-localizes with Rab10 in the periphery and Rab8 near the nucleus. wn are HeLa cells stained for transfected R1441G-LRRK2 (red) and either GFP-Rab10 (A) or GFP-Rab8 (B) in green. Scale bar, 10 lm. The second row in both els shows enlarged portions boxed in the rows above. Scale bar, 2 lm. ent co-localization of R1441G-LRRK2 with the indicated Rab in the indicated cell regions was determined from a Mander’s coefficient after automatic sholding. Error bars represent mean SEM. P < 0.0001; P = 0.0076 by Student’s unpaired, two-tailed t-test (n = 13 from two experiments). For pheral quantitation, boxes of the size indicated were generated (see A) that included the plasma membrane and excluded the nucleus. For perinuclear ntitation (see B), the boxes included half of the nucleus. A Rab10 R1441G LRRK2 A B Rab8 R1441G LRRK2 B Rab10 Rab8 0 20 40 60 80 Co-localized puncta at cell periphery (percent) C ** Rab10 Rab8 0 20 40 60 Co-localized puncta near nucleus (percent) D C C D Figure 2. The EMBO Journal ª 2017 The Authors Rab29 activates LRRK2 on Golgi membranes R1441G-LRRK2 co-localizes with Rab10 in the periphery and Rab8 near the nucleus. A, B Shown are HeLa cells stained for transfected R1441G-LRRK2 (red) and either GFP-Rab10 (A) or GFP-Rab8 (B) in green. Scale bar, 10 lm. The second row in both panels shows enlarged portions boxed in the rows above. Scale bar, 2 lm. C, D Percent co-localization of R1441G-LRRK2 with the indicated Rab in the indicated cell regions was determined from a Mander’s coefficient after automatic thresholding. Error bars represent mean SEM. **P < 0.0001; P = 0.0076 by Student’s unpaired, two-tailed t-test (n = 13 from two experiments). For peripheral quantitation, boxes of the size indicated were generated (see A) that included the plasma membrane and excluded the nucleus. For perinuclear quantitation (see B), the boxes included half of the nucleus. Co-localization of LRRK2 G2019S (Fig 3B) and R1441G proteins (see Fig 7H below) with Rab29 supports a model in which Rab29 can activate pathogenic LRRK2 proteins on Rab29-containing membranes. somewhat perinuclear, reticular structure (Fig 3B). This structure is likely to represent a disrupted Golgi complex, as staining became much more concentrated in the perinuclear region upon treatment of cells with the MLI-2 LRRK2 kinase inhibitor (Fig 3C and D). 4 ª 2017 The Authors Published online: December 6, 2017 The EMBO Journal Elena Purlyte et al Activation of LRRK2 by Rab29 +MLI2 LRRK2 G2019S Rab29 Rab29 G2019S+Rab29 G2019S+Rab29 +MLI2 0 20 40 60 80 100 Cells with compact Rab29 staining (percent) A C B D +MLI2 Rab29/p115 * E LRRK2 G2019S Figure 3. Pathogenic LRRK2 mutants co-localize with Rab29 and disperse Golgi membranes. HeLa cells were transfected with LRRK2-G2019S or Myc-Rab29 or LRRK2-G2019S and 24 h later transfected with Myc-Rab29. After 48 h, cells were permeabilized by liquid nitrogen freeze–thaw to deplete cytosol and then fixed and stained with mouse anti-p115, mouse anti-Myc, and rabbit anti-GFP or rabbit anti- LRRK2 antibodies. A Myc-Rab29 (green) and p115 (red) show co-localization at the Golgi. Left and right panels were treated with or without MLI2 (200 nM, 4 h) as indicated. B Myc-Rab29 (red) and eGFP-LRRK2-G2019S (green) show co-localization and dispersed Rab29-labeled Golgi membranes. Rab29 activates LRRK2 on Golgi membranes C C ll t t d ith MLI 2 (200 M 4 h) h t R b29 iti A +MLI2 Rab29/p115 +MLI2 LRRK2 G2019S Rab29 A C B +MLI2 Rab29/p115 A Disruption of the Golgi by pathogenic LRRK2 proteins has been previously reported (MacLeod et al, 2013; Beilina et al, 2014), and light microscopic analysis of the trans-Golgi network in mouse embryonic fibroblasts (MEFs) (as monitored using anti-GCC185 antibodies) confirmed this finding (Fig 4A and C); MLI2 treatment restored compact Golgi localization for GCC185-stained compart- ments (compare Fig 4B and C right and left panels). Note that the extent of Golgi fragmentation correlated with the relative kinase activation of the LRRK2 mutant proteins. Together, these data show that LRRK2 co-localizes with its substrates, Rab10 and Rab8, and also co-localizes with an important key activator, Rab29 GTPase. +MLI2 LRRK2 G2019S Rab29 C B LRRK2 G2019S Rab29 B B Rab29 activation of LRRK2 would be predicted to occur on membrane surfaces harboring active Rab29 protein. To test this, cells expressing Rab29 and R1441G LRRK2 were fractionated into membrane and cytosol fractions and analyzed for their content of total and activated LRRK2 protein, using anti-LRRK2 and anti- pS1292 antibodies. Expression of Rab29 increased the amount of total membrane-associated LRRK2 (Fig 5A and B) and also led to a greater than threefold enrichment of activated, pS1292 LRRK2 on membranes (Fig 5C). Similar data were obtained for G2019S-LRRK2 (see Fig 7 below). Note that about 10% of LRRK2-R1441G is also detected in membrane fractions obtained from Rab29 knockout 293 T cells (Fig 5B). This Rab29-independent pool may represent associ- ation of LRRK2 with another Rab GTPase such as Rab8A or Rab38, or could represent R1441G LRRK2 aggregates. +MLI2 C C Ankyrin domain residues permit activation of LRRK2 by Rab29 Rab29 G2019S+Rab29 G2019S+Rab29 +MLI2 0 20 40 60 80 100 Cells with compact Rab29 staining (percent) D * E LRRK2 G2019S Rab29 G2019S+Rab29 G2019S+Rab29 +MLI2 0 20 40 60 80 100 Cells with compact Rab29 staining (percent) D * D D In an initial attempt to define the region of LRRK2 required for Rab29 activation, we generated a truncation mutant of LRRK2 lack- ing the N-terminal, 969 non-catalytic residues encompassing the armadillo and ankyrin domains. Although this mutant is expressed at lower levels than full-length LRRK2 in HEK293 cells, it was clearly not activated by Rab29 overexpression (Fig 6A), indicating that the Rab29 effector region lies within the LRRK2 N-terminal fragment, which was also suggested in a previous study (Beilina et al, 2014). Rab32 and Rab38, the Rab proteins most closely related to Rab29, interact directly with the ankyrin domain of an effector called VARP (VPS9-ankyrin repeat protein, also known as ANKRD27), a regulator of endosomal trafficking (Fukuda, 2016). The crystal structure of the VARP ankyrin domain/Rab32 complex (PDB 4CYM) reveals a large interface of interacting residues, encom- passing several clusters of adjacent Leu residues on VARP that make hydrophobic interactions with Rab32 (Hesketh et al, 2014). Muta- tion of these VARP ankyrin domain Leu residues decreased binding to Rab32 (Hesketh et al, 2014). These studies prompted us to explore whether the LRRK2 ankyrin domain might comprise the Rab29 binding site. Inspection of the LRRK2 ankyrin domain reveals that it possesses three Leu-rich motifs that are conserved in human, chicken, Xenopus, Zebrafish, and Drosophila LRRK2, which we termed Region A, B, and C (Fig 6B). We mutated representative Leu residues within each of these regions to Asp, as this corresponds to the mutations that were designed to prevent the interaction of VARP with Rab32 (Hesketh et al, 2014). We studied how these mutations impacted Rab29-mediated LRRK2 activation. This revealed that in particular, Region A (Cys727Asp, Leu728Asp, and Leu729Asp) mutations strikingly prevented Rab29-mediated activation of Figure 3. Pathogenic LRRK2 mutants co-localize with Rab29 and disperse Golgi membranes. HeLa cells were transfected with LRRK2-G2019S or Myc-Rab29 or LRRK2-G2019S and 24 h later transfected with Myc-Rab29. After 48 h, cells were permeabilized by liquid nitrogen freeze–thaw to deplete cytosol and then fixed and stained with mouse anti-p115, mouse anti-Myc, and rabbit anti-GFP or rabbit anti- LRRK2 antibodies. The EMBO Journal The EMBO Journal ª 2017 The Authors Ankyrin domain residues permit activation of LRRK2 by Rab29 Wild type Wild type + MLI2 GCC185 A B R1441G R1441G + MLI2 Wild type Wild type + MLI2 GCC185 A Wild A B R1441G R1441G + MLI2 B R1441G R1441G+MLI2 WT WT+MLI2 0 20 40 60 80 100 Cells with compact trans Golgi (percent) C * Figure 4. LRRK2-R1441G expression disrupts Golgi morphology. A Wild-type MEFs were stained with rabbit anti-GCC185 (green) and DAPI (blue); left and right panels were treated with or without MLI2 as indicated. B Knock-in R1441G MEF cells MLI-2 (200 nM, 4 h) stained with rabbit anti-GCC185 antibody (green) and DAPI (blue). C Quantitation of the percent of cells showing a compact trans-Golgi network as seen in (A). Error bars represent SEM from two experiments with > 50 cells per condition in each experiment. P = 0.0184 with Student’s unpaired, two-tailed t-test. Differences between WT, WT+MLI2, and R1441G+MLI2 were not significant (P > 0.5). Data information: Scale bars, 10 lm. R1441G R1441G+MLI2 WT WT+MLI2 0 20 40 60 80 100 Cells with compact trans Golgi (percent) C R1441G R1441G+MLI2 WT WT+MLI2 0 20 40 60 80 100 Cells with compact trans Golgi (percent) C * R1441G R1441G+MLI2 WT WT+MLI2 0 20 40 60 80 100 Cells with compact trans Golgi (percent) C * C Figure 4. LRRK2-R1441G expression disrupts Golgi morphology. g p p g p gy A Wild-type MEFs were stained with rabbit anti-GCC185 (green) and DAPI (blue); left and right panels were treated with or without MLI2 as indicated. B Knock-in R1441G MEF cells MLI-2 (200 nM, 4 h) stained with rabbit anti-GCC185 antibody (green) and DAPI (blue). C Quantitation of the percent of cells showing a compact trans-Golgi network as seen in (A). Error bars represent SEM from two experiments with > 50 cells per condition in each experiment. P = 0.0184 with Student’s unpaired, two-tailed t-test. Differences between WT, WT+MLI2, and R1441G+MLI2 were not significant (P > 0.5). Data information: Scale bars, 10 lm. wild-type LRRK2 (Fig 6C) and LRRK2[R1441G] mutant (Fig 6D). These ankyrin domain mutations reduced basal levels of Ser1292 phosphorylation, consistent with these enzymes being less active due to their inability to bind Rab29 (Fig 6C and D). Mutations in Region B and Region C also suppressed Rab29-stimulated Ser1292 autophosphorylation of wild-type LRRK2, further supporting a role for the ankyrin repeat domain playing a critical role in controlling LRRK2 activation (Fig 6C). Ankyrin domain residues permit activation of LRRK2 by Rab29 HeLa cells were transfected with LRRK2-G2019S or Myc-Rab29 or LRRK2-G2019S and 24 h later transfected with Myc-Rab29. After 48 h, cells were permeabilized by liquid nitrogen freeze–thaw to deplete cytosol and then fixed and stained with mouse anti-p115, mouse anti-Myc, and rabbit anti-GFP or rabbit anti- LRRK2 antibodies. A Myc-Rab29 (green) and p115 (red) show co-localization at the Golgi. Left and right panels were treated with or without MLI2 (200 nM, 4 h) as indicated. B Myc-Rab29 (red) and eGFP-LRRK2-G2019S (green) show co-localization and dispersed Rab29-labeled Golgi membranes. C Cells treated with MLI-2 (200 nM, 4 h) show compact, Rab29-positive Golgi, and associated LRRK2-G2019S (green). D Percent of cells with compact Rab29 staining; P = 0.0002; P = 0.002 with Student’s unpaired, two-tailed t-test. Error bars represent SEM for three experiments with > 30 cells per condition in each experiment. E LRRK2-G2019S alone showing punctate staining throughout the cell. Data information: Scale bars, 10 lm. 5 The EMBO Journal ª 2017 The Authors The EMBO Journal Published online: December 6, 2017 Activation of LRRK2 by Rab29 Elena Purlyte et al LRRK2 (Fig 6C) and LRRK2[R1441G] mutant (Fig 6D). kyrin domain mutations reduced basal levels of Ser1292 ylation, consistent with these enzymes being less active Region B and Region C also suppressed Rab29-stimulated Ser1 autophosphorylation of wild-type LRRK2, further supporting a for the ankyrin repeat domain playing a critical role in contro Wild type Wild type + MLI2 GCC185 R1441G R1441G+MLI2 WT WT+MLI2 0 20 40 60 80 100 Cells with compact trans Golgi (percent) A C B R1441G R1441G + MLI2 * LRRK2-R1441G expression disrupts Golgi morphology. ype MEFs were stained with rabbit anti-GCC185 (green) and DAPI (blue); left and right panels were treated with or without MLI2 as indicated. -in R1441G MEF cells MLI-2 (200 nM, 4 h) stained with rabbit anti-GCC185 antibody (green) and DAPI (blue). itation of the percent of cells showing a compact trans-Golgi network as seen in (A). Error bars represent SEM from two experiments with > 50 cells per conditi h experiment. P = 0.0184 with Student’s unpaired, two-tailed t-test. Differences between WT, WT+MLI2, and R1441G+MLI2 were not significant (P > 0.5). rmation: Scale bars, 10 lm. Ankyrin domain residues permit activation of LRRK2 by Rab29 - MycRab29 25 - Fraction total LRRK2 on membranes Myc-Rab29: - + B p=.0025 0 0.1 0.2 Rab29KO 0.3 * Fraction total LRRK2 on membranes Myc-Rab29: - + B p=.0025 0 0.1 0.2 Rab29KO 0.3 B pS1292/Total LRRK2(relative) Rab29: - + - + membrane cytosol C 0 1 2 3 p=.00003 ns Ankyrin domain residues permit activation of LRRK2 by Rab29 6 ª 2017 The Authors ª 2017 The Authors Elena Purlyte et al Activation of LRRK2 by Rab29 Published online: December 6, 2017 The EMBO Journal - pS1292 - tubulin - MycRab29 - FLAG-LRRK2 R1441G - LAMP2 250 - 25 - 50 - 100 - 250 - membrane cytosol Myc-Rab29: - - + + - - + + Fraction total LRRK2 on membranes Myc-Rab29: - + A B pS1292/Total LRRK2(relative) Rab29: - + - + membrane cytosol C 0 1 2 3 p=.0025 p=.00003 0 0.1 0.2 Rab29KO 0.3 ** ns Figure 5. Rab29 increases membrane association of LRRK2-R1441G. HEK293T Rab29/ (KO) and WT cells were transfected with LRRK2-R1441G and 24 h later transfected with Myc Rab29 After 48 h cells were harvested and - pS1292 - tubulin - MycRab29 - FLAG-LRRK2 R1441G - LAMP2 250 - 25 - 50 - 100 - 250 - membrane cytosol Myc-Rab29: - - + + - - + + A A experiments demonstrated that the Region A mutations (Cys727Asp, Leu728Asp, and Leu729Asp) inhibited LRRK2 kinase activity toward Nictide and Rab8A by four- to fivefold (Fig 6E and F). At the end of in vitro kinase assays, we also assessed LRRK2 autophosphorylation at Ser1292 by immunoblot, which also revealed that Region A muta- tions also substantially inhibit autophosphorylation (Fig 6E and F). The crystal structure of the VARP:Rab32 complex reveals that Rab32 binding to the VARP ankyrin domain is controlled by two conserved Met91 and Arg93 residues that lie within the Rab32 effector-binding switch II motif (Hesketh et al, 2014). Mutation of these residues to Ser abolished VARP binding (Hesketh et al, 2014). Interestingly, Rab29, Rab32, and Rab38 are the only Rab proteins that possess Met and Arg residues at the equivalent positions within their effector-binding loops (Fig EV3A), perhaps suggesting that this family of Rab proteins binds ankyrin domains via a common mecha- nism. Although we found mutation of equivalent residues in Rab29 (Met73 and Arg75) prevented activation of LRRK2 (Fig EV3B), the Rab29[M73S, R75S] mutant was localized in the cytosol, indicating that these mutations likely disrupt Rab29 nucleotide binding and C-terminal prenylation and should not be employed in future studies (Fig EV3C). ª 2017 The Authors The EMBO Journal Ankyrin domain residues influence LRRK2 membrane association and Rab29 co-localization C If the ankyrin domain is important for Rab GTPase interaction, it would be predicted to be important for LRRK2 membrane associa- tion. To test this, cells expressing LRRK2 R1441G or LRRK2 R1441G protein also carrying Region A mutations were fractionated and analyzed for their content of membrane-associated LRRK2 protein by immunoblot. As shown in Fig 7A, approximately 10% of total R1441G LRRK2 was detected on membranes; the individual L728D or L729D LRRK2 proteins showed only a slight decrease in overall membrane association, and the L728D/L729D mutant protein led to a 60% decrease in membrane association determined by this method (Fig 7B). In addition, the ankyrin domain mutants failed to respond to Rab29 co-expression in terms of their activation on membranes, as monitored using anti-LRRK2 pS1292 antibodies (Fig 7C, E and F) or cytosol (Fig 7D); Rab29 overexpression enhanced the membrane association of total (Fig 7E) and pS1292-LRRK2 (Fig 7F) on membranes. Further support for the importance of ankyrin domain sequences in Rab29 interaction comes from light microscopy experi- ments, in which R1441G LRRK2 ankyrin domain mutant proteins showed significantly less co-localization with Rab29 upon expres- sion in HeLa cells (Fig 7G and H). R1441G LRRK2 staining was much more punctate than that seen for G2019S (compare with Fig 3E); 40% of R1441G LRRK2 punctae co-localized with Rab29, and most Rab29 remained associated with a disrupted Golgi complex (Fig 7G and H). Importantly, R1441G L728D/L729D failed to disrupt the Golgi (Fig 7I), consistent with the requirement for Rab29 interaction to mediate this process. These experiments support a model in which ankyrin domain residues are important for Rab29 interaction, LRRK2 kinase activation, and Golgi complex disruption. Figure 6. Ankyrin domain residues permit activation of LRRK2 by Rab29. Similar results were obtained in two experiments. E As in (C) except that the indicated forms of LRRK2 were immunoprecipitated from cell extracts and then subjected to an LRRK2 kinase activity by measuring phosphorylation of the Nictide peptide substrate in the presence of 0.2 mM 32PcATP and in the absence () or presence (+) of 1 lM MLi-2 LRRK2 in a 30-min kinase reaction. After the kinase assay, phosphorylation of Nictide was quantified by Cherenkov counts and data presented as average SEM for three independent experiments each undertaken in triplicate. Cherenkov counts recorded for no LRRK2 (-) controls were subtracted from all values. There was a statistically significant difference between groups (P < 0.0001, one-way ANOVA, F(9, 20) = 95.87) P < 0.001 by one-way ANOVA with Dunnett’s multiple comparison with mean difference 95% confidence intervals of groups compared to WT: WT MLI-2 0.8668–1.126; C727D 0.7428–1.002; C727D MLI-2 0.8731–1.132; L728D 0.7602–1.019; L728D MLI-2 0.8559–1.115; L729D 0.7570–1.016, L729D MLI-2 0.8708–1.130, L728D+L729D 0.7466–1.006; L728D+L729D MLI-2 0.8655–1.124. Assay mixtures were subjected to immunoblot analysis with the indicated antibodies. E As in (C) except that the indicated forms of LRRK2 were immunoprecipitated from cell extracts and then subjected to an LRRK2 kinase activity by measuring phosphorylation of the Nictide peptide substrate in the presence of 0.2 mM 32PcATP and in the absence () or presence (+) of 1 lM MLi-2 LRRK2 in a 30-min kinase reaction. After the kinase assay, phosphorylation of Nictide was quantified by Cherenkov counts and data presented as average SEM for three independent experiments each undertaken in triplicate. Cherenkov counts recorded for no LRRK2 (-) controls were subtracted from all values. There was a statistically significant difference between groups (P < 0.0001, one-way ANOVA, F(9, 20) = 95.87) P < 0.001 by one-way ANOVA with Dunnett’s multiple comparison with mean difference 95% confidence intervals of groups compared to WT: WT MLI-2 0.8668–1.126; C727D 0.7428–1.002; C727D MLI-2 0.8731–1.132; L728D 0.7602–1.019; L728D MLI-2 0.8559–1.115; L729D 0.7570–1.016, L729D MLI-2 0.8708–1.130, L728D+L729D 0.7466–1.006; L728D+L729D MLI-2 0.8655–1.124. Assay mixtures were subjected to immunoblot analysis with the indicated antibodies. F As in (E) except phosphorylation of Rab8A by LRRK2 was assessed using a phospho-specific antibody. Similar results were obtained in two experiments each undertaken in duplicate. horylation of Rab8A by LRRK2 was assessed using a phospho-specific antibody. Similar results were obtained in two experiments each e. Source data are available online for this figure. Figure 5. Rab29 increases membrane association of LRRK2-R1441G. Figure 5. Rab29 increases membrane association of LRRK2-R1441G. Figure 5. Rab29 increases membrane association of LRRK2-R1441G. HEK293T Rab29/ (KO) and WT cells were transfected with LRRK2-R1441G and 24 h later transfected with Myc-Rab29. After 48 h, cells were harvested and fractionated into cytosol and membrane fractions. Figure 5. Rab29 increases membrane association of LRRK2-R1441G. HEK293T Rab29/ (KO) and WT cells were transfected with LRRK2-R1441G and 24 h later transfected with Myc-Rab29. After 48 h, cells were harvested and fractionated into cytosol and membrane fractions. HEK293T Rab29/ (KO) and WT cells were transfected with LRRK2-R1441G and 24 h later transfected with Myc-Rab29. After 48 h, cells were harvested and fractionated into cytosol and membrane fractions. A Immunoblot of membrane protein (75 lg) and the 50% of equivalent volume of cytosolic proteins, Rab29 as indicated. Numbers at left indicate mobility of marker proteins in kDa; proteins were detected with rabbit anti- pS1292, rabbit anti-LRRK2 UDD3, mouse anti-LRRK2, mouse anti-LAMP2, mouse anti-tubulin, and mouse anti-Myc antibodies. B Quantitation of the fraction of total LRRK2-R1441G on membranes Rab29 (transfected and endogenous). B Quantitation of the fraction of total LRRK2-R1441G on membranes Rab29 (transfected and endogenous). C Amount of active (pS1292) LRRK2 in membrane and cytosol fractions Rab29 expression normalized to the amount of total LRRK2 in the fraction. Data information: Error bars represent SEM from two experiments. P = 0.0025; **P = 0.00013; ns = not significant (P = 0.1968) by Student’s unpaired, two- tailed t-test. Differences between Rab29 KO and (minus) Myc-Rab29 are not significant. Source data are available online for this figure. To study further how Rab29 binding controls LRRK2, we immuno- precipitated wild-type LRRK2 and Region A mutations from HEK293 cells and assayed kinase activity employing the Nictide peptide substrate (Dzamko et al, 2010) (Fig 6E) or recombinant Rab8A (Steger et al, 2016) (Fig 6F). Assays were undertaken in the presence or absence of the MLi-2 LRRK2 inhibitor to ensure activity measured was mediated by LRRK2 rather than a contaminating kinase. These Ankyrin domain mutant proteins showed decreased overall kinase activity, and the protein was somewhat less stable in cells, as determined by monitoring its turnover after cycloheximide addition (Fig 7J). Loss of a binding partner interaction often leads to decreased protein stability. Figure 5. Rab29 increases membrane association of LRRK2-R1441G. Rab29 Rab32 A B LRRK2-total LRRK2-pS1292 Rab29-pT71 Rab29-total (HA) FL 970-end Rab10-pT73 (endogenous) Rab10-total (endogenous) FL 970-end FL 970-end FL 970-end FL 970-end FL 970-end 25kDa 25kDa 25kDa 25kDa C Rab29 LRRK2 D2017A WT R1441G Y1699C LRRK2 Rab29 B A C 250kDa 250kDa 25kDa 25kDa 50kDa - - + + - - + + - - + + - - + + - - + + - - + + LRRK2-pS1292 LRRK2-total Rab29-total (HA) tubulin Rab29-pT71 WT C727D+ R1441G L728D+ R1441G L729D+ R1441G R1441G R1441G + KD 25kDa 25kDa Rab10-pT73 (endogenous) Rab10-total (endogenous) D ) LRRK2 Rab29 A LRRK2-total LRRK2-pS1292 Rab29-pT71 Rab29-total (HA) FL 970-end Rab10-pT73 (endogenous) Rab10-total (endogenous) FL 970-end FL 970-end FL 970-end FL 970-end FL 970-end 25kDa 25kDa 25kDa 25kDa Rab29 LRRK2 D2017A WT R1441G Y1699C D A 50kDa tubulin Rab8A-total LRRK2-total LRRK2-pS1292 Rab8A-pT72 LRRK2 WT C727D L728D L729D MLi-2 L728D+ L729D F E MLi-2 LRRK2 WT C727D L728D L729D L728D+ L729D IP: LRRK2 IB: LRRK2 IP: LRRK2 IB: LRRK2-pS1292 LRRK2 Kinase Activity (Relative to wild-type) * * * * E MLi-2 LRRK2 WT C727D L728D L729D L728D+ L729D IP: LRRK2 IB: LRRK2 IP: LRRK2 IB: LRRK2-pS1292 LRRK2 Kinase Activity (Relative to wild-type) * * * * E 250kDa 250kDa 25kDa 25kDa - + - + - + - + - + - + - + - + - + - + LRRK2-pS1292 LRRK2-total Rab29-total (HA) tubulin Rab29-pT71 WT 50kDa KD L728D L729D L760D L761D L762D L789D L790D L791D Region 25kDa 25kDa Rab10-pT73 (endogenous) Rab10-total (endogenous) Region A: L728D, L729D Region B: L760D, L761D, L762D Region C: L789D, L790D, L791D LRRK2: Human Chicken Xenopus Zebrafish Drosophila EC LL LLG EC LL LLG AE LL LLG EC LI QLG QS LL QHG VE LLL NS VE LLL AN VE LLL NN LE LLV SS LD LLL KH IS LLL RR IS LLL KK IS LLL KK VT LIL RR LS RLL AR ANK LL ANK VARP LRRK2 ? Rab29 Rab32 B C LRRK2 Rab29 B A C B C Rab8A-total LRRK2-total LRRK2-pS1292 Rab8A-pT72 LRRK2 WT C727D L728D L729D MLi-2 L728D+ L729D F F F Figure 5. Rab29 increases membrane association of LRRK2-R1441G. 7 The EMBO Journal The EMBO Journal Published online: December 6, 2017 Activation of LRRK2 by Rab29 Elena Purlyte et al The EMBO Journal 250kDa 250kDa 25kDa 25kDa - + - + - + - + - + - + - + - + - + - + LRRK2-pS1292 LRRK2-total Rab29-total (HA) tubulin Rab29-pT71 WT 50kDa KD L728D L729D L760D L761D L762D L789D L790D L791D Region 25kDa 25kDa Rab10-pT73 (endogenous) Rab10-total (endogenous) Region A: L728D, L729D Region B: L760D, L761D, L762D Region C: L789D, L790D, L791D LRRK2: Human Chicken Xenopus Zebrafish Drosophila EC LL LLG EC LL LLG AE LL LLG EC LI QLG QS LL QHG VE LLL NS VE LLL AN VE LLL NN LE LLV SS LD LLL KH IS LLL RR IS LLL KK IS LLL KK VT LIL RR LS RLL AR ANK LL ANK VARP LRRK2 ? Figure 5. Rab29 increases membrane association of LRRK2-R1441G. Rab29 Rab32 A B 250kDa 250kDa 25kDa 25kDa 50kDa - - + + - - + + - - + + - - + + - - + + - - + + LRRK2-pS1292 LRRK2-total Rab29-total (HA) tubulin Rab29-pT71 WT C727D+ R1441G L728D+ R1441G L729D+ R1441G R1441G R1441G + KD 25kDa 25kDa Rab10-pT73 (endogenous) Rab10-total (endogenous) D LRRK2-total LRRK2-pS1292 Rab29-pT71 Rab29-total (HA) FL 970-end Rab10-pT73 (endogenous) Rab10-total (endogenous) FL 970-end FL 970-end FL 970-end FL 970-end FL 970-end 25kDa 25kDa 25kDa 25kDa C Rab8A-total LRRK2-total LRRK2-pS1292 Rab8A-pT72 LRRK2 WT C727D L728D L729D MLi-2 L728D+ L729D F E MLi-2 LRRK2 WT C727D L728D L729D L728D+ L729D LRRK2 Rab29 Rab29 LRRK2 D2017A WT R1441G Y1699C LRRK2 Rab29 B A C IP: LRRK2 IB: LRRK2 IP: LRRK2 IB: LRRK2-pS1292 LRRK2 Kinase Activity (Relative to wild-type) * * * * Figure 6 Ankyrin domain residues permit activation of LRRK2 by Rab29 250kDa 250kDa 25kDa 25kDa - + - + - + - + - + - + - + - + - + - + LRRK2-pS1292 LRRK2-total Rab29-total (HA) tubulin Rab29-pT71 WT 50kDa KD L728D L729D L760D L761D L762D L789D L790D L791D Region 25kDa 25kDa Rab10-pT73 (endogenous) Rab10-total (endogenous) Region A: L728D, L729D Region B: L760D, L761D, L762D Region C: L789D, L790D, L791D LRRK2: Human Chicken Xenopus Zebrafish Drosophila EC LL LLG EC LL LLG AE LL LLG EC LI QLG QS LL QHG VE LLL NS VE LLL AN VE LLL NN LE LLV SS LD LLL KH IS LLL RR IS LLL KK IS LLL KK VT LIL RR LS RLL AR ANK LL ANK VARP LRRK2 ? Figure 6. Ankyrin domain residues permit activation of LRRK2 by Rab29. A HEK293 cells were transfected with the indicated wild-type human full length (FL) or a fragment lacking the N-terminal 969 residues (LRRK2[residues 970-end]) pathogenic LRRK2 variants with either HA-empty vector () or HA-tagged Rab29 (+). 24 h post-transfection, cells were lysed and analyzed by immunoblotting with the indicated antibodies. WT is wild-type and D2017A corresponds to the kinase-inactive LRRK2 mutant. Similar results were obtained in two independent experiments, each performed in duplicate. A HEK293 cells were transfected with the indicated wild-type human full length (FL) or a fragment lacking the N-terminal 969 residues (LRRK2[residues 970-end]) pathogenic LRRK2 variants with either HA-empty vector () or HA-tagged Rab29 (+). 24 h post-transfection, cells were lysed and analyzed by immunoblotting with the indicated antibodies. WT is wild-type and D2017A corresponds to the kinase-inactive LRRK2 mutant. Similar results were obtained in two independent experiments, each performed in duplicate. p p p B Upper panel: Schematic representation of how Rab29 might interact with the ankyrin domain (ANK) of LRRK2 by analogy with how Rab32 binds VARP. Lower panel: Sequence alignments of the three Leu-rich regions in the ankyrin domain of LRRK2 in the indicated species. B Upper panel: Schematic representation of how Rab29 might interact with the ankyrin domain (ANK) of LRRK2 by analogy Sequence alignments of the three Leu-rich regions in the ankyrin domain of LRRK2 in the indicated species. B Upper panel: Schematic representation of how Rab29 might interact with the ankyrin domain (ANK) of LRRK2 by analogy with how Rab32 binds VARP. Lower panel Sequence alignments of the three Leu-rich regions in the ankyrin domain of LRRK2 in the indicated species. C, D As in (A) except that HEK293 cells were transfected with the wild type and indicated LRRK2 ankyrin domain mutations with either HA-empty vector () or HA- tagged Rab29 (+) KD is the Kinase Dead LRRK2[D2017A] mutant Similar results were obtained in two experiments q g g y p C, D As in (A) except that HEK293 cells were transfected with the wild type and indicated LRRK2 ankyrin domain mutations with either HA-empty vector () or HA- tagged Rab29 (+). KD is the Kinase Dead LRRK2[D2017A] mutant. Similar results were obtained in two experiments. q g g y p C, D As in (A) except that HEK293 cells were transfected with the wild type and indicated LRRK2 ankyrin domain mutation tagged Rab29 (+). KD is the Kinase Dead LRRK2[D2017A] mutant. Figure 7. Membrane association of ankyrin domain mutant LRRK2 proteins. mutations (Cys727Asp, Leu728Asp and Leu729Asp) that disrupt Rab29-mediated activation of LRRK2 tested blocked phosphorylation of LRRK2 at Ser910, Ser935, Ser955, and Ser973 (Fig 8A). Rab29 knockout decreases endogenous LRRK2 activity and biomarker site phosphorylation We generated two independent Rab29 knockout A549 cell lines employing a CRISPR/CAS9 approach (Fig 8B). Rab29 knockout mark- edly inhibited LRRK2-mediated phosphorylation of endogenous Rab10 by around twofold, measured with two different phospho-Rab10 monoclonal antibodies (Fig 8B). Consistent with Rab29 controlling biomarker phosphorylation sites, knockout of Rab29 moderately reduced LRRK2 phosphorylation at Ser935 and Ser973 (Fig 8B). We were unable to detect phosphorylation of endogenous LRRK2 at Ser1292 in these cells with available antibodies possibly due to low stoichiometry of phosphorylation of wild-type LRRK2 in these cells. GTP binding to LRRK2 is required for Rab29-mediated activation of LRRK2 To further study the role that LRRK2 GTP binding might play in controlling Rab29-mediated activation and biomarker phosphoryla- tion, we employed the well-characterized LRRK2 T1348N mutation that blocks GTP binding (Ito et al, 2007; Taymans et al, 2011). Introduction of the T1348N mutation into wild-type, R1441G, Y1699C, and G2019S LRRK2 completely inhibited Ser1292 autophos- phorylation, as well as Rab10 phosphorylation that was induced following overexpression of Rab29 (Fig 9A). Consistent with a previous report (Doggett et al, 2011), we also confirm that the T1348N mutation ablates phosphorylation of LRRK2 at the biomarker sites (Fig 9A). We also find that in endogenous homozy- gous LRRK2[T1348N] knock-in MEFs, although the mutation decreases LRRK2 expression, Rab10 phosphorylation and biomarker site phosphorylation are clearly abolished (Fig 9B). Evidence that Rab29 may regulate phosphorylation of LRRK2 biomarker sites by LRRK2 kinase activity, as these residues become dephosphory- lated following administration of LRRK2 inhibitors (Dzamko et al, 2010; Nichols et al, 2010; Doggett et al, 2011). We studied whether ankyrin domain mutations that prevent Rab29 from activating LRRK2 influence phosphorylation of these sites. Strikingly, all Region A LRRK2 possesses a cluster of well-studied, constitutively phosphory- lated residues (Ser910, Ser935, Ser955 and Ser973) that are controlled 8 ª 2017 The Authors The EMBO Journal Elena Purlyte et al Activation of LRRK2 by Rab29 Published online: December 6, 2017 Elena Purlyte et al Activation of LRRK2 by Rab29 Published online: December 6, 2017 The EMBO Journal Figure 7. Membrane association of ankyrin domain mutant LRRK2 proteins. I Quantitation of cells with compact Rab29 morphology; error bars represent SEM of three experiments with > 20 cells per experiment. P = 0.0011 for R1441G alone compared with Rab29 alone; P = 0.0049 for R1441G-L728/L729D. Differences between R1441G-LRRK2-L728/729D + Rab29 and Rab29 alone were not significant (P > 0.5). H Light microscopy of HeLa cells transfected as in Fig 3 with LRRK2-R11441G or LRRK2-R1441G+L728D/L729D (red) and Rab29 (green). LRRK2-R1441G (red) co- localization on distinct Rab29 (green)-positive puncta representing dispersed Golgi membranes requires ankyrin domain sequences. Scale bar, 2 lm. I Quantitation of cells with compact Rab29 morphology; error bars represent SEM of three experiments with > 20 cells per experiment. P = 0.0011 for R1441G alone compared with Rab29 alone; P = 0.0049 for R1441G-L728/L729D. Differences between R1441G-LRRK2-L728/729D + Rab29 and Rab29 alone were not significant (P > 0.5). J Quantitation of the relative stability of LRRK2 R1441G protein (blue) in comparison with its ankyrin domain mutated form (red) in HEK293T cells transfected with LRRK2 R1441G or R1441G-LRRK2-L728/729D. After 24 h, cells were treated with 50 lg/ml cycloheximide for 0, 3, or 6 h. Error bars represent SEM from three combined experiments carried out in duplicate. Source data are available online for this figure. microtubule association is enhanced upon LRRK2 inhibitor addi- tion, a condition that is documented to enhance LRRK2 turnover (Blanca Ramirez et al, 2017). Using low LRRK2 expression levels and gentle release of cytosol with liquid nitrogen treatment, we have never seen microtubule association for LRRK2 G2019S or R1441G proteins. However, certain mutants appear to form what appear to be non-specific aggregates, including T1348N (Fig 9C). We believe these structures represent concentration-dependent “aggresomes” that are microtubule-associated protein aggregates— a finding that would be consistent with the previous microtubule association seen by others. Consistent with the inability of Rab29 to activate LRRK2 T1348N, co-expression with Rab29 did not increase the amount of membrane-associated LRRK2 T1348N, as determined by cell fractionation (Fig 9D). Note that the centrifuga- tion protocol employed may pellet some LRRK2 T1348N protein aggregates that may also be present in the “membrane” fraction; nevertheless, the amount present in the fraction (16% of total LRRK2 protein) was unchanged upon Rab29 co-expression. Even though this mutant forms aggresomes, the 84% of the molecules that remain in the cytosol do not appear to be recruited to the Golgi by Rab29 GTPase (Fig 9D). ª 2017 The Authors The EMBO Journal Figure 7. Membrane association of ankyrin domain mutant LRRK2 proteins. 1G LRRK2 in membrane (top two rows) or cytosol (bottom two rows) fractions after 48-h expression in HEK293T cells. TfR (transferrin oading controls are included. A Immunoblots of R1441G LRRK2 in membrane (top two A Immunoblots of R1441G LRRK2 in membrane (top two rows) or cytosol (bottom two rows) fractions after 48-h expression in H receptor) and tubulin loading controls are included. B Relative membrane association of the constructs analyzed in (A). Error bars represent SEM from duplicate samples of a representative experiment. P = 0.001 B Relative membrane association of the constructs analyzed in (A). Error bars represent SEM from duplicate samples of a rep using Student’s unpaired t-test. Differences between WT and single mutations in ankyrin domain were not significant. C, D Membrane and cytosol fractionation of extracts from cells expressing G2019S LRRK2 or its derived ankyrin domain mutant B Relative membrane association of the constructs analyzed in (A). Error bars represent SEM from duplicate samples of a representative experiment. P = 0.001 i St d t’ i d t t t Diff b t WT d i l t ti i k i d i t i ifi t B Relative membrane association of the constructs analyzed in (A). Error bars represent SEM from duplicate samples of a representative experiment. P = 0.0 using Student’s unpaired t-test. Differences between WT and single mutations in ankyrin domain were not significant. C D Membrane and cytosol fractionation of extracts from cells expressing G2019S LRRK2 or its derived ankyrin domain mutant forms Rab29 as in Fig 5 E, F Quantitation of the relative membrane association of each construct, normalized for total LRRK2 expression. Error bars indicate SEM from duplicate determinations. ns = not significant, P > 0.05; (E) P = 0.003; (F) P = 0.0086 by Student’s unpaired two-tailed t-test. G Quantitation of the fraction of the indicated LRRK2 proteins that co-localize with Rab29. Co-localization was measured using Mander’s coefficient after automatic thresholding in FIJI. Error bars represent SEM from two experiments (15 cells); *P = 0.0002 by Student’s unpaired t-test. Scale bar, 2 lm. H Light microscopy of HeLa cells transfected as in Fig 3 with LRRK2-R11441G or LRRK2-R1441G+L728D/L729D (red) and Rab29 (green). LRRK2-R1441G (red) co- localization on distinct Rab29 (green)-positive puncta representing dispersed Golgi membranes requires ankyrin domain sequences. Scale bar, 2 lm. Discussion However, in cel R1441G R1441G-L728D R1441G-L729D R1441G- L728/729D 0 20 40 60 Membrane Associat 250 - 25 - 100 - 250 - Rab29: - - + + - - + + - pS1292 - tubulin - - MycRab29 - LRRK2 - - LAMP2 G2019S G2019S L728/729D Rab29: - - + + - - + + G2019S G2019S L728/729D membrane cytosol - tubulin - LRRK2 R1441G R1441G - TfR 250 - 50 - 100 - cytosol membra C D G2019S G2019S+Rab29 G2019S-L728/729D G2019S-L728/729D +Rab29 0.0 0.5 1.0 1.5 2.0 Membrane LRRK2 (relative) G2019S G2019S+Rab29 G2019S-L728/729D G2019S-L728/729D +Rab29 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Membrane pS1292 LRRK2 (relative) F E R1441G R1441G + L728D/L729D 0 0.2 0.4 0.6 Fraction of LRRK2 colocalizing with Rab29 G H 0 2 4 6 0 0.5 1.0 LRRK2 (normalized) cycloheximide chase (hr) R1441G + L728D/L729D LRRK2 R1441G ns ns * LRRK2 R1441G R1441G + L728D/L729D Rab29 Rab29 Rab29 R1441G 0 20 40 60 80 100 Cells with compact Rab29 staining (percent) R1441G- L728/729D I J e 7. Discussion We have shown here that in addition to comprising an LRRK2 substrate, Rab29 operates as a master upstream regulator of LRRK2, controlling Golgi localization, kinase activity, and potentially N- terminal biomarker phosphorylation (Fig 10). We also demonstrate that pathogenic mutations such as R1441G/C or Y1699C that enhance GTP binding to the ROC domain are recruited to the Golgi apparatus and activated more efficiently than wild-type LRRK2. These observations are consistent with previous studies that have suggested that LRRK2 and Rab29 operate as part of a common signaling pathway (Dodson et al, 2012; MacLeod et al, 2013; Pihlstrom et al, 2015; Zhang et al, 2015; Kuwahara et al, 2016). Our work also reveals an intriguing interplay between Rab proteins and the LRRK2 kinase that also possesses a Rab-like GTPase domain. Discussion Our data are consistent with a model in which GTP binding to Hilfiker and colleagues have recently analyzed the localization of a large set of LRRK2 mutant proteins and found that in about 20% of cells expressing certain mutant forms (but not wild type or G2019S), LRRK2 appears to associate with microtubules; 9 The EMBO Journal The EMBO Journal Published online: December 6, 2017 ublished online: December 6, 2017 Activation of LRRK2 by Rab29 Elena Purlyte et al The EMBO Journal R1441G R1441G-L728D R1441G-L729D R1441G- L728/729D 0 20 40 60 80 100 Membrane Associated LRRK2 250 - 25 - 100 - 250 - Rab29: - - + + - - + + - pS1292 - tubulin - - MycRab29 - LRRK2 - - LAMP2 G2019S G2019S L728/729D Rab29: - - + + - - + + G2019S G2019S L728/729D membrane cytosol WT L728D L729D L728/L729D - tubulin - LRRK2 R1441G - LRRK2 R1441G - TfR 250 - 50 - 100 - 250 - cytosol membrane A B C D G2019S G2019S+Rab29 G2019S-L728/729D G2019S-L728/729D +Rab29 0.0 0.5 1.0 1.5 2.0 Membrane LRRK2 (relative) G2019S G2019S+Rab29 G2019S-L728/729D G2019S-L728/729D +Rab29 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Membrane pS1292 LRRK2 (relative) F E R1441G R1441G + L728D/L729D 0 0.2 0.4 0.6 Fraction of LRRK2 colocalizing with Rab29 G H 0.5 1.0 RK2 (normalized) R1441G + L728D/L729 LRRK2 R1441G ns ns * LRRK2 R1441G R1441G + L728D/L729D Rab29 Rab29 20 40 60 80 100 with compact Rab29 taining (percent) I J WT L728D L729D L728/L729D - tubulin - LRRK2 R1441G - LRRK2 R1441G - TfR 250 - 50 - 100 - 250 - cytosol membrane A B R1441G R1441G-L728D R1441G-L729D R1441G- L728/729D 0 20 40 60 80 100 Membrane Associated LRRK2 B B A K2’s ROC domain promotes Rab29 activation of LRRK2. Once ated, LRRK2 kinase phosphorylates and influences the function series of other downstream Rab proteins (Steger et al, 2016, ). It was previously unclear how the LRRK2[R1441G/C] and LRR [Y1699C] variants activated LRRK2, as these mutants possess simi kinase activity as wild-type LRRK2 in in vitro experiments (Jale et al, 2007; Nichols et al, 2010; Steger et al, 2016). Discussion R1441G R1441G-L728D R1441G-L729D R1441G- L728/729D 0 20 40 Membrane Asso 250 - 25 - 100 - 250 - Rab29: - - + + - - + + - pS1292 - tubulin - - MycRab29 - LRRK2 - - LAMP2 G2019S G2019S L728/729D Rab29: - - + + - - + + G2019S G2019S L728/729D membrane cytosol - tubulin - LRRK2 R1441G - TfR 250 - 50 - 100 - cytosol mem C D R1 R1 250 - 25 - 100 - 250 - Rab29: - - + + - - + + - pS1292 - tubulin - - MycRab29 - LRRK2 - - LAMP2 G2019S G2019S L728/729D Rab29: - - + + - - + + G2019S G2019S L728/729D membrane cytosol C D 250 - 25 - 100 - 250 - Rab29: - - + + - - + + - pS - Myc - LR - LAM G2019S G2019S L728/729D membrane C 1292 - tubulin - Rab29 RK2 - P2 Rab29: - - + + - - + + G2019S G2019S L728/729D cytosol D D C G2019S G2019S+Rab29 G2019S-L728/729D G2019S-L728/729D +Rab29 0.0 0.5 1.0 1.5 2.0 Membrane LRRK2 (relative) G2019S G2019S+Rab29 G2019S-L728/729D G2019S-L728/729D +Rab29 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Membrane pS1292 LRRK2 (relative) F E R1441G R1441G + L728D/L729D 0 0.2 0.4 0.6 Fraction of LRRK2 colocalizing with Rab29 G ns ns * 9D R1441G R1441G + L728D/L729D 0 0.2 0.4 0.6 Fraction of LRRK2 colocalizing with Rab29 G * G F E LRRK2’s ROC domain promotes Rab29 activation of LRRK2. Once activated, LRRK2 kinase phosphorylates and influences the function of a series of other downstream Rab proteins (Steger et al, 2016, 2017). It was previously unclear how the LRRK2[R1441G/C] and LRRK2 [Y1699C] variants activated LRRK2, as these mutants possess similar kinase activity as wild-type LRRK2 in in vitro experiments (Jaleel et al, 2007; Nichols et al, 2010; Steger et al, 2016). However, in cells, G G H 0 2 4 6 0 0.5 1.0 LRRK2 (normalized) cycloheximide chase (hr) R1441G + L728D/L729D LRRK2 R1441G LRRK2 R1441G R1441G + L728D/L729D Rab29 Rab29 Rab29 R1441G 0 20 40 60 80 100 Cells with compact Rab29 staining (percent) R1441G- L728/729D I J Figure 7. Discussion H 0 2 4 6 0 0.5 1.0 LRRK2 (normalized) cycloheximide chase (hr) R1441G + L728D/L729D LRRK2 R1441G LRRK2 R1441G R1441G + L728D/L729D Rab29 Rab29 Rab29 R1441G 0 20 40 60 80 100 Cells with compact Rab29 staining (percent) R1441G- L728/729D I J Rab29 R1441G 0 20 40 60 80 100 Cells with compact Rab29 staining (percent) R1441G- L728/729D I 0 2 4 6 0 0.5 1.0 LRRK2 (normalized) cycloheximide chase (hr) R1441G + L728D/L729D LRRK2 R1441G J H LRRK2 R1441G R1441G + L728D/L729D Rab29 Rab29 J H Figure 7. Figure 7. LRRK2’s ROC domain promotes Rab29 activation of LRRK2. Once activated, LRRK2 kinase phosphorylates and influences the function of a series of other downstream Rab proteins (Steger et al, 2016, 2017). It was previously unclear how the LRRK2[R1441G/C] and LRRK2 [Y1699C] variants activated LRRK2, as these mutants possess similar kinase activity as wild-type LRRK2 in in vitro experiments (Jaleel et al, 2007; Nichols et al, 2010; Steger et al, 2016). However, in cells, 10 The EMBO Journal ª 2017 The Authors Published online: December 6, 2017 Published online: December 6, 2017 The EMBO Journal al Activation of LRRK2 by Rab29 Elena Purlyte et al Activation of LRRK2 by Rab29 A B 250kDa 25kDa 25kDa 25kDa 25kDa 250kDa 250kDa 37kDa Rab29 KO-1 WT Rab29 KO-2 MLi-2 - + - + - + - + - - + Rab10-pT73 (108-10) LRRK2-pS935 LRRK2-pS973 LRRK2-total Rab29-total Rab10-total GAPDH Rab10-pT73 (83-4) + Rab29: WT C727D L728D L729D - - + + - - + + - - + + - - + + LRRK2: LRRK2-pS1292 LRRK2-total Rab29 (HA) tubulin Rab29-pT71 LRRK2-pS973 LRRK2-pS935 LRRK2-pS910 LRRK2-pS955 Rab10-pT73 (108-10) (endogenous) Rab10-total (endogenous) 250kDa 250kDa 250kDa 250kDa 250kDa 25kDa 25kDa 50kDa 250kDa 25kDa 25kDa WT DMSO WT MLI-2 Rab29-KO clone 1 DMSO Rab29-KO clone 1 MLI-2 Rab29-KO clone 2 DMSO Rab29-KO clone 2 MLI-2 0.0 0.5 1.0 1.5 pT73-Rab10/totla Rab10 +/-SEM * * * * * pT73-Rab10 108-10 pS935-LRRK2 WT DMSO WT MLI-2 Rab29-KO clone 1 DMSO Rab29-KO clone 1 MLI-2 Rab29-KO clone 2 DMSO Rab29-KO clone 2 MLI-2 0.0 0.5 1.0 1.5 pS935-LRRK2/total LRRK2 +/-SEM * * *** * ns pS973-LRRK2 WT DMSO WT MLI-2 Rab29-KO clone 1 DMSO Rab29-KO clone 1 MLI-2 Rab29-KO clone 2 DMSO Rab29-KO clone 2 MLI-2 0.0 0.5 1.0 1.5 ns ns pS973-LRRK2/total LRRK2 +/- SEM Figure 8. Discussion Our data suggest that the LRRK2[R1441G/C] and LRRK2[Y1699C] mutations are activated in vivo, due to their increased ability to bind GTP, thereby promoting Rab29-mediated recruitment and activation of LRRK2 on the Golgi apparatus. Consistent with this model, introduction of the T1348N mutation that blocks GTP binding prevents Rab29-mediated recruitment of LRRK2 to the Golgi, and concomitant LRRK2 activation (Fig 9). In future work, it will be important to better define the mechanism by which the R1441G/C and Y1699C mutations promote GTP binding and how this influences Rab29 binding and LRRK2 kinase activation. A 3D model of the structure of dimeric, full-length LRRK2 has been generated based on homology models, chemical cross-linking, negative-stain EM, and small-angle X-ray scattering (Guaitoli et al, 2016). Interestingly, this model indicates that the ankyrin domain lies in close proximity to the kinase domain (Guaitoli et al, 2016). Such an arrangement could explain the potent activation of the kinase upon Rab29 binding to the active site-adjacent ankyrin domain. Higher resolution structural analysis and further mechanis- tic studies are required to more precisely define how Rab29 binding to the ankyrin domain is coupled to LRRK2 kinase activity. This might also help design improved ankyrin mutants of LRRK2 that are unable to bind to Rab29 that might not destabilize the protein to better study the role that this biological interaction plays. Thus far, we have not been able to reconstitute activation of LRRK2 by Rab29 in an in vitro system. These experiments have been hampered by challenges in expressing fully active and mono-dispersed, the LRRK2[R1441G/C] and LRRK2[Y1699C] mutations are clearly more active and phosphorylate Rab proteins to a greater extent that the LRRK2[G2019S] variant (Ito et al, 2016; Steger et al, 2016). Consistent with this, the average age of onset of Parkinson’s is report- edly earlier with patients with R1441G/C mutations compared to those carrying the G2019S mutation (Gonzalez-Fernandez et al, 2007; Healy et al, 2008). Our data suggest that the LRRK2[R1441G/C] and LRRK2[Y1699C] mutations are activated in vivo, due to their increased ability to bind GTP, thereby promoting Rab29-mediated recruitment and activation of LRRK2 on the Golgi apparatus. Consistent with this model, introduction of the T1348N mutation that blocks GTP binding prevents Rab29-mediated recruitment of LRRK2 to the Golgi, and concomitant LRRK2 activation (Fig 9). Discussion 24 h post-transfection, cells were lysed and analyzed by immunoblotting with the indicated antibodies. WT is wild type. Similar results were obtained in two experiments A HEK293 cells were transfected with the indicated wild-type and ankyrin domain mutant forms of LRRK2 with either HA-empty vector () or HA-tagged Rab29 (+). 24 h post-transfection, cells were lysed and analyzed by immunoblotting with the indicated antibodies. WT is wild type. Similar results were obtained in two experiments. B Wild-type A549 cells and two independent clones of CRISPR/CAS9 Rab29 knockout (KO-1 and KO-2) were lysed and analyzed by immunoblotting with the indicated antibodies. Two separate phospho-Rab10 rabbit monoclonal antibodies were employed (108–10 and 83–4). Blots were signals quantified by LiCor and presented as average SEM. Similar results were obtained in three experiments. There was a statistically significant difference between groups for pT73-Rab10/total Rab10 signal (P < 0.0001, one-way ANOVA, F(5, 12) = 41.56). P < 0.001 by one-way ANOVA with Dunnett’s multiple comparison with mean difference 95% confidence intervals of groups compared to WT DMSO control: WT MLI-2 0.6725–1.129; Rab29-KO CL1 DMSO 0.3348–0.7916; Rab29-KO CL1 MLI-2 0.6953–1.152; Rab29-KO CL2 DMSO 0.3103–0.7671; Rab29-KO CL2 MLI-2 0.6754–1.132. There was a significant difference between groups for pS935-LRRK2/total LRRK2 signal (P < 0.0001, one-way ANOVA, F(5, 12) = 18.00). nsP > 0.05; P < 0.05, *P < 0.001 by one-way ANOVA with Dunnett’s multiple comparison test with mean difference 95% confidence intervals of groups compared to WT DMSO control: WT MLI-2 0.5319–1.271; Rab29-KO CL1 DMSO 0.01298 to 0.7261; Rab29-KO CL1 MLI-2 0.5353–1.274; Rab29-KO CL2 DMSO 0.01820–0.7573; Rab29-KO CL2 MLI-2 0.5235–1.263. There was a significant difference between groups for pS973-LRRK2/total LRRK2 signal as well (P = 0.003, one-way ANOVA, F(5, 12) = 6.903). nsP > 0.05, P < 0.01 by one-way ANOVA with Dunnett’s multiple comparison test with mean difference 95% confidence intervals of groups compared to WT DMSO control: WT MLI-2 0.1955–1.300; Rab29-KO CL1 DMSO 0.1599 to 0.9449; Rab29-KO CL1 MLI-2 0.2619–1.367; Rab29-KO CL2 DMSO 0.4202 to 0.6846; Rab29-KO CL2 MLI-2 0.2159–1.321. the LRRK2[R1441G/C] and LRRK2[Y1699C] mutations are clearly more active and phosphorylate Rab proteins to a greater extent that the LRRK2[G2019S] variant (Ito et al, 2016; Steger et al, 2016). Consistent with this, the average age of onset of Parkinson’s is report- edly earlier with patients with R1441G/C mutations compared to those carrying the G2019S mutation (Gonzalez-Fernandez et al, 2007; Healy et al, 2008). Discussion Endogenous Rab29 activates LRRK2 and is required for biomarker site phosphorylation. A HEK293 cells were transfected with the indicated wild-type and ankyrin domain mutant forms of LRRK2 with either HA-empty vector () or HA-tagged Rab29 (+). B 250kDa 25kDa 25kDa 25kDa 25kDa 250kDa 250kDa 37kDa Rab29 KO-1 WT Rab29 KO-2 MLi-2 - + - + - + - + - - + Rab10-pT73 (108-10) LRRK2-pS935 LRRK2-pS973 LRRK2-total Rab29-total Rab10-total GAPDH Rab10-pT73 (83-4) + pS935-LRRK2 WT DMSO WT MLI-2 Rab29-KO clone 1 DMSO Rab29-KO clone 1 MLI-2 Rab29-KO clone 2 DMSO Rab29-KO clone 2 MLI-2 0.0 0.5 1.0 1.5 pS935-LRRK2/total LRRK2 +/-SEM * * *** * ns A B Rab29: WT C727D L728D L729D - - + + - - + + - - + + - - + + LRRK2: LRRK2-pS1292 LRRK2-total Rab29 (HA) tubulin Rab29-pT71 LRRK2-pS973 LRRK2-pS935 LRRK2-pS910 LRRK2-pS955 Rab10-pT73 (108-10) (endogenous) Rab10-total (endogenous) 250kDa 250kDa 250kDa 250kDa 250kDa 25kDa 25kDa 50kDa 250kDa 25kDa 25kDa B A pS973-LRRK2 WT DMSO WT MLI-2 Rab29-KO clone 1 DMSO Rab29-KO clone 1 MLI-2 Rab29-KO clone 2 DMSO Rab29-KO clone 2 MLI-2 0.0 0.5 1.0 1.5 ns ns pS973-LRRK2/total LRRK2 +/- SEM WT DMSO WT MLI-2 Rab29-KO clone 1 DMSO Rab29-KO clone 1 MLI-2 Rab29-KO clone 2 DMSO Rab29-KO clone 2 MLI-2 0.0 0.5 1.0 1.5 pT73-Rab10/totla Rab10 +/-SEM * * * * * pT73-Rab10 108-10 pS973-LRRK2 WT DMSO WT MLI-2 Rab29-KO clone 1 DMSO Rab29-KO clone 1 MLI-2 Rab29-KO clone 2 DMSO Rab29-KO clone 2 MLI-2 0.0 0.5 1.0 1.5 ns ns pS973-LRRK2/total LRRK2 +/- SEM WT DMSO WT MLI-2 Rab29-KO clone 1 DMSO Rab29-KO clone 1 MLI-2 Rab29-KO clone 2 DMSO Rab29-KO clone 2 MLI-2 0.0 0.5 1.0 1.5 pT73-Rab10/totla Rab10 +/-SEM * * * * * pT73-Rab10 108-10 gure 8. Endogenous Rab29 activates LRRK2 and is required for biomarker site phosphorylation. Figure 8. Endogenous Rab29 activates LRRK2 and is required for biomarker site phosphorylation. A HEK293 cells were transfected with the indicated wild-type and ankyrin domain mutant forms of LRRK2 with either HA-empty vector () or HA-tagged Rab29 (+). 24 h post-transfection, cells were lysed and analyzed by immunoblotting with the indicated antibodies. WT is wild type. Similar results were obtained in two i A HEK293 cells were transfected with the indicated wild-type and ankyrin domain mutant forms of LRRK2 with either HA-empty vector () or HA-tagged Rab29 (+). Discussion Journal Activation of LRRK2 by Rab29 Elena Pur LRRK2-pS1292 LRRK2-pS955 LRRK2-pS973 LRRK2-pS935 LRRK2-pS910 LRRK2-total Rab10-pT73 (endogenous) Rab10 (endogenous) Rab29-pT71 Rab29 (HA) tubulin WT R1441G Y1699C G2019S 250kDa 250kDa 250kDa 250kDa 250kDa 250kDa 25kDa 25kDa 25kDa 25kDa 50kDa A HEK293 cells Rab29: T1348N: + - + - + - + - + - + - + - + - - - - - - - - - + + + + + + + + LRRK2-pS1292 LRRK2-pS955 LRRK2-pS973 LRRK2-pS935 LRRK2-pS910 LRRK2-total Rab10-pT73 (endogenous) Rab10 (endogenous) Rab29-pT71 Rab29 (HA) tubulin WT R1441G Y1699C G2019S 250kDa 250kDa 250kDa 250kDa 250kDa 250kDa 25kDa 25kDa 25kDa 25kDa 50kDa A HEK293 cells Rab29: T1348N: + - + - + - + - + - + - + - + - - - - - - - - - + + + + + + + + WT/WT WT/TN TN/TN MLI-2 + - - + + + - - + + - - Rab10-pT73 Rab10 LRRK2-pS955 LRRK2-pS935 Tubulin LRRK2 Nterm 250kDa 250kDa 250kDa 25kDa 25kDa 50kDa B TN=T1348N Mouse embryonic fibroblasts A B HEK293 cells Rab29 tubulin LRRK2 250kDa 50kDa Rab29 (Myc) 25kDa D Membrane Cytosol - + - + T1348N-LRRK2 / DAPI C HeLa cells D C HeLa cells HeLa cells on of the T1348N mutation that ablates GTP binding to LRRK2 abolishes Rab10 and biomarker phosphorylation. nding to LRRK2 abolishes Rab10 and biomarker phosphorylatio Figure 9. Knock-in mutation of the T1348N mutation that ablates GTP binding to LRRK2 abolishes Rab10 and biomarker phosphorylation. A HEK293 cells were transfected with the indicated wild-type and mutant forms of LRRK2 with either HA-empty vector () or HA-tagged Rab29 (+). 24 h post- transfection, cells were lysed and analyzed by immunoblotting with the indicated antibodies WT is wild type. Similar results were obtained in two experiments. B Wild-type LRRK2, heterozygous LRRK2[T1348N/+], and homozygous LRRK2[T1348N/T1348N] knock-in MEFs derived from littermate embryos were lysed immunoblotted with the indicated antibodies. Similar results were obtained in two experiments. WT is Wild type and TN corresponds to T1348N. C HeLa cells were transfected with FLAG-T1348N-LRRK2. After 48 h, cells were permeabilized by liquid nitrogen freeze–thaw to deplete cytosol, then fixed, and stained with rabbit anti-LRRK2 antibody. Nuclear DAPI stain (blue); LRRK2 (red). Scale bar, 10 lm. Dotted line represents cell outlines. D HEK293T cells were transfected with FLAG-T1348N-LRRK2 and 24 h later with Myc-Rab29. Discussion In future work, it will be important to better define the mechanism by which the R1441G/C and Y1699C mutations promote GTP binding and how this influences Rab29 binding and LRRK2 kinase activation. 11 The EMBO Journal ª 2017 The Authors The EMBO Journal Published online: December 6, 2017 Activation of LRRK2 by Rab29 Elena Purlyte et al The EMBO Journal T1348N-LRRK2 / DAPI WT/WT WT/TN TN/TN MLI-2 + - - + + + - - + + - - Rab10-pT73 Rab10 LRRK2-pS955 LRRK2-pS935 Tubulin LRRK2 Nterm 250kDa 250kDa 250kDa 25kDa 25kDa 50kDa B LRRK2-pS1292 LRRK2-pS955 LRRK2-pS973 LRRK2-pS935 LRRK2-pS910 LRRK2-total Rab10-pT73 (endogenous) Rab10 (endogenous) Rab29-pT71 Rab29 (HA) tubulin WT R1441G Y1699C G2019S 250kDa 250kDa 250kDa 250kDa 250kDa 250kDa 25kDa 25kDa 25kDa 25kDa 50kDa A C Rab29 tubulin LRRK2 250kDa 50kDa Rab29 (Myc) 25kDa D Membrane Cytosol TN=T1348N Mouse embryonic fibroblasts HEK293 cells HeLa cells Rab29: T1348N: + - + - + - + - + - + - + - + - - - - - - - - - + + + + + + + + - + - + Knock-in mutation of the T1348N mutation that ablates GTP binding to LRRK2 abolishes Rab10 and biomarker phosphorylation. cells were transfected with the indicated wild-type and mutant forms of LRRK2 with either HA-empty vector () or HA-tagged Rab29 (+). 24 h post- ction, cells were lysed and analyzed by immunoblotting with the indicated antibodies WT is wild type. Similar results were obtained in two experiment pe LRRK2, heterozygous LRRK2[T1348N/+], and homozygous LRRK2[T1348N/T1348N] knock-in MEFs derived from littermate embryos were lysed oblotted with the indicated antibodies. Similar results were obtained in two experiments. WT is Wild type and TN corresponds to T1348N. ells were transfected with FLAG-T1348N-LRRK2. After 48 h, cells were permeabilized by liquid nitrogen freeze–thaw to deplete cytosol, then fixed, and s bbit anti-LRRK2 antibody. Nuclear DAPI stain (blue); LRRK2 (red). Scale bar, 10 lm. Dotted line represents cell outlines. T cells were transfected with FLAG-T1348N-LRRK2 and 24 h later with Myc-Rab29. After 24 h of Rab29 expression, cytosol and membrane fractions we d. Immunoblot of membrane protein (50 lg) and cytosolic protein (40% of equivalent volume) Rab29 as indicated. Numbers at left indicate mobility proteins in kDa; proteins were detected with rabbit anti-LRRK2, mouse anti-tubulin, and mouse anti-Myc antibodies. a are available online for this figure. Discussion There has been much interest in how the phosphorylation of the N-terminal LRRK2 biomarker (Ser910, Ser935, Ser955 and Ser973) sites are controlled. To our knowledge, every LRRK2 kinase inhi- bitor tested (> 100 compounds) induces efficient dephosphorylation of these biomarker sites in around 1–2 h, suggesting that LRRK2 is somehow controlling the phosphorylation of these sites either directly through autophosphorylation or indirectly via another kinase or phosphatase (Dzamko et al, 2010; Doggett et al, 2011). Our finding that all Rab29 binding-deficient ankyrin domain LRRK2 mutants we have tested are not phosphorylated at the biomarker sites (Fig 8A), and that knockout of endogenous Rab29 in A549 cells moderately reduced phosphorylation of these sites (Fig 8B), strongly suggests that Rab29 recruitment to the Golgi is required for the phosphorylation of the biomarker sites. This is further supported by the finding that GTP binding-deficient LRRK2[T1348N] mutants that cannot be activated by Rab29, are also not phosphorylated at the biomarker sites (Fig 9A and B). This is also consistent with previous studies showing that T1348N inhibited phosphorylation of Ser935, Ser955, and Ser973 (Doggett et al, 2011). Overall, the data suggest that highly active LRRK2 associated with the Rab29 at the Golgi may become capable of autophosphorylation at the biomarker sites. However, our data do not exclude the possibility that another Golgi- resident, LRRK2-controlled kinase or phosphatase regulates phos- phorylation of these sites. It will be vital to reconstitute activation of LRRK2 by Rab29 in vitro and establish whether or not this is accom- panied by ability of LRRK2 to autophosphorylate at the biomarker sites. recombinant Rab29. It is also possible that membrane association of Rab29 and/or other factors located on the Golgi are required for Rab29-mediated LRRK2 activation. The present study focused on Rab29 due to the previous genetic links between Rab29, LRRK2, and Parkinson’s disease. However, it is possible that other Rab proteins regulate LRRK2 localization and activity in a similar manner by binding to the ankyrin domain. Indeed, when a panel of 11 Rab proteins was tested, we observed a moderate activation of wild-type LRRK2 by Rab12 (Fig EV2A) and LRRK2[R1441G] by Rab8A and Rab38 (Fig EV2B). Recruit- ment of LRRK2 to membranes by different Rab proteins could comprise a general mechanism to activate LRRK2 at different loca- tions within the cell. Discussion Recruitment of LRRK2 to Rab29 at the Golgi also promotes phosphorylation of a cluster of highly studied biomarker phosphorylation sites (Ser910, Ser935, Ser955, and Ser973). More work is needed to define whether these biomarker residues are phosphorylated by autophosphorylation or by a Golgi-resident upstream kinase. Finally, our data suggest that LRRK2-mediated phosphorylation of Rab29 might act as a negative feedback loop and prevent Rab29 from activating LRRK2 Figure 10. Model of how Rab29 activates and recruits LRRK2 to the trans-Golgi network greatly stimulating its kinase activity. necessity of Rab29 recruitment and activation for Golgi disruption (Fig 7). It will be important to determine what substrate(s) LRRK2 phosphorylates on the Golgi to trigger its disruption of the structure and whether these are additional Rab proteins. It will also be inter- esting to evaluate the consequences of LRRK2-mediated Golgi frag- mentation in relation to Parkinson’s disease. Further work is also needed to define the consequences of Rab29 phosphorylation by LRRK2. Substituting the two LRRK2 phosphorylation sites with Glu to mimic phosphorylation appears to suppress activation of LRRK2 [R1441G] (Fig 1B), potentially indicating that this could serve as a mechanism to release activated LRRK2 from the Golgi once it becomes activated. Release of Rab29-activated LRRK2 could explain its co-localization on post-Golgi structures with Rab8 in the perinu- clear region and Rab10 near the periphery, two compartments that presumably lack Rab29 protein. In this regard, it is important to note that loss of Rab29 decreased Rab10 phosphorylation significantly, linking Rab29 activation with Rab10 substrate phosphorylation. Figure 10. Model of how Rab29 activates and recruits LRRK2 to the trans-Golgi network greatly stimulating its kinase activity. Our data suggest that Rab29 binds to the LRRK2 ankyrin domain and that GTP binding to the ROC domain of LRRK2 promotes Rab29-mediated activation. This explains why pathogenic LRRK2 R1441G/C and Y1699C mutants that promote GTP binding are more readily recruited to the Golgi and activated by Rab29 than wild-type LRRK2. Recruitment of LRRK2 to Rab29 at the Golgi also promotes phosphorylation of a cluster of highly studied biomarker phosphorylation sites (Ser910, Ser935, Ser955, and Ser973). More work is needed to define whether these biomarker residues are phosphorylated by autophosphorylation or by a Golgi-resident upstream kinase. Finally, our data suggest that LRRK2-mediated phosphorylation of Rab29 might act as a negative feedback loop and prevent Rab29 from activating LRRK2. ª 2017 The Authors Discussion After 24 h of Rab29 expression, cytosol and membrane fractions were prepared. Immunoblot of membrane protein (50 lg) and cytosolic protein (40% of equivalent volume) Rab29 as indicated. Numbers at left indicate mobility of marker proteins in kDa; proteins were detected with rabbit anti-LRRK2, mouse anti-tubulin, and mouse anti-Myc antibodies. Source data are available online for this figure. Figure 9. Knock-in mutation of the T1348N mutation that ablates GTP binding to LRRK2 abolishes Rab10 and biomarker phosphorylation. A HEK293 cells were transfected with the indicated wild-type and mutant forms of LRRK2 with either HA-empty vector () or HA-tagged Rab29 (+). 24 h post- transfection, cells were lysed and analyzed by immunoblotting with the indicated antibodies WT is wild type. Similar results were obtained in two experiments. B Wild-type LRRK2, heterozygous LRRK2[T1348N/+], and homozygous LRRK2[T1348N/T1348N] knock-in MEFs derived from littermate embryos were lysed immunoblotted with the indicated antibodies. Similar results were obtained in two experiments. WT is Wild type and TN corresponds to T1348N. C HeLa cells were transfected with FLAG-T1348N-LRRK2. After 48 h, cells were permeabilized by liquid nitrogen freeze–thaw to deplete cytosol, then fixed, and stained with rabbit anti-LRRK2 antibody. Nuclear DAPI stain (blue); LRRK2 (red). Scale bar, 10 lm. Dotted line represents cell outlines. D HEK293T cells were transfected with FLAG-T1348N-LRRK2 and 24 h later with Myc-Rab29. After 24 h of Rab29 expression, cytosol and membrane fractions were prepared. Immunoblot of membrane protein (50 lg) and cytosolic protein (40% of equivalent volume) Rab29 as indicated. Numbers at left indicate mobility of marker proteins in kDa; proteins were detected with rabbit anti-LRRK2, mouse anti-tubulin, and mouse anti-Myc antibodies. Source data are available online for this figure. Source data are available online for this figure. 12 The EMBO Journal ª 2017 The Authors Published online: December 6, 2017 The EMBO Journal Elena Purlyte et al Activation of LRRK2 by Rab29 Figure 10. Model of how Rab29 activates and recruits LRRK2 to the trans-Golgi network greatly stimulating its kinase activity. Our data suggest that Rab29 binds to the LRRK2 ankyrin domain and that GTP binding to the ROC domain of LRRK2 promotes Rab29-mediated activation. This explains why pathogenic LRRK2 R1441G/C and Y1699C mutants that promote GTP binding are more readily recruited to the Golgi and activated by Rab29 than wild-type LRRK2. Discussion The Rab29-related proteins, Rab32 and Rab38, are obvious candidates for potential interactors, as reported in a recent study (Waschbusch et al, 2014) and indeed Rab38 can modestly activate LRRK2[R1441G] (Fig EV2A). Consis- tent with other Rab proteins potentially controlling LRRK2 activity, we find that in A549 cells, knockout of Rab29 significantly reduces but does not abolish LRRK2-mediated phosphorylation of Rab10 (Fig 8B). It is possible that remaining LRRK2 activity observed under these conditions is controlled by other Rab proteins binding to LRRK2. In future work, it will also be important to study how Rab29 expression, localization, and nucleotide binding are controlled in vivo and to explore further, whether overexpression or activation of Rab29 is linked to Parkinson’s disease. It would also be important to obtain more detailed structural information on how Rab29 binds to LRRK2. This would enable the design of improved mutants that disable binding of LRRK2 and Rab29 to better probe biological of this interaction. It would also be interesting to investigate whether Parkinson’s patients with PARK16 mutations display elevated LRRK2 kinase activity and Rab10 phosphorylation. If this is the case, it would suggest that patients with PARK16 locus mutations might benefit from a future LRRK2 inhibitor therapeutic. Our data also suggest that inhibitors targeting the LRRK2 ankyrin domain Our data are consistent with previous work (MacLeod et al, 2013; Beilina et al, 2014), showing that recruitment of LRRK2 to the Golgi by Rab29 significantly effects Golgi apparatus integrity and induces its fragmentation in a manner that can be ameliorated by treatment with LRRK2 inhibitors (Fig 3). We also find that LRRK2 ankyrin domain mutants that are unable to interact with Rab29 do not fully disperse the Golgi apparatus, emphasizing the 13 The EMBO Journal The EMBO Journal Published online: December 6, 2017 Activation of LRRK2 by Rab29 Elena Purlyte et al phosphorylated). The phospho-Rab8 antibody was raised against two phospho-T72-Rab8A/Rab8B peptides (C-Ahx-AGQERFRTIT- TAYYR-amide, corresponding to residues 65–79 and Ac-AGQERFR- TITTAYYR-Ahx-C-amide corresponding to residues 65–79 of human Rab8, indicates the phosphorylated residue). For immu- nization, the peptides were coupled to KLH via the Cys residue. would be expected to block Rab29 binding and inhibit activity of LRRK2 in cells, thereby offering therapeutic potential for the treat- ment of Parkinson’s disease. Antibodies Rabbit monoclonal antibodies for total LRRK2 (N-terminus) (UDD3) and phospho-Ser935 LRRK2 (UDD2) were generated at the University of Dundee. Mouse monoclonal antibody against total LRRK2 (C-terminus) was from NeuroMab (clone N241A/34). Rabbit monoclonal antibodies detecting phospho-Ser1292 LRRK2 [MJFR-19-7-8] (ab203181), phospho-Ser910 LRRK2 [UDD1 (15-3)] (ab133449), phospho-Ser955 [MJF-R11 (75-1)] (ab169521), and phospho-Ser973 LRRK2 [MJF-R12 (37-1)] (ab181364) were from Abcam. Anti-Rab10 total antibody was from Cell Signaling Tech- nology (#8127) and anti-HA High Affinity (clone 3F10) from Roche. Sheep polyclonal antibody for phospho-Thr71 Rab29 (S877D) was purified at the University of Dundee and used at a final concentration of 1 lg/ml in the presence of 10 lg/ml non- phosphorylated peptide. Sheep polyclonal antibody detecting total Rab29 was purified at the University of Dundee (S984D) which can be requested via our reagents website (https://mrcppureagents. dundee.ac.uk/). Plasmids The following constructs were used: HA-empty vector (DU49303); HA-Rab29 wt/T71E/S72E/T71A+S72A/T71E+S72E/M73S+R75S (DU50222, DU50242, DU50243, DU52690, DU27422, DU52670, DU27495, DU27918); 6His-SUMO-Rab8a (DU47363); Flag-tagged LRRK2 wt full-length (DU6841), LRRK2 wt 970-end (DU26764), LRRK2 D2017A full-length (DU10128), LRRK2 D2017A 970-end (DU26689), LRRK2 R1441C full-length (DU13078), LRRK2 R1441G full-length (DU13077), LRRK2 R1441G 970-end (DU26770), LRRK2 R1441G+D2017A full-length (DU52702), LRRK2 R1441H full-length (DU13287), LRRK2 Y1699C full-length (DU13165), LRRK2 Y1699C 970-end (DU26763), LRRK2 Y1699C+D2017A full-length (DU52703), LRRK2 R1728H full-length (DU17138), LRRK2 G2019S full-length (DU10129), LRRK2 G2019S 970-end (DU19006), LRRK2 G2019S+D2017A full-length (DU52723), LRRK2 I2020T full-length (DU13081), LRRK2 T2031S full-length (DU17135), LRRK2 G2385R full-length (DU13083). LRRK2 C727D full-length (DU26942), LRRK2 L728D full-length (DU26916), LRRK2 L729D full-length (DU26929), LRRK2 L728D L729D full-length (DU26925), LRRK2 L760D full- length (DU27224), LRRK2 L761D full-length (DU27240), LRRK2 L762D full-length (DU27225), LRRK2 L789D full-length (DU27229), LRRK2 L790D full-length (DU27226), LRRK2 L791D full-length (DU27227), LRRK2 C727D R1441G full-length (DU27040), LRRK2 L728D R1441G full-length (DU27042), and LRRK2 L729D R1441G full-length (DU27022). Rab29 KO N-terminal antisense guide and Cas9 D10A (DU52630), Rab29 KO N-terminal sense guides (DU52626). eGFP-LRRK2-G2019S was cloned into modified pSLQ1371 with eGFP at the N-terminus. Rab29 was subcloned into pcDNA3.1 with Myc-tag and modified pSLQ1371 with eGFP at the N-terminus. All cDNA clones generated for the present study can be requested via our reagents website (https://mrcppureagents. dundee.ac.uk/). MLi-2 LRRK2 inhibitor (Scott et al, 2017) was synthesized as described in Miller et al (2014). All recombinant proteins, DNA constructs, and antibodies generated for the present study and more detailed information on these can be requested via our reagents website (https://mrcppureagents.dundee.ac.uk/). LRRK2 [R1441G] knock-in MEFs were kindly provided by Dr Shu-Leong Ho (Division of Neurology, Department of Medicine, University of Hong Kong, Hong Kong) and have been described previously (Ito et al, 2016). General methods DNA constructs were amplified in Escherichia coli DH5a and puri- fied using a Hi-Speed Plasmid Maxi Kit (Qiagen). DNA cloning procedures were undertaken using standard protocols. DNA sequence verification of the DNA constructs used in the present study was performed by our Sequencing Service (http://www.dnase q.co.uk). Purification of Rab proteins The coding sequence for human Rab8A (accession number: NM_005370.4) was cloned into pET15b (DU47363), expressed in E. coli BL21 and purified as described previously (Steger et al, 2016). Horseradish peroxidase-conjugated anti-mouse (#31450), anti- rabbit (#31460), and anti-rat (#31470) were from Thermo Fisher Scientific. Rabbit monoclonal antibody recognizing phospho-Thr72 Rab8A/8B and phospho-Thr73 Rab10 were custom-made by Abcam in collaboration with the Michael J Fox Foundation and Abcam (Burlingame, California) (Lis et al, 2017). The Phospho-Rab10 anti- body was raised against two phospho-T73-Rab10 peptides C-Ahx- AGQERFHTITTSYYR-amide (corresponds to residues 66–80 of human Rab10 in which Thr73 marked as T* is phosphorylated) and Ac-AGQERFHTITTSYYR-Ahx-C-amide (corresponds to residues 66–80 of human Rab10 in which Thr73 marked as T is The EMBO Journal Generation of mouse embryonic fibroblasts To generate Rab29 knockout cells, a modified Cas9 nickase system was used. Guides were chosen following careful transcript analysis using both NCBI and Ensembl and that the guides themselves were identified using the Sanger center’s CRISPR finder (http://www.sa nger.ac.uk/htgt/wge/find_crisprs). Optimal sgRNA pairs were iden- tified with a low combined off-targeting score ((Rab29 KO-sgRNA1: GCACACTACCCAATGGAGAGC (DU52626); sgRNA2: GCTAGGTCC TGTTTCCACCTC (DU52630). Complementary oligos with BbsI- compatible overhangs were designed for each and the dsDNA guide inserts ligated into BbsI-digested target vectors; the antisense guides (sgRNA2) were cloned onto the spCas9 D10A-expressing pX335 vector (Addgene plasmid no. 42335) and the sense guides (sgRNA1) into the puromycin-selectable pBABED P U6 plasmid (Dundee-modified version of the original Cell Biolabs pBABE plasmid). A549 and HEK293Trex cells at ~80% confluency were co-transfected in a six-well plate with DU52630 and DU52626 plas- mids (for the Rab29 knockout) using for A549 cells Lipofectamine LTX according to the manufacturer’s instructions, with the final amount of 9 ll Lipofectamine LTX and 2.5 lg of DNA per well in a 6 well plate, and Polyethylenimine HCl MAX 4000 (Polysciences, Inc.) for HEK293Trex cells with 6 lg of Polyethylenimine, and 2.5 lg of DNA per well in a six-well plate followed by 24-h incu- bation in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. Medium was then replaced with fresh medium supplemented with 2 lg/ml of puromycin. After 24 h of puromycin selection, medium was replaced again with fresh medium without puromycin and the cells were left to recover for 48 h before performing single-cell sorting. LRRK2[T1348N] knock-in mice were obtained from The Jackson Laboratory and maintained on a C57BL/6J background (for further information see http://jaxmice.jax.org/strain/021829.html). Litter- mate-matched wild-type and homozygous LRRK2[T1348N] MEFs were isolated from mouse embryos at day E12.5 resulting from crosses between heterozygous LRRK2[T1348N/WT] mice as described previously (Wiggin et al, 2002). Genotyping of mice and MEFs was performed by PCR using genomic DNA isolated from ear biopsies and KOD Hot Start DNA Polymerase. Primer 1 (50-ACAAT CATGAGCTTCATTCGGTTGTAGGGT-30) and Primer 2 (50-ACATAT GTGTATATAACACAACCAAGGCTGC-30) were used to detect the wild-type and knock-in alleles. DNA sequencing was used to con- firm the knock-in mutation and performed by DNA Sequencing & Services (MRC–PPU; http://www.dnaseq.co.uk) using Applied Biosystems Big-Dye version 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer. Cell culture, transfection, treatment, and lysis HEK293, HeLa, A549, and mouse embryonic fibroblast cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 2 mM Glutamine, and penicillin (100 U/ml)/ streptomycin (100 lg/ml). Media for HEK293Trex cells before knock-in also contained 15 lg/ml Blasticidin and 50 lg/ml Zeocin. Flp-In T-REx 293 cells knock-in for eGFP-LRRK2-R1441G were main- tained in 15 lg/ml Blasticidin and 100 lg/ml Hygromycin B (Thermo Scientific). LRRK2 expression was induced with 1 lg/ml 14 The EMBO Journal ª 2017 The Authors Elena Purlyte et al Activation of LRRK2 by Rab29 Published online: December 6, 2017 Elena Purlyte et al Activation of LRRK2 by Rab29 Published online: December 6, 2017 The EMBO Journal through a 0.22-lm-pore-size Spinex column and added with 2-Mercaptoethanol to 1% (v/v). Samples were incubated for 5 min at 70°C before being subjected to SDS–PAGE and Western blotting. For the peptide substrate phosphorylation assay, kinase reactions were set up in a total volume of 50 ll with immunoprecipitated LRRK2 in 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 10 mM MgCl2, and 0.2 mM [c-32P]ATP (~300–500 c.p.m./pmol) in the presence of 40 lM Nictide (RLGWWRFYTLRRARQGNTKQR). Reactions were undertaken for 30 min at 30°C and terminated by applying 45 ll of the reaction mixture on to P81 phosphocellulose paper and immersing in 50 mM phosphoric acid. After extensive wash- ing, the radioactivity in the reaction products was quantified by Cherenkov counting. LRRK2 was then eluted from the beads by addition of LDS before being subjected to SDS–PAGE and Western blotting. Tetracycline for 24 h. HeLa cells were transfected with Fugene 6 (Promega), and HEK293T cells were transfected with Polyethylen- imine HCl MAX 4000 (Polysciences, Inc.) as described previously (Reed et al, 2006). Cells were lysed 24 h after transfection in an ice- cold lysis buffer containing 50 mM Tris/HCl, pH 7.5, 1% (v/v) Triton X-100, 1 mM EGTA, 1 mM sodium orthovanadate, 50 mM NaF, 10 mM 2-glycerophosphate, 5 mM sodium pyrophosphate, 0.1 lg/ml mycrocystin-LR (Enzo Life Sciences), 270 mM sucrose, and Complete EDTA-free protease inhibitor cocktail (Roche). Lysates were centrifuged at 20,800 g for 15 min at 4°C, and super- natants were quantified by Bradford assay (Thermo Scientific) and subjected to immunoblot analysis. Treatment of cells with MLi-2 was for 60 min at a final concentration of 100 nM, unless otherwise specified. Cell culture, transfection, treatment, and lysis All cell lines used in this study were tested for myco- plasma contamination and confirmed as negative for experimental analysis. Generation of mouse embryonic fibroblasts Wild-type, heterozygous, and homozygous T1348N knock-in MEFs isolated from the same littermate were selected for subsequent experiments. Cells cultured in parallel at passage 6 were used for the immunoblotting experiments presented in this paper. ª 2017 The Authors Immunoblot determination of membrane-associated LRRK2 Cells were chilled on ice, washed with ice-cold PBS, and swelled in hypotonic buffer (10 mM HEPES pH 7.4). After 15 min, 5× buffer was added to achieve a final concentration of resuspension buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.5 mM DTT, 100 nM GDP, 1× protease inhibitor cocktail (Sigma) and 2 mM sodium orthovanadate, 5 mM sodium fluoride, 5 mM sodium pyrophosphate, 10 mM beta-glycerophosphate, 0.1 lg/ml Microcystin-LR), and the suspension was passed 20 times through 25-G syringe. Nuclei were pelleted by centrifugation at 1,000 g for 5 min at 4°C. The post-nuclear supernatant was spun 100,000 g for 20 min in a table top ultracentrifuge in TLA100.2 rotor; the resulting supernatant was the cytosol fraction. Membrane pellets were solubilized in 1% Triton X-100-containing 1× resuspension buffer. Protein concentrations were estimated by Bradford assay (Bio-Rad, Richmond, CA). Samples containing 50 lg of membrane protein, or the equivalent volume of cytosolic protein, were heated at 37°C for 10 min after addition of 5× SDS–PAGE sample buffer. For experiments utilizing FLAG-LRRK2-G2019S, FLAG-LRRK2-R1441G, and derivative mutants, expression was for 48 h and Myc-Rab29 expression was for 24 h. Samples were loaded in duplicate onto TGX mini-PROTEAN 4–20% precast gradient gels (Bio-Rad) or NuPAGE 3–8% precast gradient Tris-acetate gels (Invitrogen). Gels were transferred onto nitrocellulose membrane using Trans-blot turbo system, blocked in 5% milk in TBS-T. Antibodies used were rabbit anti-LRRK2 UDD3 1:1,000 (MRC PPU University of Dundee), mouse anti-Myc (Cell signaling, 1:1,000), chicken anti-GFP (Aves, 1:1,000), mouse anti-GFP (Neuromab, 1:1,000), Mouse anti-tubulin DM1A (Sigma, 1:1,000), rabbit anti-phosphoserine 1292 (Abcam, 1:500), mouse anti-LAMP2 (DSHB 1:1,000), and mouse anti-Trans- ferrin receptor (BD Bioscience, 1:1,000). Primary antibody incubations were overnight in blocking buffer. Secondary donkey anti-Rabbit 800 and donkey anti-mouse 680 antibodies (Licor, 1:10,000) were incubated for 1 h, imaged using an Odyssey Infrared scanner (Licor), and quantified using ImageJ software. LRRK2 immunoprecipitation kinase assays FLAG-tagged LRRK2 wild-type and mutant variants of LRRK2 were transiently overexpressed in HEK293 cells using Polyethylenimine transfection (Reed et al, 2006), and 24 h post-transfection, cells were lysed in lysis buffer as described above and LRRK2 immuno- precipitated using anti-FLAG M2-agarose for 1 h (10 ll resin per 1 mg of cell extract). A control was also included where HEK293 cells were transfected with FLAG-empty vector. Immunoprecipi- tates were then washed three times with lysis buffer supplemented with 300 mM NaCl, and twice with 50 mM Tris/HCl (pH 7.5). Kinase assays were set up in a total volume of 50 ll with immuno- precipitated LRRK2 in 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2, and 1 mM ATP in the presence of 5 lg recombinant Rab8A. Assays were carried out at 30°C for 45 min with shaking. Reac- tions were terminated by adding LDS (lithium dodecyl sulfate) loading buffer to the beads. The mixture was then incubated at 100°C for 10 min, and the eluent was collected by centrifugation Cell sorting was performed using Influx cell sorter (Becton Dickin- son). Single cells were placed in individual wells of a 96-well plate containing DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin and 100 mg/ ml Normocin (InvivoGen). For HEK293Trex cells, the wells were coated by gelatin and the media contained Blasticidin and Zeocin as described above. After reaching ~80% confluency, individual clones were transferred into six-well plates. After reaching ~80% conflu- ency, the clones were screened for presence of Rab29 by immunoblotting. A549 Rab29-KO were further confirmed by 15 The EMBO Journal The EMBO Journal Published online: December 6, 2017 Activation of LRRK2 by Rab29 Elena Purlyte et al Activation of LRRK2 by Rab29 Elena Purlyte et al sequencing. For this purpose, genomic DNA was isolated using GenElute TM Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich). PCR was performed using PfuUltra High-Fidelity DNA Polymerase (Agilent Technologies) using 50-CAGGAGCGCTTCACCTCTATG and 50-GTCTCACTCACCCTCAACATCC primers to amplify the region targeted for knockout. The PCR products were then cloned into pSC- A-amp/kan vector using StrataClone PCR Cloning Kit (Agilent Tech- nologies). For each cloning reaction, 20 positive bacterial colonies were selected and amplified for plasmid DNA isolation using QIAprep Spin Miniprep Kit (Qiagen). Statistics Graphs were made using Graphpad Prism 5 software. Error bars indicate SEM. Student’s t-test was used to test significance. Two-tailed P-values < 0.05 were considered statistically significant. Figures EV2, 6E and 8B were analyzed using one-way ANOVA with Dunnett’s multiple comparison test to test significance. Acknowledgements We thank Dr. Martin Steger (Department of Proteomics and Signal Transduc- tion, Max Planck Institute of Biochemistry, Martinsried, Germany) as well as Kalpana Merchant (TransThera Consulting), Marco Baptista, and Shalini Padmanabhan (Michael J Fox Foundation for Parkinson’s research) for helpful discussions. We also thank Thineskrishna Anbarasan for performing CRISPR/ Cas9 knockouts of Rab29 in HEK293Trex cells, Philip Wing-Lok Ho and Shu-Leong Ho (Division of Neurology, Department of Medicine, University of Hong Kong, Hong Kong) for provision of the LRRK2[R1441G] knock-in MEFs and the excellent technical support of the MRC-Protein Phosphorylation and Ubiquitylation Unit (PPU) DNA Sequencing Service (coordinated by Nicholas Helps), the MRC PPU tissue culture team (coordinated by Laura Fin), MRC LRRK2 immunoprecipitation kinase assays The inserts in each individual clone were then sequenced using M13 primers (DNA sequencing facility of Division of Signal Transduction Therapy at the University of Dundee) to confirm that there were no wild-type alleles of Rab29 gene present in the genome of selected clones. chilled on ice, washed twice with cold PBS, and incubated in gluta- mate buffer (25 mM KCl, 25 mM HEPES pH 7.4, 25 mM magnesium acetate, 5 mM EGTA, 150 mM potassium glutamate). Excess buffer was removed by blotting with a tissue; the coverslip was then dipped in liquid nitrogen for 5 s and allowed to thaw for few seconds. Coverslips were then gently washed with glutamate buffer and rehydrated for 5 min in cold PBS. Cells were then fixed (cold 3% PFA for 20 min on ice), permeabilized with 0.1% Trixon X-100, and blocked with 2% BSA in PBS. Antibodies were diluted as follows: anti-LRRK2 UDD3 antibody (1:1,000, MRC PPU University of Dundee), mouse anti-GFP (Neuromab, 1:1,000), and mouse anti- Myc (9E10 Hybridoma culture supernatant—undiluted). Secondary antibodies (Thermo Scientific) were goat anti-rabbit Alexa 568 (1:2,000), goat anti-mouse Alexa 488 (1:1,000), goat anti-mouse Alexa 555 (1:2,000), and goat anti-rabbit Alexa 488 (1:2,000). Images were acquired using a laser scanning confocal microscope (Leica SP8) fitted with a 63× 1.4NA objective and acousto-optical beam splitter with hybrid detector, or a spinning disk confocal microscope (Yokogawa) with an electron multiplying charge- coupled device (EMCCD) camera (Andor, UK) and a 100× 1.4NA oil immersion objective. Images were analyzed using Fiji (https://fiji. sc/). Co-localization was quantified using JACoP, a Fiji plugin, and Mander’s coefficients were calculated using an automatic threshold. MEF-R1441G or WT cells were fixed (3% PFA, RT, 15 min), perme- abilized using Triton X-100 (0.1%), blocked with 2% BSA, and stained with rabbit anti-GCC185 (1:1,000), (Cheung et al, 2015) and goat anti-rabbit Alexa 488 (1:1,000) antibody. Nuclei were stained using 0.1 lg/ml DAPI (Sigma). Data availability All primary data are available to anyone requesting this. We confirm that this study does not contain protein, DNA, RNA sequence, macro- molecular structure, crystallographic, functional genomic, or proteomic data that are subject to the “EMBO Data Deposition policy”. Expanded View for this article is available online. References main contacts. Proc Natl Acad Sci USA 113: E4357 – E4366 Beilina A, Rudenko IN, Kaganovich A, Civiero L, Chau H, Kalia SK, Kalia LV, Lobbestael E, Chia R, Ndukwe K, Ding J, Nalls MA, International Parkinson’s Disease Genomics C, North American Brain Expression C, Olszewski M, Hauser DN, Kumaran R, Lozano AM, Baekelandt V, Greene LE et al (2014) Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease. 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Author contributions EP designed and executed experiments (Figs 6B–D and 8A, and 9A and B, and EV2, and EV3A and B); HSD designed and executed experiments (Figs 2 and 3 and 4 and 5 and 7A–I, and 9C and D, and EV3C); FT designed and executed experiments (Figs 1, and EV1A, and 6A, E and F) and was the first to discover that Rab29 stimulated LRRK2 Ser1292 phosphorylation; ARS executed experi- ment Fig 8B; RG executed experiment Fig 7J; PL generated Rab29 KO A549 cells and helped generate the phospho Rab8 and Rab10 antibodies and executed experiment Fig EV1B and C; MW undertook most of the cloning studies; TNM designed, organized, and oversaw the effort required for development the rabbit phospho Rab8 and Rab10 antibodies; SRP and DRA supervised the project and wrote the manuscript. All authors were involved in discussing and interpreting the data. Gonzalez-Fernandez MC, Lezcano E, Ross OA, Gomez-Esteban JC, Gomez- Busto F, Velasco F, Alvarez-Alvarez M, Rodriguez-Martinez MB, Ciordia R, Zarranz JJ, Farrer MJ, Mata IF, de Pancorbo MM (2007) Lrrk2-associated Parkinsonism is a major cause of disease in Northern Spain. Parkinsonism Relat Disord 13: 509 – 515 Greggio E, Jain S, Kingsbury A, Bandopadhyay R, Lewis P, Kaganovich A, van der Brug MP, Beilina A, Blackinton J, Thomas KJ, Ahmad R, Miller DW, Kesavapany S, Singleton A, Lees A, Harvey RJ, Harvey K, Cookson MR (2006) Kinase activity is required for the toxic effects of mutant LRRK2/ dardarin. Neurobiol Dis 23: 329 – 341 Light microscopy Parkinsonism Relat Disord 13: 509 – 515 Dzamko N, Inesta-Vaquera F, Zhang J, Xie C, Cai H, Arthur S, Tan L, Choi H, Gray N, Cohen P, Pedrioli P, Clark K, Alessi DR (2012) The IkappaB kinase family phosphorylates the Parkinson’s disease kinase LRRK2 at Ser935 and Ser910 during Toll-like receptor signaling. PLoS One 7: e39132 Fukuda M (2016) Multiple roles of VARP in endosomal trafficking: Rabs, Retrome components and R-SNARE VAMP7 meet on VARP. Traffic 17: 709– 719 References Small GTPases 4: 16 – 21 Hesketh GG, Perez-Dorado I, Jackson LP, Wartosch L, Schafer IB, Gray SR, McCoy AJ, Zeldin OB, Garman EF, Harbour ME, Evans PR, Seaman MN, Luzio JP, Owen DJ (2014) VARP is recruited on to endosomes by direct interaction with retromer, where together they function in export to the cell surface. Dev Cell 29: 591 – 606 Cherfils J, Zeghouf M (2013) Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiol Rev 93: 269 – 309 Ito G, Okai T, Fujino G, Takeda K, Ichijo H, Katada T, Iwatsubo T (2007) GTP binding is essential to the protein kinase activity of LRRK2, a causative gene product for familial Parkinson’s disease. Biochemistry 46: 1380 – 1388 Cheung PY, Limouse C, Mabuchi H, Pfeffer SR (2015) Protein flexibility is required for vesicle tethering at the Golgi. 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Biochem J 473: 2671 – 2685 Daniels V, Vancraenenbroeck R, Law BM, Greggio E, Lobbestael E, Gao F, De Maeyer M, Cookson MR, Harvey K, Baekelandt V, Taymans JM (2011) Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant. J Neurochem 116: 304 – 315 Jaleel M, Nichols RJ, Deak M, Campbell DG, Gillardon F, Knebel A, Alessi DR (2007) LRRK2 phosphorylates moesin at threonine-558: characterization of how Parkinson’s disease mutants affect kinase activity. Biochem J 405: 307 – 317 Dodson MW, Zhang T, Jiang C, Chen S, Guo M (2012) Roles of the Drosophila LRRK2 homolog in Rab7-dependent lysosomal positioning. Doggett EA, Zhao J, Mork CN, Hu D, Nichols RJ (2011) Phosphorylation of LRRK2 serines 955 and 973 is disrupted by Parkinson’s disease mutations and LRRK2 pharmacological inhibition. J Neurochem 120: 37 – 45 Dzamko N, Deak M, Hentati F, Reith AD, Prescott AR, Alessi DR, Nichols RJ (2010) Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization. Biochem J 430: 405 – 413 Dzamko N, Inesta-Vaquera F, Zhang J, Xie C, Cai H, Arthur S, Tan L, Choi H, Gray N, Cohen P, Pedrioli P, Clark K, Alessi DR (2012) The IkappaB kinase family phosphorylates the Parkinson’s disease kinase LRRK2 at Ser935 and Ser910 during Toll-like receptor signaling. PLoS One 7: e39132 Fukuda M (2016) Multiple roles of VARP in endosomal trafficking: Rabs, Retromer components and R-SNARE VAMP7 meet on VARP. Traffic 17: 709– 719 Gonzalez-Fernandez MC, Lezcano E, Ross OA, Gomez-Esteban JC, Gomez- Busto F, Velasco F, Alvarez-Alvarez M, Rodriguez-Martinez MB, Ciordia R, Zarranz JJ, Farrer MJ, Mata IF, de Pancorbo MM (2007) Lrrk2-associated Parkinsonism is a major cause of disease in Northern Spain. Parkinsonism Relat Disord 13: 509 – 515 Light microscopy HeLa cells were plated on collagen coated coverslips, transfected with indicated plasmids using Fugene 6 (Promega). After 48 h for LRRK2 and 24 h for Rab expression, cells were cytosol depleted by liquid nitrogen freeze-thaw (Seaman, 2004). Briefly, cells were 16 ª 2017 The Authors The EMBO Journal Elena Purlyte et al Activation of LRRK2 by Rab29 Published online: December 6, 2017 Elena Purlyte et al Activation of LRRK2 by Rab29 Published online: December 6, 2017 The EMBO Journal PPU Reagents and Services antibody purification teams (coordinated by Hilary McLauchlan and James Hastie). This work was supported by the Michael J. Fox Foundation for Parkinson’s research [grant number 6986 (to S.R.P. and D.R.A.)]; the Medical Research Council [grant number MC_UU_12016/2 (to D.R.A.)]; the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (Boehringer-Ingelheim, GlaxoSmithKline, and Merck KGaA, to D.R.A.); and the U.S. National Institutes of Health DK37332 (to S.R.P.). Doggett EA, Zhao J, Mork CN, Hu D, Nichols RJ (2011) Phosphorylation of LRRK2 serines 955 and 973 is disrupted by Parkinson’s disease mutations and LRRK2 pharmacological inhibition. J Neurochem 120: 37 – 45 Dzamko N, Deak M, Hentati F, Reith AD, Prescott AR, Alessi DR, Nichols RJ (2010) Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization. Biochem J 430: 405 – 413 Dzamko N, Deak M, Hentati F, Reith AD, Prescott AR, Alessi DR, Nichols RJ (2010) Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization. Biochem J 430: 405 – 413 Dzamko N, Inesta-Vaquera F, Zhang J, Xie C, Cai H, Arthur S, Tan L, Choi H, Gray N, Cohen P, Pedrioli P, Clark K, Alessi DR (2012) The IkappaB kinase family phosphorylates the Parkinson’s disease kinase LRRK2 at Ser935 and Ser910 during Toll-like receptor signaling. PLoS One 7: e39132 Fukuda M (2016) Multiple roles of VARP in endosomal trafficking: Rabs, Retromer components and R-SNARE VAMP7 meet on VARP. Traffic 17: 709– 719 Gonzalez-Fernandez MC, Lezcano E, Ross OA, Gomez-Esteban JC, Gomez- Busto F, Velasco F, Alvarez-Alvarez M, Rodriguez-Martinez MB, Ciordia R, Zarranz JJ, Farrer MJ, Mata IF, de Pancorbo MM (2007) Lrrk2-associated Parkinsonism is a major cause of disease in Northern Spain. 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J Med Chem 60: 2983 – 2992 License: This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduc- tion in any medium, provided the original work is properly cited. 18 The EMBO Journal ª 2017 The Authors
https://openalex.org/W4313477519	https://pure.eur.nl/ws/files/85479883/Proteomics_and_Metabolomics_Based_Analysis_of_Metabolic_Changes_in_a_Swine_Model_of_Pulmonary_Hypertension.pdf	English	null	Proteomics and Metabolomics Based Analysis of Metabolic Changes in a Swine Model of Pulmonary Hypertension	null	2,023	cc-by	9,599	Article Proteomics- and Metabolomics- Based Analysis of Metabolic Changes in a Swine Model of Pulmonary Hypertension Payel Sen, Bachuki Shashikadze, Florian Flenkenthaler, Esther Van de Kamp, Siyu Tian, Chen Meng, Michael Gigl, Thomas Fröhlich and Daphne Merkus Special Issue Molecular Research on Pulmonary Hypertension 4.0 Edited by Dr. Tatyana Novoyatleva Special Issue Molecular Research on Pulmonary Hypertension 4.0 Edited by Dr. Tatyana Novoyatleva Citation: Sen, P.; Shashikadze, B.; Flenkenthaler, F.; Van de Kamp, E.; Tian, S.; Meng, C.; Gigl, M.; Fröhlich, T.; Merkus, D. Proteomics- and Metabolomics-Based Analysis of Metabolic Changes in a Swine Model of Pulmonary Hypertension. Int. J. Mol. Sci. 2023, 24, 4870. https:// doi.org/10.3390/ijms24054870 Keywords: pulmonary hypertension; proteomic analysis; metabolomic analysis https://doi.org/10.3390/ijms24054870 International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences Article Proteomics- and Metabolomics-Based Analysis of Metabolic Changes in a Swine Model of Pulmonary Hypertension Payel Sen 1,2, Bachuki Shashikadze 3, Florian Flenkenthaler 3, Esther Van de Kamp 4, Siyu Tian 4, Chen Meng 5, Michael Gigl 5 , Thomas Fröhlich 3 and Daphne Merkus 1,2,4,* 1 Walter Brendel Center for Experimental Medicine (WBex), University Clinic Munich, LMU Munich, 81377 Munich, Germany 3 Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, 81377 Munich, Germany 4 Division of Experimental Cardiology, Erasmus University Medical Center, 3015 GD Rotterdam, The Netherlands 5 Bavarian Center for Biomolecular Mass Spectrometry, Technical University Munich, 85354 Freising, Germany * Correspondence: daphne.merkus@med.uni-muenchen.de; Tel.: +31-624592486 5 Bavarian Center for Biomolecular Mass Spectrometry, Technical University Munich, 85354 Freising, Germany * Correspondence: daphne.merkus@med.uni-muenchen.de; Tel.: +31-624592486 Abstract: Pulmonary vein stenosis (PVS) causes a rare type of pulmonary hypertension (PH) by im- pacting the ﬂow and pressure within the pulmonary vasculature, resulting in endothelial dysfunction and metabolic changes. A prudent line of treatment in this type of PH would be targeted therapy to relieve the pressure and reverse the ﬂow-related changes. We used a swine model in order to mimic PH after PVS using pulmonary vein banding (PVB) of the lower lobes for 12 weeks to mimic the hemodynamic proﬁle associated with PH and investigated the molecular alterations that provide an impetus for the development of PH. Our current study aimed to employ unbiased proteomic and metabolomic analyses on both the upper and lower lobes of the swine lung to identify regions with metabolic alterations. We detected changes in the upper lobes for the PVB animals mainly pertaining to fatty acid metabolism, reactive oxygen species (ROS) signaling and extracellular matrix (ECM) remodeling and small, albeit, signiﬁcant changes in the lower lobes for purine metabolism. 1. Introduction Pulmonary hypertension (PH) due to pulmonary vein stenosis (PVS) is a life-threatening disease, which mainly affects the pediatric population [1]. This type of PH, which ulti- mately results in a left ventricular inﬂow tract obstruction, is classiﬁed under type II PH [2]. PVS presents mostly with congenital heart defects (univentricular heart disease, ventricular septal defect, atrial septal defect or persistent arterial duct), lung disease (bron- chopulmonary dysplasia) or Down syndrome or other trisomy [3,4]. In rare cases, PVS can also occur in adults because of radiofrequency ablation therapy after atrial ﬁbrillation [5]. This particular type of PH is characterized by an initial passive increase in pulmonary artery pressure brought on by increased resistance due to the banding. The increased mean pulmonary artery pressure results in vascular remodeling, which further raises pulmonary vascular resistance and causes an additional increase in pressure. Surgical inter- ventions and/or stenting of the lesions in patients with PVS frequently lead to restenosis, and the use of vasodilators comes with the risk for pulmonary edema [6]. The complex molecular mechanisms involved in PH are limiting factors in the development of novel therapeutic interventions. Academic Editor: Tatyana Novoyatleva Received: 30 December 2022 Revised: 14 February 2023 Accepted: 18 February 2023 Published: 2 March 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Previous work by our group has shown that endothelial factors are important in the development of PH in a swine model using pulmonary vein banding (PVB) of the lower lobes for 12 weeks to induce type II PH [7]. This procedure results in areas of the lung with https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 4870. https://doi.org/10.3390/ijms24054870 Int. J. Mol. Sci. 2023, 24, 4870 2 of 15 2 of 15 a varied hemodynamic proﬁle within the lung; the lower lobes experience high pressure and low ﬂow (HF/LF) whereas the upper lobes experience high pressure and high ﬂow (HP/HF). High and low shear stress have very striking effects on the endothelial cells of the lung vasculature. Endothelial cells typically respond to high shear stress with strong nitric oxide synthesis, but they “activate” a pro-inﬂammatory proﬁle at low shear stress, characterized by low nitric oxide production [8]. In this study, we conducted a quantitative LC-MS/MS-based proteomic analysis of lung samples along with untargeted metabolomics from swine with PVB and control (Cntrl) swine. We analyzed tissues from the upper and lower lobes to investigate how different hemodynamic proﬁles impact protein and metabolite expression due to PH in the lobes. 2.1. Characteristics of Pulmonary Hypertension Using LC-MS/MS-based proteomics, we identiﬁed 5112 proteins with high conﬁdence (false discovery rate < 0.01) (Supplementary Data 1, Table S1). The dataset has been submitted to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identiﬁer PXD038982 [9]. Differential abundance analysis revealed signiﬁcant differences between groups. The principal component analysis showed clustering of the PVB animals compared to the Cntrls in the upper (HP/HF) lobe, whereas similar clustering was absent in the lower lobe (HP/LF) (Figure 2A,B). Figure 2. Proteomic analysis of lung tissue of PVB vs. Cntrl animals. (A,B) Principal component analysis (PCA) of proteome proﬁles from upper (A) and lower (B) lobe of Cntrl (n = 7) and PVB animals (n = 6). Colored circles indicate individual animals. Red-ﬁlled circles are Cntrl and blue-ﬁlled circles are PVB animals. Figure 2. Proteomic analysis of lung tissue of PVB vs. Cntrl animals. (A,B) Principal component analysis (PCA) of proteome proﬁles from upper (A) and lower (B) lobe of Cntrl (n = 7) and PVB animals (n = 6). Colored circles indicate individual animals. Red-ﬁlled circles are Cntrl and blue-ﬁlled circles are PVB animals. In total, 104 proteins were found to be differentially abundant (Benjamini–Hochberg corrected p-value < 0.05 and fold change ≥1.5) between PVB and Cntrl in the upper lobes (Supplementary Data S1, Table S2). In the lower lobes, 52 proteins differed signiﬁcantly in abundance between PVB and Cntrl (Supplementary Data S1, Table S3). Volcano plots were used to visualize proteome alterations between conditions (Figure 3A,B). In the upper lobe, several apolipoproteins (APOF, APOA, APOC3), complement cascades (C6, C7, C8, C9) and coagulation proteins (Serpins, HRG, PROC) were found to be downregulated in the PVB animals, while several membrane transport proteins (SCGB1A1, SFTPA1) were found to be upregulated. In the lower lobe, DNA binding and ribosomal proteins (H1-2, H1-1, RPS6) were upregulated, while membrane remodeling proteins (GMPR, ALF1, SIGLEC1) were downregulated. g We performed a STRING preranked functional enrichment analysis of proteome pro- ﬁles from the upper and lower lobe to reveal lobe-speciﬁc signatures for PVB and Cntrl animals. 2.1. Characteristics of Pulmonary Hypertension Pulmonary vein banding in the PVB group animals resulted in signiﬁcant stenosis in the inferior pulmonary conﬂuence as shown in the angiogram (Figure 1A). Twelve weeks after banding, this resulted in a signiﬁcantly higher mean pulmonary artery pressure (38 ± 8 mmHg) in the PVB animals compared to the control (mean of 20 ± 4 mmHg, p < 0.05) (Figure 1D) as well as an increased pulmonary vascular resistance and reduced pulmonary artery compliance (Figure 1E,F). Figure 1. Pulmonary vein banding causes PH. (A) Angiogram of the inferior venous conﬂuence of a representative Cntrl (top) and PVB animal (bottom). (B,C) Picrosirius red staining of the lung upper and lower lobe in Cntrl and PVB animals. (D–F) Mean pulmonary artery pressure (mPAP), total pulmonary vascular resistance index (tPVRi) and pulmonary artery compliance in awake, resting swine. PA—pulmonary artery, PV—pulmonary vein, Br—bronchiole. Values are means ± SEM. * p ≤0.05, * ≤0.001, ** ≤0.0001. Scale bar: 100 µm. PVB vs. Cntrl by Student’s t-test, Cntrl n = 7; PVB n = 6. Figure 1. Pulmonary vein banding causes PH. (A) Angiogram of the inferior venous conﬂuence of a representative Cntrl (top) and PVB animal (bottom). (B,C) Picrosirius red staining of the lung upper and lower lobe in Cntrl and PVB animals. (D–F) Mean pulmonary artery pressure (mPAP), total pulmonary vascular resistance index (tPVRi) and pulmonary artery compliance in awake, resting swine. PA—pulmonary artery, PV—pulmonary vein, Br—bronchiole. Values are means ± SEM. * p ≤0.05, * ≤0.001, ** ≤0.0001. Scale bar: 100 µm. PVB vs. Cntrl by Student’s t-test, Cntrl n = 7; PVB n = 6. Histology of the lung tissue revealed more picrosirius red staining in the PVB animals in the upper and lower lobe, depicting more ﬁbrosis compared to the Cntrl (Figure 1B,C). Histology of the lung tissue revealed more picrosirius red staining in the PVB animals in the upper and lower lobe, depicting more ﬁbrosis compared to the Cntrl (Figure 1B,C). Int. J. Mol. Sci. 2023, 24, 4870 3 of 15 3 of 15 2.2. Proteomic Analysis of PVB vs. Control in the Upper and Lower Lobe To explore the chronic effects of ﬂow and pressure alterations in the lung tissue, we performed a label-free liquid chromatography–tandem mass spectrometry analysis (LC- MS/MS) of PVB vs. Cntrl samples from the upper as well as the lower lobes (n = 6 for PVB; n = 7 for Cntrl). 2.1. Characteristics of Pulmonary Hypertension (A,B) The quantitative proteome alterations are visualized via volcano plots in upper and lower lobes, respectively, from PVB vs. Cntrl pigs. Color-ﬁlled circles (blue—upregulated, red—downregulated) indicate differentially abundant proteins (Benjamini–Hochberg corrected p-value ≤0.05 and fold change ≥1.5). (C) Functional characterization of differences between upper and lower lobes in PVB and Cntrl animals. Heatmap shows GO-term enrichment in PVB compared to Cntrl. Cntrl (n = 7); PVB (n = 6). 2.1. Characteristics of Pulmonary Hypertension From the Gene Ontology (GO) biological processes database, 67 and 7 signiﬁcantly enriched terms were found in the upper and lower lobe, respectively (enrichment factor >1) (Figure 3C, Supplementary Data S1, Tables S4 and S5), PVB upper lobes showed a distinct downregulation of proteins related to humoral immune regulation, lipoprotein particle organization, cholesterol esteriﬁcation and triglyceride homeostasis and an upregulation of Int. J. Mol. Sci. 2023, 24, 4870 4 of 15 4 of 15 proteins related to platelet degranulation, coagulation, cholesterol efﬂux and intermem- brane lipid transport. Proteins related to the extracellular matrix were found to be both up- as well as downregulated in PVB animals, indicating altered matrix turnover. In the lower lobe, PVB showed fewer enriched pathways compared to the upper lobe. The majority of the pathways were related to blood coagulation, extracellular matrix reorganization, actin cytoskeletal organization and carbohydrate metabolism. Since ECM-related proteins were altered in both lobes, we also compared the proteins in this pathway in both upper and lower PVB lobes with the established lung matrix gene set and found several proteins (collagens, serpins, etc.) that were differentially regulated (Supplementary Figure S1A) [10]. Figure 3. Quantitative and functional analysis of upper and lower lobe of PVB vs. Cntrl anim (A,B) The quantitative proteome alterations are visualized via volcano plots in upper and lower lo respectively, from PVB vs. Cntrl pigs. Color-ﬁlled circles (blue—upregulated, red—downregula indicate differentially abundant proteins (Benjamini–Hochberg corrected p-value ≤0.05 and change ≥1.5). (C) Functional characterization of differences between upper and lower lobes in and Cntrl animals. Heatmap shows GO-term enrichment in PVB compared to Cntrl. Cntrl (n PVB (n = 6). 2.3. Metabolomic Proﬁling of the Lower and Upper Lobe versus Control Next, we performed untargeted metabolomics on these lung tissues to better un Figure 3. Quantitative and functional analysis of upper and lower lobe of PVB vs. Cntrl animals. (A,B) The quantitative proteome alterations are visualized via volcano plots in upper and lower lobes, respectively, from PVB vs. Cntrl pigs. Color-ﬁlled circles (blue—upregulated, red—downregulated) indicate differentially abundant proteins (Benjamini–Hochberg corrected p-value ≤0.05 and fold change ≥1.5). (C) Functional characterization of differences between upper and lower lobes in PVB and Cntrl animals. Heatmap shows GO-term enrichment in PVB compared to Cntrl. Cntrl (n = 7); PVB (n = 6). Figure 3. Quantitative and functional analysis of upper and lower lobe of PVB vs. Cntrl animals. 2.3. Metabolomic Proﬁling of the Lower and Upper Lobe versus Control Next, we performed untargeted metabolomics on these lung tissues to better under- stand the ongoing metabolic alterations caused by variations in ﬂow and pressure. To detect the relevant metabolites, we used statistical analysis with XCMS and MetaboAnalyst 5:0 software. Supplementary Data S2, Table S1 lists the metabolites that were detected Int. J. Mol. Sci. 2023, 24, 4870 5 of 15 5 of 15 in the HILIC-negative mode MS. On the MetaboAnalyst platform, a 3D PCA analysis (Figure 4A,B) and a supervised orthogonal partial least squares discriminant analysis (OPLS-DA) (Figure 4C,D) were performed for both the upper and lower lobes. In the upper lobe, 3D PCA and OPLS-DA analysis revealed separation between the PVB and Cntrl groups. Similar to the case for our proteomics ﬁndings, the lower lobe groups did not show a clear separation in metabolomics either. The OPLS-DA analysis also allowed for the identiﬁcation of the metabolites that contributed the most to group segregation, known as variable importance in the projection (VIP) scores, and they were ranked accordingly (Figure 5A). Metabolites with a VIP score of ≥1 were interpreted as highly inﬂuential (Supplementary Data S2, Tables S2 and S4), and we performed an enrichment analysis of metabolites with p < 0.05 to differentiate control from PVB animals (Figure 5B). In compari- son to Cntrl, we found 82 such metabolites in the PVB upper lobe (Supplementary Data S2, Table S3) and 29 metabolites in the PVB lower lobe (Supplementary Data S2, Table S5). Enrichment analysis for the PVB upper lobe revealed 25 metabolic pathways, of which the following six pathways had a p-value of <0.05: linoleic acid metabolism, ubiquinone biosyn- thesis pathway, transfer of acetyl groups into mitochondria, arginine, proline metabolism and glycerolipid metabolism. The lower lobe showed enrichment of 11 pathways, but none were signiﬁcantly altered (p < 0.05). We detected the pathway for purine metabolism (p = 0.06) to be the most differentially regulated (Supplementary Figure S2). Figure 4. Metabolomic analysis in the upper and lower lobe of PVB vs. Cntrl animals. (A,B) Thre dimensional PCA analysis based on metabolic proﬁles of PVB and Cntrl animals in the upper an lower lobe in negative mode (HILIC). (C,D) OPLS-DA analysis based on metabolic proﬁles of PV and Cntrl animals in upper and lower lobe in negative mode (HILIC). n = 6 in each group. Figure 4. Metabolomic analysis in the upper and lower lobe of PVB vs. Cntrl animals. 2.3. Metabolomic Proﬁling of the Lower and Upper Lobe versus Control (A,B) Three- dimensional PCA analysis based on metabolic proﬁles of PVB and Cntrl animals in the upper and lower lobe in negative mode (HILIC). (C,D) OPLS-DA analysis based on metabolic proﬁles of PVB and Cntrl animals in upper and lower lobe in negative mode (HILIC). n = 6 in each group. Int. J. Mol. Sci. 2023, 24, 4870 6 of 15 Figure 5. Metabolomic analysis in the upper lobe of PVB vs. Cntrl animals. (A) Metabolites ranked by Variable importance in the projection score (VIP) > 1 in PVB vs. Cntrl pigs in upper lobes based on OPLS-DA analysis. (B) Pathway analysis based on KEGG database of the enriched metabolites (VIP > 1 and p-value < 0.05 in the upper lobe of PVB vs. Cntrl swine. n = 6 in each group. The size of the dots represents the enrichment ratio and the shade of the color represent the −log 10 (p value). 2 4 N k A l i f P i d M b l i D Figure 5. Metabolomic analysis in the upper lobe of PVB vs. Cntrl animals. (A) Metabolites ranked by Variable importance in the projection score (VIP) > 1 in PVB vs. Cntrl pigs in upper lobes based on OPLS-DA analysis. (B) Pathway analysis based on KEGG database of the enriched metabolites (VIP > 1 and p-value < 0.05 in the upper lobe of PVB vs. Cntrl swine. n = 6 in each group. The size of the dots represents the enrichment ratio and the shade of the color represent the −log 10 (p value). 2.4. Network Analysis of Proteomics and Metabolomics Datasets They functionally connected with the downregulated the enzyme guanosine monophos- phate reductase (GMPR) and HPRT1 in the PVB lower lobe. Furthermore, the metabolite guanosine monophosphate was connected to the interferon-induced guanylate binding protein (GBP1). Figure 6. Interaction network analysis of the metabolites and proteins from metabolomics and proteomics. (A) Metabolite–metabolite interaction network for the most enriched metabo- lites in the upper lobe of PVB vs. Cntrl. Labeled and red-filled circles represent the upregu- lated metabolites, and green-filled circles represent the downregulated metabolites detected in the PVB group. Metabolites clustered in green background represent metabolites partici- pating in fatty acid metabolism, red background—ROS pathway; blue—ECM remodeling and yellow—glucose metabolism. (B) Gene–metabolite network analysis for the significantly altered proteins and enriched metabolites for upper lobe of PVB vs. Cntrl. The red-filled squares repre- sent the downregulated proteins, and green-filled circles represent the upregulated metabolites detected in the PVB group. Metabolites of VIP > 1 with p-value < 0.05 and proteins with fold change > 1.3 and Benjamini–Hochberg-adjusted p-value < 0.05 were used for the analysis. The proteins and metabolites belonging to one GO term are encircled together. Figure 6. Interaction network analysis of the metabolites and proteins from metabolomics and proteomics. (A) Metabolite–metabolite interaction network for the most enriched metabo- lites in the upper lobe of PVB vs. Cntrl. Labeled and red-filled circles represent the upregu- lated metabolites, and green-filled circles represent the downregulated metabolites detected in the PVB group. Metabolites clustered in green background represent metabolites partici- pating in fatty acid metabolism, red background—ROS pathway; blue—ECM remodeling and yellow—glucose metabolism. (B) Gene–metabolite network analysis for the significantly altered proteins and enriched metabolites for upper lobe of PVB vs. Cntrl. The red-filled squares repre- sent the downregulated proteins, and green-filled circles represent the upregulated metabolites detected in the PVB group. Metabolites of VIP > 1 with p-value < 0.05 and proteins with fold change > 1.3 and Benjamini–Hochberg-adjusted p-value < 0.05 were used for the analysis. The proteins and metabolites belonging to one GO term are encircled together. 2.4. Network Analysis of Proteomics and Metabolomics Datasets A combined analysis of the two omics datasets was carried out in order to identify com- monly altered pathways and to provide additional insight into the process of pulmonary vascular remodeling. The metabolite–metabolite and the gene–metabolite interaction net- works provide an overview of functionally related metabolites and proteins found to be most differentially abundant in metabolomics and proteomics. The metabolite–metabolite pathway interaction network derived from the KEGG database is shown in Figure 6A and highlights functional interactions among the top altered metabolites such as oleic acid, linoleic acid, palmitic acid, prostaglandin E2 and L-malic acid, butyric acid, NADP, proline, threonine, S-adenosylhomocysteine and arachidonic acid. Next, the most signiﬁcantly altered proteins and metabolites identiﬁed were mapped to the gene–metabolite molecu- lar interactions to create a network (Figure 6B). The network includes 31 nodes (protein, metabolites) and shows that the metabolites (squares) are upregulated whereas the proteins (ﬁlled circles) are downregulated. The metabolite chondroitin sulfate, a major component of the extracellular matrix (ECM), is upregulated in the upper lobe of the PVB group and formed a network with proteins important for wound healing such as serpinc1, serpinD1, F12, PROC, VTN, AMBP and TNC. Plasminogen (PLG), another prominent protein in- volved in wound healing and ECM remodeling, is functionally linked to both chondroitin sulfate and oleic acid. The PVB upper lobe was enriched in oleic acid, linoleic acid, palmitic acid, butyric acid and arachidonic acid, which formed a network with downregulated proteins in the PVB group such as ApoA1, ApoB, AdipoQ and ALB. These proteins and metabolites together participate in fatty acid metabolism. Prostaglandin E2, a common byproduct of arachidonic acid, is also upregulated in the PVB upper lobe and has formed a network with complement cascade members C8A and C3 as well as the chemokine PPBP, which are involved in inﬂammation. Finally, the monosaccharide metabolite glucose was downregulated in the upper lobe and functionally linked with the surfactant protein SFPTD and the blood coagulation protein HBB, indicating altered glucose metabolism. The PVB lower lobe presented with a metabolite–metabolite interaction network involving only Int. J. Mol. Sci. 2023, 24, 4870 7 of 15 7 of 15 four metabolites: guanosine monophosphate, inosinic acid, glyceric acid and dodecanoic acid (Supplementary Figure S3A). The gene metabolite network showed a simple network involving only guanosine monophosphate and inosinic acid (Supplementary Figure S3B). 2.5. Protein and Transcriptional Regulation of Fatty Acid Uptake in the Upper Lobe The protein abundance of members of apolipoproteins in the upper lobe as well as the lower lobe was further compared in dot plot analysis, and members of this fatty acid uptake pathway were validated at a transcriptional level (Figure 7A–C, Supplementary Figure S4A). ApoE was found to be significantly altered in both proteomics as well as at the transcrip- tional level in the upper lobe of the PVB group. In addition, further transcripts coding for proteins important for fatty acid uptake such as CD36 (p = 0.1) showed a trend to- ward a decrease in the PVB upper lobe compared to Cntrls along with significant up- regulation of the LDLR (low-density lipoprotein receptor). In contrast, we did not see Int. J. Mol. Sci. 2023, 24, 4870 8 of 15 similar changes at the mRNA level for these proteins in the lower lobe of PVB compared to Cntrls. Figure 7. Fatty acid uptake pathway in the upper lobe at the protein and mRNA level. (A) Protein abundance of apolipoproteins that were signiﬁcantly changed (Benjamini–Hochberg corrected p- value ≤0.05) between PVB compared to Cntrls in the upper and lower lobes are depicted as dots. n = 6 per group. (B–E) mRNA level of APOA, APOE, CD36 and LDLR, respectively, are expressed relative to the mean expression of the sham animals (values are mean +/−SEM. * p < 0.05, ** p < 0.01 Students’s t-test n = 5 animals per group). Figure 7. Fatty acid uptake pathway in the upper lobe at the protein and mRNA level. (A) Protein abundance of apolipoproteins that were signiﬁcantly changed (Benjamini–Hochberg corrected p- value ≤0.05) between PVB compared to Cntrls in the upper and lower lobes are depicted as dots. n = 6 per group. (B–E) mRNA level of APOA, APOE, CD36 and LDLR, respectively, are expressed relative to the mean expression of the sham animals (values are mean +/−SEM. * p < 0.05, ** p < 0.01 Students’s t-test n = 5 animals per group). 3. Discussion The main ﬁndings in this study were that (i) chronic alterations in ﬂow and pressure induced by PVB impact the proteomic and metabolomic proﬁle in the lung tissue and result in increased ECM and collagen production in lobes with both HP/HF and HP/LF; (ii) upper lung lobes with HP/HF adapt by altering the fatty acid metabolism as well as ROS signaling and (iii) the lower lobes with HP/LF increase their purine metabolism in order to cope with the increased demand of cellular proliferation (Figure 8). p p g We have previously demonstrated that PH caused by pulmonary vein stenosis results in a progressive increase in pulmonary vascular resistance, which is accompanied by func- tional (increased contribution of endothelin, phosphodiesterase 5) as well as structural (increased media thickness) pulmonary vascular remodeling [7,11]. Banding of the conﬂu- ence of veins from the lower lobes results in areas of the lungs with distinct hemodynamic proﬁles: HP/HF in the unbanded upper lobes and HP/LF in the banded lobes. This model is, therefore, well suited for the study of mechano-metabolic coupling and its role in pulmonary vascular remodeling in PH, as it has been demonstrated that metabolic and structural changes are coupled to each other [12]. Here, we present a thorough proteome and metabolome proﬁle analysis of lung tissue with these distinct mechanical proﬁles. Pathway enrichment analysis in PVB animals demonstrated changes in several pathways that have been associated with the progression of PH. Thus, alterations were observed for extracellular matrix proteins involving integrins, matrix metalloproteases, collagen, vitronectin, serpins and others observed in both lobes of the animals. This is in accordance Int. J. Mol. Sci. 2023, 24, 4870 9 of 15 9 of 15 with our histological data, suggesting increased ECM deposition around the vessels. We also detected high amounts of phosphatidylcholine (PC) as well as prostaglandins, which indicate plasma membrane break and inﬂammatory signaling due to high shear stress [13]. In line with these ﬁndings, the comparison with the lung matrix database revealed that further extracellular matrix proteins were altered in the upper and lower lobes of PVB swine (Supplementary Figure S1A). ECM proteins such as FGB, COL1A and COL15A1 were signiﬁcantly upregulated in the PVB lower lobe, whereas in the upper lobe, the analysis revealed synergistic downregulation of extracellular proteolytic proteins such as MMP9 and serpins [14,15]. Figure 8. Schematic illustration of the metabolic pathways altered in PVB. 3. Discussion 2023, 24, 4870 10 of 15 10 of 15 reﬂects inhibition of mitochondrial fatty acid β-oxidation and has been previously shown to be involved in the development of PH [22]. p In keeping with studies from Umar et al. who showed higher oxidized LDL in the lungs and plasma in PH with a decrease in CD36, our proteomic analysis did detect modiﬁcations of pathways regulating cholesterol levels consisting primarily of the down- regulation of fatty acid transporters such CD36 and LDLRAP1 [23]. Along with this, we detected signiﬁcant upregulation of the protein LDLR in the PVB upper lobe. LDLR mainly binds to apolipoprotein B100 (APOB) and APOE to clear cholesterol from the blood [24]. Both ApoB and ApoE are high-afﬁnity ligands for LDLR and are expressed in various immune and vascular cells [24,25]. Negative feedback inhibition from transcriptional and posttranscriptional mechanisms closely controls the LDLR pathway, and disruption of this tightly controlled pathway can inﬂuence lipid and cholesterol regulation [26]. These data are also in accordance with integrated proteomic and metabolomics data on HUVECs presented by Venturini et al., showing that high shear stress upregulates the lipoprotein metabolism and increases the expression of LDLR [27]. p Additionally, we found metabolites such as oxaloacetic acid and L-malic acid, both intermediate products of the TCA cycle, to be enriched in the PVB upper lobe along with decreased glucose. These metabolites take part in anaplerotic reactions in which the intermediate metabolites exit the TCA cycle and are used by proliferating cells due to an increased demand for protein and fatty acids in PH [12]. These data support the presence of the Warburg effect, showing that glucose metabolism is increased in PH [28–31]. Further evidence for this Warburg effect is the lower amount of NADP in the PVB upper lobe. NADP maintains the redox balance in the cells and supports the biosynthesis of the fatty acids and is essential for maintaining a large number of biological processe [32]. In agreement with this ﬁnding, Nukula et al. reported a lower NADPH/NADP ratio in CTEPH patients’ endothelial cells compared to healthy subjects, implying increased oxidative stress and endothelial cell dysfunction [30]. A key metabolite that was downregulated in the PVB upper lobe and deemed impor- tant from our network analysis was S-adenosylhomocysteine (SAH). 3. Discussion The blue-labeled metabo- lites and proteins are differentially expressed in the upper lobe, which is under high shear stress, and the green-labeled ones are differentially expressed in the lower lobe, which is under low shear stress. Pathways or metabolites/proteins labeled in red are found to be altered in both the lobes. 3PG—3- phosphoglyceric acid, SAM—S-adenosyl methionine, SAH—S-adenosylhomocysteine, NO—nitric oxide, PC—phosphatidyl choline, OAA—oxaloacetic acid. Figure 8. Schematic illustration of the metabolic pathways altered in PVB. The blue-labeled metabo- lites and proteins are differentially expressed in the upper lobe, which is under high shear stress, and the green-labeled ones are differentially expressed in the lower lobe, which is under low shear stress. Pathways or metabolites/proteins labeled in red are found to be altered in both the lobes. 3PG—3- phosphoglyceric acid, SAM—S-adenosyl methionine, SAH—S-adenosylhomocysteine, NO—nitric oxide, PC—phosphatidyl choline, OAA—oxaloacetic acid. Strikingly, proteins of the apolipoprotein family were signiﬁcantly altered in abun- dance in PVB animals. The key protein component of HDL-C, apolipoprotein A (APOA), which was downregulated in the upper lobe, was not shown to be differentially regulated in the lower lobe. Downregulation of APOA1 is in accordance with data showing that ApoA-1 is less prevalent in PH, which contributes to oxidative stress and endothelial dysfunction [16]. Furthermore, administration of a peptide mimetic of ApoA-1 reduced pulmonary hypertension in rodent models with PH [17]. Along with ApoA, we also de- tected signiﬁcantly reduced levels of ApoE at the proteomics as well as at the transcription level in the PVB upper lobe. The metabolomics data in the upper lobe further point to an ongoing alteration of lipid homeostasis and detected increased fatty acids such as oleic acid, linoleic acid, arachidonic acid and palmitic acid in the PVB group, indicating a reduced uptake of fatty acids due to decreased levels of apolipoproteins [18]. High amounts of linoleic and oleic acids have been found to signiﬁcantly lower nitric oxide (NO) levels in endothelial cells and exert their deleterious effects via ROS [11,19,20]. It has been shown that HIF-1α activation, a common dysregulated pathway in PH and lung diseases, can inhibit β-oxidation of long-chain fatty acids leading to accumulation of fatty acids [21]. However, we did not detect increased accumulation of carnitine and acyl-carnitine, which Int. J. Mol. Sci. 3. Discussion Asymmetric dimethyl arginine (ADMA), a negative regulator of endothelial nitric oxide synthase, is formed by the hydrolysis of methylated proteins, and the methylated proteins are derived when S-adenosyl methionine (SAM) is converted to SAH. We also simultaneously observed increased aspartic arginine (VIP > 1, Supplementary Data S2, Table S2), which is a source of NO in endothelial cells, in the upper lobe of the PVB group [23,24]. In our previous work, we have shown that NO production is increased in HP/HF areas, likely as a compensatory mechanism to maintain vasodilation [33]. Our current data suggest that low SAH, and hence low ADMA, in combination with high arginine, the substrate for endothelial NO synthesis, facilitates NO synthesis in the PVB upper lobe vasculature. y y pp Notably, proteomic and metabolic alterations were less pronounced between PVB and Cntrls in the lower lobes. The STRING analysis points to reduced glucose synthesis in the PVB lower lobes, which supports the notion that glycolysis predominates over other metabolic activities in PH in the lower lobes as well [22]. Another intriguing observation was that the purine pathway metabolites adenosine monophosphate (AMP) and guanosine monophosphate (GMP) were signiﬁcantly enriched, and the enzyme guanosine monophos- phate reductase (GMPR), which converts GMP to inosine monophosphate (IMP), was downregulated. Additionally, our proteome data revealed that the PVBs had a decreased abundance of the enzyme HPRT, which transforms hypoxanthine into IMP and is crucial for the salvage pathway for recycling nucleotides [34]. We also found more inosine in the metabolome of PVB lower lobes, which indicated that the cells increased de novo purine production rather than using the standard active salvage pathway. These data are consistent with the study by Hautbergue et al., wherein modiﬁcations to the purine metabolic pathway in the right ventricle and plasma of PH rats were shown [35]. The purine metabolite levels in endothelial cells from PAH patients have also been found to be higher, the same was true for the serine to glycine ratio, which is mediated by the mitochondrial enzyme serine Int. J. Mol. Sci. 2023, 24, 4870 11 of 15 11 of 15 hydroxymethytransferase (SHMT) [36]. Although SHMT was unchanged in our lung tissue samples, we did observe an increase in the metabolite serine (VIP > 1) in the PVB lower lobes (Supplementary Data 2, Table S4). 4. Materials and Methods Lung tissue was used from experiments that have previously been published [7,11,33]. These experiments followed the guiding principles in the care and use of laboratory animals, which are endorsed by the Council of the American Physiological Society, and the protocol was approved by the Animal Care Committee at Erasmus University Medical Center (EMC3158, 109-13-09). 3. Discussion Moreover, in atherosclerosis models, it has been shown that vessels with low ﬂow and shear stress have decreased endothelial nitric oxide synthase (eNOS) along with increased cell proliferation and collagen deposition [31]. 4.2. Real-Time Quantitative PCR of Lung Tissue Lung tissue was snap-frozen and 30 mg of tissue was homogenized, and mRNA was extracted using the RNeasy Fibrous Tissue Mini kit (Qiagen, Hilden, Germany). cDNA was synthesized using 500 ng of mRNA and the SenSi FAST cDNA synthesis kit (Bioline, London, UK). Target genes were normalized against beta-actin and cyclophilin using the CFX manager software 3.1 (BioRad, CA, USA). Relative gene expression was calculated using the delta–delta Ct method. 4.1. Outline of Study For all surgical procedures, swine were sedated with an intramuscular injection of a mixture of tiletamine/zolazepam (5 mg kg−1, Virbac, Barneveld, The Netherlands), xy- lazine (2.25 mg kg−1, AST Pharma, Oudewater, The Netherlands) and atropine (0.5 mg) and intubated and ventilated (O2:N2 (1:2)). Isoﬂurane (2% vol/vol, Pharmachemie, Haarlem, The Netherlands) was added to the gas mixture to induce anesthesia. Post- surgical analgesia was administered by means of an i.m. injection (0.3 mg buprenorphine i.m. Indivior, Slough, UK) and a fentanyl slow-release patch (6 or 12 µg h−1 depending on body weight, 72 h). Crossbred Landrace x Yorkshire pigs of either sex (8 ± 2 kg) underwent non-restrictive inferior pulmonary vein banding (n = 6) via the third right intercostal space or a sham pro- cedure (n = 7). All 13 animals, underwent chronic instrumentation 4 weeks later, enabling hemodynamic assessments on awake animals. Following a left-sided thoracotomy in the fourth intercostal space, ﬂuid-ﬁlled catheters (Braun Medical Inc., Bethlehem, PA, USA), were inserted in the aorta, the pulmonary artery, the left and right ventricle and the left atrium for the measurement of blood pressure. A ﬂow probe (20PAU, Transonic systems, Ithaca, NY, USA) was placed around the ascending aorta for the measurement of cardiac output. Aorta ﬂow was indexed to bodyweight. The total pulmonary vascular resistance index was calculated as the ratio of mean PAP and cardiac index, while pulmonary vascular compliance was calculated as stroke volume index/(systolic PAP −diastolic PAP). Hemo- dynamics were recorded (WinDaq, Dataq Instruments, Akron, OH, USA) in the awake state, with swine standing quietly, and analyzed ofﬂine using a custom written program (Matlab, version R2007b, The MathWorks). Twelve weeks after the PVB procedure, swine were re-anesthetized; the thorax was opened using sternotomy, and the heart and lungs were excised, snap-frozen in liquid nitrogen and processed for further analysis. 4.2. Real-Time Quantitative PCR of Lung Tissue 4.3. Proteomics 4.3.1. Sample Preparation for Proteome Analysis 4.3.2. Nano-Liquid Chromatography (LC)–Tandem Mass Spectrometry (MS) Analysis and Bioinformatics 4.3.2. Nano-Liquid Chromatography (LC)–Tandem Mass Spectrometry (MS) Analysis and Bioinformatics 1 µg of the digest was injected on an UltiMate 3000 nano-LC system coupled online to a Q Exactive HF-X instrument operated in the data-dependent acquisition (DDA) mode. Peptides were transferred to a PepMap 100 C18 trap column (100 µm × 2 cm, 5 µM parti- cles, Thermo Fisher Scientiﬁc) and separated on an analytical column (PepMap RSLC C18, 75 µm × 50 cm, 2 µm particles, Thermo Fisher Scientiﬁc) at a 250 nL/min ﬂow rate with a 160 min gradient of 3–25% of solvent B followed by a 10 min ramp to 40% and a 5 min ramp to 85%. Solvent A consisted of 0.1% formic acid in water and solvent B of 0.1% FA in acetonitrile. MS spectra were acquired using a top-15 data-dependent acquisition method on a Q Exactive HF-X mass spectrometer. Protein identiﬁcation was carried out using MaxQuant (v.1.6.7.0) [37] and the NCBI RefSeq Sus scrofa database (v.7-5-2020). All statisti- cal analyses and data visualization were performed using R (https://www.r-project.org/) (accessed on 29 December 2022). Prior to statistical analysis, potential contaminants, only identiﬁed by site and reverse hits were excluded. Proteins with at least two pep- tides detected in at least three samples of each condition were quantiﬁed using the MS-EmpiRe algorithm as previously described [38,39]. The R script used for quantita- tive analysis is available at https://github.com/bshashikadze/pepquantify (accessed on 7 September 2022). Proteins with a Benjamini–Hochberg corrected p-value ≤0.05 and fold change ≥1.5 were regarded as signiﬁcantly altered. Preranked gene set enrichment anal- ysis using STRING was employed to reveal biological processes related to differentially abundant proteins [40]. Signed (based on fold change) and log-transformed p-values were used as ranking metrics and the false discovery rate was set to 1%. The redundancy of the signiﬁcantly enriched biological processes was minimized using REVIGO tool [41]. 4.3.1. Sample Preparation for Proteome Analysis Frozen lung tissue samples were placed into precooled tubes and cryopulverized in a CP02 Automated Dry Pulverizer (Covaris, Woburn, MA, USA) with an impact level of 5 according to the manufacturer’s instructions. Tissue lysis was performed in 8 M urea/ 0.5 M NH4HCO3 with ultrasonication (18 cycles of 10 s) using a Sonopuls HD3200 (Bandelin, Int. J. Mol. Sci. 2023, 24, 4870 12 of 15 Berlin, Germany). Total protein concentration was quantiﬁed using a Pierce 660 nm Protein Assay (Thermo Fisher Scientiﬁc, Rockford, IL, USA). Fifty micrograms of protein were digested sequentially, ﬁrstly with Lys-C (FUJIFILM Wako Chemicals Europe GmbH, Neuss, Germany) for 4 h and, subsequently, with modiﬁed porcine trypsin (Promega, Madison, WI, USA) for 16 h at 37 ◦C. Berlin, Germany). Total protein concentration was quantiﬁed using a Pierce 660 nm Protein Assay (Thermo Fisher Scientiﬁc, Rockford, IL, USA). Fifty micrograms of protein were digested sequentially, ﬁrstly with Lys-C (FUJIFILM Wako Chemicals Europe GmbH, Neuss, Germany) for 4 h and, subsequently, with modiﬁed porcine trypsin (Promega, Madison, WI, USA) for 16 h at 37 ◦C. 4.4. Metabolomics Approximately 50 mg of sample material was weighed in a 2 mL bead beater tube (CKMix, Bertin Technologies, Montigny-le-Bretonneux, France) ﬁlled with 2.8 mm and 5.0 mm ceramic beads. Then, 1 mL of a methanol/water mixture (70/30, v/v) was added, and the samples were extracted with a bead beater (Precellys Evolution, Bertin Technolgies, Montigny-le-Bretonneux, France) supplied with a Cryolys cooling module 3 times each for 20 s with 15 s breaks in between at 10,000 rpm. After centrifugation at 13,000 U/min for 10 min, the supernatants were dried by vacuum centrifugation, suspended in 150 µL of methanol/water (70/30, v/v) and subjected to MS analysis. j y Untargeted analysis was carried out on a Nexera UHPLC system connected to a Q-TOF mass spectrometer (TripleTOF 6600, AB Sciex, MA, USA). Chromatographic separation was achieved by using a HILIC UPLC BEH Amide 2.1 × 100, 1.7 µm column with a 0.4 mL/min flow rate. The mobile phase consisted of 5 mM ammonium acetate in water (eluent A) and 5 mM ammonium acetate in acetonitrile/water (95/5, v/v) (eluent B). The following gradient profile was used: 100% B from 0 to 1.5 min, 60% B at 8 min, 20% B at 10 min to 11.5 min and 100% B at 12 to 15 min. Aliquots of 5 µL per sample were injected into the UHPLC-TOF-MS. The autosampler was cooled to 10 ◦C, and the column oven was heated to 40 ◦C. A quality control (QC) sample was pooled from all samples and injected after every 10 samples. MS settings in the positive mode were as follows: gas 1 55, gas 2 65, curtain gas 35, temperature 500 ◦C, ion spray voltage 5500, declustering potential 80. The mass range of the TOF-MS scans was 50–2000 m/z, and the collision energy was ramped from 15 to 55 V. MS settings in the negative mode were as follows: gas 1 55, gas 2 65, cur 35, temperature 500 ◦C, ion spray voltage −4500, Int. J. Mol. Sci. 2023, 24, 4870 13 of 15 13 of 15 declustering potential −80. The mass range of the TOF-MS scans was 50–2000 m/z, and the collision energy was ramped from −15 to −55 V. gy p The “msconvert” tool from ProteoWizard [42] was used to convert raw ﬁles to mzXML (denoised by centroid peaks). The bioconductor/R package xcms [43] was used for data processing and feature identiﬁcation. 4.4. Metabolomics More speciﬁcally, the matched ﬁlter algorithm was used to identify peaks (full width at half maximum set to 7.5 s). Then the peaks were grouped into features using the “peak density” method. The area under the peak was integrated to represent the abundance of features. The retention time was adjusted based on the peak groups presented in most samples. To annotate features with the names of metabolites, the exact mass and MS2 fragmentation pattern of the measured features were compared to the records in HMBD [44] and the public MS/MS spectra in MSDIAL [45], referred to as MS1 and MS2 annotation, respectively. Missing values were imputed with half of the limit of detection (LOD) methods, i.e., for every feature, the missing values were replaced with half of the minimal measured value of that feature in all measurements. To conﬁrm that an MS2 spectrum was well annotated, we manually reviewed our MS2 fragmentation pattern and compared it with records in the public database or previously measured reference standards to evaluate the correctness of the annotation. The MetaboAnalyst 5.0 platform was utilized to conduct multivariate data analysis, for PCA and OPLS-DA. The contribution of each variable to the classiﬁcation was indicated by the VIP value that was calculated in the OPLS-DA model after Pareto scaling. The Student’s t-test at the univariate level was further employed to measure the signiﬁcance of metabolites with VIP > 1.0. Metabolites with a p-value < 0.1 were considered as differential metabolites, while those with a p-value < 0.05 were recognized as statistically signiﬁcant differential metabolites. Enrichment analysis and network analysis was performed using only the signiﬁcant metabolites and signiﬁcant genes using the KEGG pathway database. 5. Conclusions In conclusion, our combined omics study showed PVB-related key metabolic alter- ations in a compartment-speciﬁc manner. The combination of a model of PH, with speciﬁc changes in shear stress in different areas of the lung, with proteome and metabolomic data shows that particular metabolic pathways, including fatty acid absorption and purine synthesis, are altered in early PH. Such a deeper understanding of the metabolic changes in lung tissue may provide new targets for therapy and may, thereby, pave the way for new avenues in precision medicine for PH. Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/ijms24054870/s1. Author Contributions: Conceptualization, P.S. and D.M.; methodology, P.S., B.S., F.F., M.G. and C.M.; validation, P.S., B.S., E.V.d.K.; data curation, P.S., B.S.; S.T. and C.M.; draft preparation, P.S. and D.M.; reviewing, T.F., B.S., D.M. and M.G. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement No. 812660 (DohART-NET) (for B.S. and T.F.), the Friedrich Bauer Foundation Reg Nr-28/21 (for P.S.) as well as by the German Center for Cardiovascular Research (DZHK81Z0600207 to D.M. and P.S.) and the Dutch CardioVascular Alliance: An initiative with support of the Dutch Heart Foundation (Grants 2020B008 RECONNEXT to D.M.). Institutional Review Board Statement: These experiments followed the guiding principles in the care and use of laboratory animals, which are endorsed by the Council of the American Physiological Society, and the protocol was approved by the Animal Care Committee at Erasmus University Medical Center (EMC3158, 109-13-09). Int. J. Mol. Sci. 2023, 24, 4870 14 of 15 14 of 15 Data Availability Statement: The mass spectrometry proteomics dataset has been deposited to the PRIDE repository, dataset identiﬁer PXD038982. The untargeted metabolomics data have been uploaded to the MassIVE database https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp with ID: MSV000090966 (accessed on 24 December 2022). Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Proteomics 2012, 11, M111.0 11. Du, X.; Edelstein, D.; Obici, S.; Higham, N.; Zou, M.-H.; Brownlee, M. Insulin resistance reduces arterial prostacyclin synthase and eNOS activities by increasing endothelial fatty acid oxidation. J. Clin. Investig. 2006, 116, 1071–1080. [CrossRef] [PubMed] 11. Du, X.; Edelstein, D.; Obici, S.; Higham, N.; Zou, M.-H.; Brownlee, M. Insulin resistance reduces arterial prostacyclin synthase and eNOS activities by increasing endothelial fatty acid oxidation. J. Clin. Investig. 2006, 116, 1071–1080. [CrossRef] [PubMed] 12. Raﬁkova, O.; Al Ghouleh, I.; Raﬁkov, R. 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https://openalex.org/W4281627708	https://zenodo.org/records/6611674/files/%D0%90%D0%BA%D0%BC%D0%B0%D0%BB%D0%B6%D0%BE%D0%BD%20%D0%98%D0%BA%D1%80%D0%BE%D0%BC%D0%BE%D0%B2.pdf	Russian	null	ЗАМОНАВИЙ ШАҲАРЛАР ЙЎЛ-КЎЧА ТАРМОҒИДАГИ ЧОРРАҲАЛАРНИ ТАРТИБГА СОЛИШ ТУРЛАРИНИНГ ТАҲЛИЛИ	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	1,709	ЗАМОНАВИЙ ШАҲАРЛАР ЙЎЛ-КЎЧА ТАРМОҒИДАГИ ЧОРРАҲАЛАРНИ ТАРТИБГА СОЛИШ ТУРЛАРИНИНГ ТАҲЛИЛИ https://doi.org/10.5281/zenodo.6611674 Акмалжон Икромов Тошкент давлат транспорт университети, PhD Кучкаров Хуршид Тошкент давлат транспорт университети, ассистент Шаҳар йўллари тармоғи учун светофорни бошқариш бир неча ўн йиллар давомида энг юқори самарадорликни таъминлашнинг муҳим йўналишларидан бири бўлиб келган. Бу даврда унинг турли услуб ва воситаларини ишлаб чиқиш бўйича бир қатор тадқиқотлар олиб борилган. Аммо автомобиллар сонининг доимий кўпайиши ва бунинг натижасида йўл шароитлари ёмонлашиши ва тирбандликлар сонининг ортиши, мавжуд муаммоларни ҳал қилишга имкон берадиган янги, илғор усулларни ҳамда келгусидаги вазиятни башорат қилишни талаб қилади. Замонавий шаҳарлар ривожланишида йўл-кўча тармоғидаги тирбандликнинг ортиб бораётгани муаммо бўлиб қолмоқда. Шахсий транспорт воситаларидан фойдаланиш даражасини камайтириш ёки қопламали йўллар ҳажмини ошириш эвазига тирбандликлар сонини қисқартиришга эришиш мумкин [25]. Йўл инфратузилмасини инобатга олган ҳолда йўллар сонини доимий равишда ошириш мумкин эмас. Шунга кўра, транспорт оқимини бошқариш эвазига тирбандликни камайтириш ва бунинг натижасида шаҳарларнинг транспорт мобиллигини ошириш зарур. Йирик шаҳарларнинг йўл-кўча тармоғида транспорт воситаларининг давомий кечикиши, йўл-кўча тармоғининг геометрик параметрлари билан доимий равишда ортиб бораётган ҳаракат интенсивлиги ўртасидаги номувофиқлик оқибати ҳисобланади. Маълумки, йўллар ҳамда кўчалар сонининг кўпайиши ва бир сатҳдаги чорраҳалар кесишмаларини турли сатҳли чорраҳаларга қайта қуриш йўл тармоғи бўйлаб транспорт оқимларининг қайта тақсимланишига ва ўтказувчанлик қобилияти кичик бўлган йўл-кўча тармоғи ҳудудлари камайишига олиб келади. Лекин янги йўллар ва уларнинг элементларини қуриш жуда катта харажат талаб қилади. Шунга кўра, биринчи навбатда йўл-кўча тармоғи ҳаракат сиғимининг мавжуд захираларини аниқлаш ва улардан фойдаланиш, сўнгра йўл-кўча тармоғи участкаларида конструктив ўзгаришлар бўйича қарор қабул қилиш талаб этилади. 1 6 – расм Бир сатҳли чорраҳаларни қўлланиш соҳаси [24] 1.6 – расм. Бир сатҳли чорраҳаларни қўлланиш соҳаси [24] 1.6 – расм. Бир сатҳли чорраҳаларни қўлланиш соҳаси [24] Узоқ муддатли тирбандликка эга бўлмаган йўл-кўча тармоғи участкалари ўтказиш қобилиятини ошириш чораларини талаб қилмайди, шунинг учун йўл тармоғининг тор жойларини аниқлаш ва уларнинг ўтказувчанлиги захираларини баҳолаш керак. Шаҳарнинг зич аҳоли яшаш худудларида айнан тартибга солинган чорраҳалар кўпроқ транспорт воситасини ўтказиш имкониятига эга бўлади. 1.6- расмда автомобиль йўлларининг бир сатҳли турли кесишмалари учун талаб этиладиган майдоннинг ҳаракат интенсивлиги келтирилган. Шаҳарнинг зич аҳоли яшаш худудларида айнан тартибга солинган чорраҳалар кўпроқ транспорт воситасини ўтказиш имкониятига эга бўлади. 1.6- расмда автомобиль йўлларининг бир сатҳли турли кесишмалари учун талаб этиладиган майдоннинг ҳаракат интенсивлиги келтирилган. Жамоат транспорти ва йўл ҳаракати иштирокчилари (велосипедлар ва пиёдалар) ҳаракатланиш сифати чорраҳалар светофор объектларини мос равишда ростлаш эвазига сезиларли даражада яхшиланади. Жамоат транспорти ва йўл ҳаракати иштирокчилари (велосипедлар ва пиёдалар) ҳаракатланиш сифати чорраҳалар светофор объектларини мос равишда ростлаш эвазига сезиларли даражада яхшиланади. ЗАМОНАВИЙ ШАҲАРЛАР ЙЎЛ-КЎЧА ТАРМОҒИДАГИ ЧОРРАҲАЛАРНИ ТАРТИБГА СОЛИШ ТУРЛАРИНИНГ ТАҲЛИЛИ Катта ҳаракат интенсивлигига эга бўлган юқори тоифали автомобиль йўлларида светофор бошқаруви ушбу йўлларга киришни чеклаш ва йўллардан чиқиш шароитларини яхшилашга қаратилади. Катта ҳаракат интенсивлигига эга бўлган юқори тоифали автомобиль йўлларида светофор бошқаруви ушбу йўлларга киришни чеклаш ва йўллардан чиқиш шароитларини яхшилашга қаратилади. Ҳаракатланиш йўналишлари бўйича интенсивлиги кескин фарқланадиган йўлларда йўлакнинг ҳаракат йўналишини ўзгарувчан қилиш учун светофор бошқарувини алоҳида йўлаклар бўйича ташкил этиш мумкин. Ҳаракатланиш йўналишлари бўйича интенсивлиги кескин фарқланадиган йўлларда йўлакнинг ҳаракат йўналишини ўзгарувчан қилиш учун светофор бошқарувини алоҳида йўлаклар бўйича ташкил этиш мумкин. Аксарият ҳолатларда светофор бошқаруви транспорт инфратузилмаси объектлари майдонини камайтиришга имкон беради. Масалан, чорраҳани ҳалқали кесишма билан алмаштириш эвазига йўл майдонини қисқартириш ва ўтказувчанлик қобилиятини ошириш [26]. Қурилмалар доимий равишда белгиланган тизимга мувофиқ ёки ўзгарувчан интервалга эга сигнал асосида ишлайди. Шунга кўра, светофорлар доимий ва мослаштирилган (адаптив) тартибга солувчи турларга ажратилади. Биринчи ҳолда қурилма ҳафтанинг куни, тирбандлиги ва куннинг вақтидан қатъи назар, доимий режимда ишлайди. Катта шаҳарларда бундай светофорлар кўпинча мослаштирилган светофорлар билан алмаштирилади. Натижада, сигнал даврларининг давомийлиги йўл ҳаракатини самарали ташкил этиш учун хизмат ф б ф У б қилади. Светофор режими тиғиз ва кундузги/тунги вақтларда фарқланади. Ушбу бошқарув тирбандликларни олдини олишга ёрдам беради. ади. Светофор режими тиғиз ва кундузги/тунги вақтларда фарқланади. Ушбу қилади. Светофор режими тиғиз ва кундузги/тунги вақтларда фарқланади. Ушбу бошқарув тирбандликларни олдини олишга ёрдам беради. Бошқариладиган чорраҳаларда светофор сигналларини автоном ва мувофиқлаштирилган ҳолда бошқариш мумкин. Автоном бошқариш битта алоҳида чорраҳадаги ҳаракатни бошқа яқин чорраҳадаги вазиятни ҳисобга олмаган ҳолда бошқаришдир. Бундай бошқариш чорраҳалар орасидаги масофа 1000 метрдан кам бўлмаганда қўлланилади. Мувофиқлаштирилган бошқариш бир нечта чорраҳадаги бошқариш бир- бири билан биргаликда вазиятига қараб ҳаракатни бошқаришдир. Бундай бошқариш чорраҳалар орасидаги масофа 150–600 м бўлганида тавсия этилади. Мувофиқлаштирилган бошқаришнинг автоном бошқаришдан афзаллиги қуйидагилардан иборат: ўтказиш қобилияти ва ҳаракат тезлиги ошади; ёнилғи сарфи, атмосферанинг булғаланиши, транспорт шовқини ва ЙТҲ камаяди; автомобилнинг тормоз тизими ва бошқа механизмларининг ишлаш муддати ортади. Мувофиқлаштирилган бошқаришнинг синхронли ва прогрессив тизими мавжуд. Синхронли тизимда барча чорраҳалардаги светофорларда бир вақтнинг ўзида бир хил сигнал ёнади ва алмашади (бу тизим «яшил кўча» деб юритилади). Прогрессив тизимда сигналлар чорраҳадаги светофорларга, ҳаракат тезлигига ва миқдорига қараб бирор вақт бирлигида суриб берилади ёки кўча «яшил тўлқин» режимида ишлайди. “Яшил тўлқин” ни фақат доимий ва мослаштирилган (адаптив) тартибга солувчи, фазалар давомийлиги ўзгаришини ростловчи светофорларда амалга ошириш мумкин. Айланма ҳаракатланиш ташкил қилинган чорраҳа – яқинлашиб келаётган автомобиллар ўнг томонлама ҳаракатланиш йўлларида соат стрелкасига тескари йўналишда ва чап томонлама ҳаракатланиш йўлларида соат стрелкаси йўналишида марказий майдон атрофидан айланиб ўтувчи чорраҳадир. Бундай чорраҳалар, одатда, светофор билан жиҳозланмаган ва тартибга солинмаган бўлади. Транспорт ҳаракатининг устуворлиги белгиланган йўл белгилари ва/ёки чорраҳаларни кесиб ўтишнинг бошқа қоидалари билан белгиланиши мумкин. Ҳалқали чорраҳалар бир сатҳли бошқа чорраҳаларга нисбатан камроқ йўл- транспорт ҳодисаси, кечикишлар ва юқори ўтказувчанлик билан тавсифланади. Кесишма элементларининг жойлашуви ҳаракат тезлигини пасайиши ва унинг ўзгариш диапазонига сезиларли таъсир кўрсатади. Автомобиль тезликларининг кичик диапазонда ўзгариши (тезликларнинг ўртача қийматдан стандарт оғиши) ҳалқа йўли ташқи қиррасининг диаметри 25–30 метрдан ошмайдиган ҳалқали кесишмага хосдир. Ушбу таъсир асосий ва иккинчи даражали йўналишдаги автомобиллар учун бир хил. Умуман олганда, йўллар учун ҳалқали кесишма транспортнинг “тинчлантирувчиси” бўлиб, чорраҳага яқинлашганда транспорт тезлигини муаммосиз пасайтириш учун тегишли чораларни талаб қилади. 1.7-расм. Оддий ва ҳалқали чорраҳаларнинг тўқнашув нуқталари [24] 1.7-расм. Оддий ва ҳалқали чорраҳаларнинг тўқнашув нуқталари [24] 1.7 расм. Оддий ва ҳалқали чорраҳаларнинг тўқнашув нуқталари [24] Бир сатҳдаги айланма чорраҳалар бир қатор афзалликларга эга. АДАБИЁТЛАР: 1. Ўзбекистон Республикаси Президентининг 11 июль 2017 йил 3127- сонли “Йўл ҳаракати ҳавфсизлигини таъминлаш тизимини янада такомиллаштириш чора-тадбирлари тўғрисида”ги қарори 2. 2018-2022 йилларда Ўзбекистон Республикасида йўл ҳаракати хавфсизлигини таъминлаш концепциясини 3. Ўзбекистон Республикаси ИИВ ЙҲХББ статистика маълумоти 4. Усманова М.Н.Подход оценки факторов навыки водителя при обеспечения безопасности движения.Вестник ТАДИ.Научно-технический журнал.4.2016г.90-93стр. 4. Усманова М.Н.Подход оценки факторов навыки водителя при обеспечения безопасности движения.Вестник ТАДИ.Научно-технический журнал.4.2016г.90-93стр. 4. Усманова М.Н.Йирик шаҳарларда йўл ҳаракатини ташкил этиш йўналишлари.Biznes-Эксперт.№8,2019 йил.42-44бет 5. Eisymont, Y., Auchynnikau, Y., Avdeychik, S., Ikramov, A., & Grigorieva, T. (2015). Mechanochemical processes in the formation of engineering materials based on polymers. Materials Science. Non-Equilibrium Phase Transformations., 1(1), 36-41. 6. Avdeychik, S., Goldade, V., Struk, V., Antonov, A., & Ikromov, A. (2020). THE PHENOMENON OF NANOSTATE IN MATERIAL SCIENCE OF FUNCTIONAL COMPOSITES BASED ON INDUSTRIAL POLYMERS. Theoretical & Applied Science, (7), 101-107. 6. Avdeychik, S., Goldade, V., Struk, V., Antonov, A., & Ikromov, A. (2020). THE PHENOMENON OF NANOSTATE IN MATERIAL SCIENCE OF FUNCTIONAL COMPOSITES BASED ON INDUSTRIAL POLYMERS. Theoretical & Applied Science, (7), 101-107. 7. Mukhitdinov A., Kutlimuratov K., Assessing the operational impacts of road intersection using ptv vissim microscopic simulation. International Journal of Advanced Research in Science, Engineering and Technology, (2021), 18682-18690, 8(12) 7. Mukhitdinov A., Kutlimuratov K., Assessing the operational impacts of road intersection using ptv vissim microscopic simulation. International Journal of Advanced Research in Science, Engineering and Technology, (2021), 18682-18690, 8(12) r A 8 c 8. Mukhitdinov A., Ruzimov S., Mavlonov J., Analysis of the powertrain component size of electrified vehicles commercially available on the market. Mechanical Engineering in Transport, (2021), 74-86, 24(1) 9. Abdurazzokov U., Sattivaldiyev B., Khikmatov R., Ziyaeva Sh., Method for assessing the energy efficiency of a vehicle taking into account the load under operating conditions, E3S Web of Conferences, (2021), DOI: 10.1051/e3sconf/202126405033 9. Abdurazzokov U., Sattivaldiyev B., Khikmatov R., Ziyaeva Sh., Method for assessing the energy efficiency of a vehicle taking into account the load under operating conditions, E3S Web of Conferences, (2021), DOI: 10.1051/e3sconf/202126405033 10. Mukhitdinov A., Ziyaev K., Omarov J., Methodology of constructing driving cycles by the synthesis, E3S Web of Conferences, (2021), DOI: 10.1051/e3sconf/202126401033 10. Mukhitdinov A., Ziyaev K., Omarov J., Methodology of constructing driving cycles by the synthesis, E3S Web of Conferences, (2021), DOI: 10.1051/e3sconf/202126401033 11. ф б Масалан: йўналишларда ҳаракат интенсивлиги нисбатларини ўзгартирган ҳолда светофорларсиз ҳаракатни ташкил этиш имконияти; чорраҳадан 4 ёки ундан ортиқ йўлни кесиб ўтишда ҳаракатни янада самарали ташкил этишга эришиш; транспорт воситалари ҳайдовчилари учун энг оддий, қулай ва тушунарли ҳаракат шароитларини шакллантириш; турли сатҳли транспорт боғламаларини қуришга нисбатан капитал харажатларнинг камлиги; кичик архитектура шакллари ва ободонлаштиришни қўллаш орқали чорраҳаларнинг меъморий ва режалаштириш сифатини ошириш; бир хил даражадаги бошқа турдаги чорраҳаларга нисбатан ҳаракат хавфсизлиги даражасини 1,5–3 баробар ошириш; айланма чорраҳалар қурилмаси йўл транспорт ҳодисалари ҳолатини ўзгартиришга ва авария оқибатлари оғирлигини камайтиришга ёрдам беради. Улардан фойдаланиш йўл-транспорт ҳодисалари 35фоизга камайишига олиб келади. Ўлим ҳолатлари – 90 фоизга, жароҳатлар – 75 фоизга, пиёдалар тўқнашуви – 35 фоизга ва велосипед билан боғлиқ ҳодисалар – 10 фоизга камаяди. Бахтсиз ҳодисалар оқибатининг оғирлиги нуқтаи назаридан айланма чорраҳалар шаҳардан ташқари йўлларда энг самарали ҳисобланади. Бу ерда бир хил даражадаги чорраҳаларда ўлим сони 2 баравардан кўпроққа камайган. Шаҳар айланмаларида бу кўрсаткич 18–20 фоизни ташкил қилади. АДАБИЁТЛАР: Kulmukhamedov Zh., Khikmatov R., Saidumarov A., Kulmukhamedova Y., Theoretical research of the external temperature influence on the traction and speed properties and the fuel economy of cargo-carrying vehicles, Journal of Applied Engineering Science, (2021), 19(1), DOI: 10.5937/jaes0-27851 11. Kulmukhamedov Zh., Khikmatov R., Saidumarov A., Kulmukhamedova Y., Theoretical research of the external temperature influence on the traction and speed properties and the fuel economy of cargo-carrying vehicles, Journal of Applied Engineering Science, (2021), 19(1), DOI: 10.5937/jaes0-27851 12. Mukhitdinov A., Kutlimuratov K., Khakimov Sh., Samatov R., Modelling traffic flow emissions at signalized intersection with PTV Vissim, E3S Web of Conferences, (2021), DOI: 10.1051/e3sconf/202126402051 13. Mukhitdinov A., Kutlimuratov K., Impact of stops for bus delays on routes, IOP Conference Series: Earth and Environmental Science, (2020), ISSN 1755- 1315, DOI: 10.1088/1755-1315/614/1/012084 12. Mukhitdinov A., Kutlimuratov K., Khakimov Sh., Samatov R., Modelling traffic flow emissions at signalized intersection with PTV Vissim, E3S Web of Conferences, (2021), DOI: 10.1051/e3sconf/202126402051 12. Mukhitdinov A., Kutlimuratov K., Khakimov Sh., Samatov R., Modelling traffic flow emissions at signalized intersection with PTV Vissim, E3S Web of Conferences, (2021), DOI: 10.1051/e3sconf/202126402051 13. Mukhitdinov A., Kutlimuratov K., Impact of stops for bus delays on routes, IOP Conference Series: Earth and Environmental Science, (2020), ISSN 1755- 1315, DOI: 10.1088/1755-1315/614/1/012084 13. Mukhitdinov A., Kutlimuratov K., Impact of stops for bus delays on routes, IOP Conference Series: Earth and Environmental Science, (2020), ISSN 1755- 1315, DOI: 10.1088/1755-1315/614/1/012084
https://openalex.org/W3113032096	https://www.aclweb.org/anthology/2020.coling-main.173.pdf	English	null	Encoding Lexico-Semantic Knowledge using Ensembles of Feature Maps from Deep Convolutional Neural Networks	null	2,020	cc-by	9,458	Encoding Lexico-Semantic Knowledge using Ensembles of Feature Maps from Deep Convolutional Neural Networks Steven Derby Paul Miller Barry Devereux Steven Derby Paul Miller Barry Devereux Steven Derby Abstract Semantic models derived from visual information have helped to overcome some of the limita- tions of solely text-based distributional semantic models. Researchers have demonstrated that text and image-based representations encode complementary semantic information, which when combined provide a more complete representation of word meaning, in particular when com- pared with data on human conceptual knowledge. In this work, we reveal that these vision-based representations, whilst quite effective, do not make use of all the semantic information available in the neural network that could be used to inform vector-based models of semantic representa- tion. Instead, we build image-based meta-embeddings from computer vision models, which can incorporate information from all layers of the network, and show that they encode a richer set of semantic attributes and yield a more complete representation of human conceptual knowledge. Proceedings of the 28th International Conference on Computational Linguistics, pages 1906–1921 Barcelona, Spain (Online), December 8-13, 2020 This work is licensed under a Creative Commons Attribution 4.0 International Licence. Licence details: http:// creativecommons.org/licenses/by/4.0/. Encoding Lexico-Semantic Knowledge using Ensembles of Feature Maps from Deep Convolutional Neural Networks Steven Derby Paul Miller Barry Devereux Queen’s University Belfast, Belfast, United Kingdom {sderby02, p.miller, b.devereux}@qub.ac.uk Encoding Lexico-Semantic Knowledge using Ensembles of Feature Maps from Deep Convolutional Neural Networks Steven Derby Paul Miller Barry Devereux Queen’s University Belfast, Belfast, United Kingdom {sderby02, p.miller, b.devereux}@qub.ac.uk 1 Introduction These insights have motivated the development of 1906 Proceedings of the 28th International Conference on Computational Linguistics, pages 1906–1921 Barcelona, Spain (Online), December 8-13, 2020 multimodal semantic representations, which learn from multiple sources of information to ground these representations in the real world – an approach most successfully demonstrated by models that combine data from text and images (Bruni et al., 2014; Kiela and Bottou, 2014; Lazaridou et al., 2015b; Silberer, 2017). Generally, when image data is incorporated into the construction of word embedding spaces, vectors are derived from the penultimate layer of a deep convolutional neural network trained on an image classiﬁcation task. Although not usually explicitly stated, the rationale for using the penultimate layer of the network is that this layer should be the layer that is maximally relevant to the predicted label for the objects, whilst also being the layer that is least inﬂuenced by the low-level visual noise due to the details of the sampled training images. However, it is possible that meaningful object knowledge is present in lower layers of the network even if such information is not directly relevant to the object labelling task that the computer vision model is trained on. For example, in human property listing data, the property is-green is reliably given for FROG. In a computer vision model trained to discriminate between object classes, however, the greenness of frogs may not be strongly relevant to discriminating between images of frogs and images of other amphibians or reptiles (which also tend to be green) and so this information – highly relevant to human semantic representations – may not be strongly represented in the penultimate layer. In this work, we demonstrate that whilst using the penultimate layer of a computer vision model is an effective way to capture concept semantics, this approach is not optimal with respect to the goal of producing representations that encode information about cognitively-relevant semantic attributes of con- cepts. Using human property norm data, we demonstrate that certain features and feature types are more decodable at particular layers of the network than others. To produce representations that can make use of all the available semantics-relevant information from the network, we merge the feature map information by constructing meta-embeddings using information from all convolutional layers. We demonstrate that these ensembles of distributional models produce more complete representations of conceptual meaning, when evaluated against human conceptual knowledge. 1 Introduction Many approaches to representing the meaning of imageable, concrete concepts (e.g. FROG, APPLE, CAR, GUITAR) have been developed in the ﬁelds of cognitive science, computational linguistics and computer vision. Most explicitly, property listing studies have been used in cognitive psychology and cognitive neuroscience to characterise word meaning in terms of discrete semantic properties (McRae et al., 2005; Devereux et al., 2014; Buchanan et al., 2019). In property listing studies, human participants enumerate as many features as they can for each concept word, and these responses are then aggregated and normalised to a set of verbal semantic descriptors that correspond to elements of concept meaning (e.g. does-croak for FROG). This gives a representation of each concept as a sparse vector which encodes the semantic properties that occur for that concept. The resulting properties can then been applied to research investigating the organisation of semantic processing across the cortex, and to studies of the speed or ease of semantic processing for different concepts and different types of concept knowledge (Fieder et al., 2019; Evans et al., 2019; Kivisaari et al., 2019a; Bruffaerts et al., 2019). A desirable trait of semantic property norms is their interpretability, since this interpretability facili- tates the design of cognitive experiments on conceptual semantics (Murphy, 2004). This interpretability has also allowed researchers in NLP interested in distributional lexical semantics to gain better insights into the kinds of information that dense vector space models attain from pretraining. Even though many state-of-the-art vector space models of word meaning perform well when evaluated on both intrinsic and downstream tasks (Mikolov et al., 2013; Pennington et al., 2014; Bojanowski et al., 2017), researchers have demonstrated that such models often fail to fully encode certain facets of conceptual meaning (Li and Gauthier, 2017). For example, taxonomic properties (i.e. properties describing the object category, such as is-an-amphibian for FROG) and properties reﬂecting encyclopedic information or information about object function tend to be well-represented in the vector space, but properties that correspond to other kinds of attributes, such as colour, form, and modes of motion, tend to be poorly encoded (Ru- binstein et al., 2015; Collell and Moens, 2016). 1 Introduction To our knowledge, ours is the ﬁrst work to con- sider and evaluate the use of all layers of computer vision models for constructing semantic models, and is the ﬁrst to consider a range of recent computer architectures in building ensembled and multi-modal representations. Finally, we demonstrate how these meta-embeddings can be used in a zero-shot property mapping task, which allows us to automate the generation of interpretable semantic properties for unseen concepts. We make our code, embeddings, and analysis pipleine openly available1. 1Github link to code at https://github.com/stevend94/Decoding-Semantic-Properties. 2 Related Work Whilst word embedding vectors derived from text data have a long history of proven utility on a wide range of downstream tasks, they have been shown to struggle with encoding certain types of semantically- relevant information. Directly analysing the relationship between explicit property knowledge found in property norm data and the information in pretrained distributional models shows that particular proper- ties relating to more sensory or perpetual information about object semantics can be poorly captured, compared with more associative and encyclopedic knowledge (Rubinstein et al., 2015; Collell and Moens, 2016; Li and Gauthier, 2017). Sommerauer and Fokkens (2018)), using probing classiﬁers, showed that many of these properties may not be decodable at all from text-based embedding spaces. Findings such as these have motivated researchers to incorporate multimodal information into represen- tations of word meaning (Bulat et al., 2016). Meta-embeddings have emerged as a useful method for combining information from different word embeddings models (Yin and Sch¨utze, 2016). Different embeddings may be trained on various corpora of text, with different sizes, vocabularies, learning methods or model architecture. Meta-embeddings can then be created that combine the complementary information from all sources (Murom¨agi et al., 2017). In the context of language modelling, different layers of a pre-trained language model may be sensitive to different kinds of linguistic information, and effectively combining embedding information across layers has been shown to improve performance on different tasks sensitive to different kinds of 1Github link to code at https://github.com/stevend94/Decoding-Semantic-Properties 1907 information (such as POS-tagging and word sense disambiguation) (Peters et al., 2018). A number of successful methods have emerged for combining word embedding spaces from different sources. Coates and Bollegala (2018) demonstrate that combining vectors using element-wise addition can be just as effective as concatenating, given that the embeddings are orthogonal. Bollegala and Bao (2018) propose a number of autoencoder type networks to combine one or more vector space models. Finally, Neill and Bollegala (2018) give a comprehensive set of empirical results for a number of models and loss functions for learning complex meta-embeddings, demonstrating that loss functions that focus on vector direction such as cosine or KL-divergence based losses give the best performance on intrinsic benchmarks. 3 Approach The most prominent method for building image-based semantic models involves using pretrained deep convolutional neural networks (DCNNs) to extract visual information from image data by retrieving the output vectors from the penultimate layer of the network. In this work, we utilise DCNNs trained for the ImageNet LSVRC competition (Deng et al., 2009), which aim to predict the correct object in an image from a set of 1000 possible labels. In general, these networks use convolutional layers to extract visual information and build increasingly complex features, before one or many fully-connected layers are used to compute the probability distribution over the classes. Distributional semantic spaces can then be constructed from the penultimate fully-connected layer. Here, we instead focus our analysis on representational spaces generated at all convolutional layers of the network. Our goal is to demonstrate that the types of semantic attributes encoded in these feature maps depend on their depth in the network, and thus using only the penultimate layer may be suboptimal for representing concept meaning. 2 Related Work In distributional semantics, powerful approaches have been developed for building functions that can map from one semantic space to another (Lazaridou et al., 2014), compelling researchers to construct cross-modal mappings from dense distributional models with much larger vocabularies onto property norm data (Fagarasan et al., 2015). For example, Derby et al. (2019) constructed distributed seman- tic representations for each property dimension used in a large set of property norms, whilst Li and Summers-Stay (2019) showed that deep neural networks provide the best performance for zero-shot mapping between semantic spaces. Motivating our work on ensembling over layers of deep convolutional neural network vision models, it has been shown that different layers of such networks learn features that reﬂect different kinds of visual properties. The lower layers of vision networks tend to capture visually basic features such as Gabor ﬁlters and colour gradients, which are then combined at later layers to construct task-speciﬁc high-level visual features that are relevant to object classiﬁcation (Yosinski et al., 2014; Zeiler and Fergus, 2014). 2https://csl.psychol.cam.ac.uk/propertynorms/ 3.1 Visual Stimulus We make use of the CSLB property norm data (Devereux et al., 2014), which includes 638 concept words together with 2725 human-elicited semantic properties2. We used a script to web scrape ten representative images for each of these concepts from a Google image search. We manually reviewed the images to check that they were appropriate and representative (for example, to ensure images for the search term APPLE do not include the logo of Apple Inc.). For each word, we feed the corresponding images into a DCNN and extract the feature map outputs at every layer of the network. Once we have retrieved the feature maps for each concept, we perform additional preprocessing steps in order to create embedding vectors at each layer. Each convolutional ﬁlter should activate if it receives certain visual patterns from the stimulus, with the resulting feature map representing the activity value of each ﬁlter at each spatial location. To obtain an overall measure of the presence of each feature in each image, we perform global max pooling across the feature maps, which takes the highest activity value at all spacial locations for each ﬁlter. We then average the max-pooled responses for each ﬁlter across each of our ten images to get the ﬁnal concept representation and ﬁnally normalise the vectors using L2 distance. 2https://csl.psychol.cam.ac.uk/propertynorms/ 1908 layer Mean F1 x 100 AlexNet VGG ResNet DenseNet Figure 1: Decodability scores on human property norm data using the convolutional layers of four deep neural networks for image classiﬁcation (AlexNet, VGG ResNet, and DenseNet. The ResNet panel in- cludes red dotted vertical lines indicating the residual connections of the network. VGG DenseNet DenseNet layer DenseNet Figure 1: Decodability scores on human property norm data using the convolutional layers of four deep neural networks for image classiﬁcation (AlexNet, VGG ResNet, and DenseNet. The ResNet panel in- cludes red dotted vertical lines indicating the residual connections of the network. 3.2 Image Classiﬁcation Models For our analysis, we evaluate a number of standard implementations of networks trained for image classi- ﬁcation3. The ﬁrst model is AlexNet, containing ﬁve convolutional layers followed by three feed-forward layers. AlexNet is the most widely used pretrained DCNN in work on multimodal distributional seman- tics. Next, we chose the VGG16 model, which has a similar architecture to AlexNet but contains 16 convolutional layers (Simonyan and Zisserman, 2014). By comparing Alexnet and VGG16, we aim to investigate how depth affects the models’ ability to encode human-relevant semantic property knowl- edge. For our third model, we chose ResNet34 which is not only deeper, with 34 convolutional layers, but also has residual connections between layers. These connections allow information from lower lay- ers to more easily ﬂow to higher layers.The ﬁnal model we consider is DenseNet; it further extends the objective of feeding low-level information as inputs to the latter layers of the network by using dense skip connections (Huang et al., 2017). As DenseNet has a vast number of layers (169), we only take the output of the ﬁrst convolutional layer, each block layer in the network, and each transition block (which perform downsampling of the feature maps). ResNet and DenseNet have been shown to be amongst the most “‘brainlike” DCNNs, insofar as their internal representations correlate well with neuroimaging data from the human visual processing stream (Jos´e Meijer and Visser, 2019; Wen et al., 2018). 3https://pytorch.org/docs/stable/torchvision/models.html 4 Analysis To measure how well each layer of a DCNN encodes salient properties of human conceptual knowledge, we train supervised models to predict the presence of a property for a given concept. We note that while a supervised classiﬁer’s ability to identify the presence of a property indicates that the property is encoded in the representations, the converse is not always true (Collell and Moens, 2016). 3https://pytorch.org/docs/stable/torchvision/models.html 3https://pytorch.org/docs/stable/torchvision/models.html 1909 layer Mean F1 x 100 AlexNet VGG ResNet DenseNet Figure 2: Decodability scores on ﬁne-grained feature categories using the convolutional layers of the four DCNNs. The ResNet panel includes red dotted lines indicating residual connections. VGG DenseNet VGG DenseNet ayer DenseNet Figure 2: Decodability scores on ﬁne-grained feature categories using the convolutional layers of the four DCNNs. The ResNet panel includes red dotted lines indicating residual connections. 4.1 Decoding Property Norms Data from DCNN layers We ﬁrst preprocessed the property norm data so as to retain only semantic properties that occur for at least 5 concepts (leaving 638 concepts and 390 properties). For each property, we perform 5-fold cross- validation with stratiﬁed sampling so that at least one positive case occurs in each test fold, and train a logistic regression classiﬁer to predict the presence of that property for each embedding, recording the average F1 score over all the folds. Since the dataset is highly imbalanced, we down-weight the negative classes in the loss function and regularise by adding the L2 norm of the weights. After obtaining the classiﬁcation result for each property, we use the partition of properties into distinct feature classes in the CSLB data to aggreggate the results by property class. These property classes are Taxonomic (e.g. is-an- amphibian), Encyclopaedic (e.g. lays-spawn), Functional (e.g. hops), Visual Perceptual (e.g. is-green) and Other perceptual (e.g. does-croak). The results are consistent across all models, with Taxonomic being the most decodable followed by Visual Perceptual, Other Perceptual, Functional and ﬁnally Encyclopaedic (Fig. 1). These results follow previous work where taxonomic features tend to be more decodable than other attributes, for many vector space models. Furthermore, all types of concept properties seem to be more decodable as we move to later layers of the network. Such results could be down to the supervised classiﬁer being unable to make use of the more image-speciﬁc information in the lower layers, or the fact that layers in the latter part of the network tend to be better for classiﬁcation tasks. The most notable exception to this is DenseNet, which has two spikes in performance at the convolutional bottleneck layers. 5 Improving Distributional Semantic Models with Visual Meta-Embeddings We have seen that particular layers of DCNNs best capture different types of semantic information. Here we investigate whether we can use this insight to obtain improved image-based semantic embedding spaces and thus build more faithful representations of conceptual meaning. (In these experiments, we focus on ResNet, since it gave the best performance on the decoding task; for results with all four models, see the Supplementary Table 1). 4.2 Fine-Grained Analysis of the Decoding Results Whilst the ﬁve property classes provide a useful distinction between types of semantic information, they tend to include a broad range of semantic attributes. For example, the visual-perceptual class includes visual features relating to colour and texture (is-green, has-smooth-skin) as well as more complex infor- mation about form (has-four-legs). To gain a deeper understanding of the kinds of semantic knowledge encoded in the DCNN layers, we divided the properties into more ﬁne-grained classes. First, we split 1910 Model Encyclo. Functional Taxonomic Visual Perceptual Other Perceptual Overall ResNet 33.09 35.97 61.56 46.84 42.83 44.05 SVD Meta ResNet 32.68 35.92 68.12 50.19 48.22 45.80 1ToN Meta ResNet 34.44 37.45 66.77 51.28 48.25 46.74 Table 1: Average cross-validation F1 scores ×100 for the ResNet embedding space based on the penul- timate layer, and the two meta-embedding approaches, for each of the ﬁve property classes. Encyclo. Functional Taxonomic Visual Perceptual Other Perceptual Overall Table 1: Average cross-validation F1 scores ×100 for the ResNet embedding space based on the penul- timate layer, and the two meta-embedding approaches, for each of the ﬁve property classes. the Visual Perceptual features into two basic types of visual information, Colour and Shape/Size. We expect the lower layers to perform well at decoding these properties since previous research has shown that the lower layers tend to learn colour gradients and Gabor ﬁlters (Zeiler and Fergus, 2014). In the CSLB property norm data, semantic properties always consist of a relation term and an attribute value. For example, FROG has the feature “has-legs”, where “has” is the relation. The property listing task prompted participants to use four such relations: “is”, “has”, “does” and “made-of” (Devereux et al., 2014). These relations relate to the type of property being described; for example, “does” relates to ac- tion or function, while “has” corresponds to object parts. We therefore use these four relations to build the other ﬁne-grained categories, for a total of six categories. Based on the previous results, we expect the later DCNN layers to have higher average F1 scores for all properties, but here we are interested in which type of property is most decodable at each layer. the Visual Perceptual features into two basic types of visual information, Colour and Shape/Size. 4.2 Fine-Grained Analysis of the Decoding Results We expect the lower layers to perform well at decoding these properties since previous research has shown that the lower layers tend to learn colour gradients and Gabor ﬁlters (Zeiler and Fergus, 2014). In the CSLB property norm data, semantic properties always consist of a relation term and an attribute value. For example, FROG has the feature “has-legs”, where “has” is the relation. The property listing task prompted participants to use four such relations: “is”, “has”, “does” and “made-of” (Devereux et al., 2014). These relations relate to the type of property being described; for example, “does” relates to ac- tion or function, while “has” corresponds to object parts. We therefore use these four relations to build the other ﬁne-grained categories, for a total of six categories. Based on the previous results, we expect the later DCNN layers to have higher average F1 scores for all properties, but here we are interested in which type of property is most decodable at each layer. In the lower layers of the DCNNs, we see that Colour tends to be the most decodable followed by “made-of” and Shape/Size, but as we move through to the middle sections of the networks “made-of” properties become the most decodable, for all DCNNs (Fig. 2). As we move further, “has” and “is-a” property decoding improves, and by the end either “is-a” or “made-of” become the most decodable property types. As we move through the networks, features related to color or shape become relatively less decodable, compared to other feature types. Overall, the results support the rationale that the penulti- mate layer (as is commonly used) should give a good correspondence to object semantics for the purpose of building distributional semantic models. But would such models also beneﬁt from having direct in- formation about different features from earlier stages of the DCNNs? 5.1 Convolutional Meta-Embeddings In order to make full use of the information generated by the network, we require a method that can effec- tively combine the feature maps from each layer, retaining only the most relevant information from each. We construct semantic representations by aggregating features from the output of each convolution layer by assembling them into a single set of representations known as a meta-embedding. Meta-embeddings are vector representations that incorporate information from a set of word embeddings that can differ in a range of aspects such as training data and training methods (Peters et al., 2018; Coates and Bollegala, 2018). Most importantly, they look to combine complementary knowledge from each embedding, and do not require that the vectors be the same dimensionality. Here we apply two common approaches. The ﬁrst approach is a simple concatenation technique to combine all embeddings. Following previous work, we also up-weight the best embeddings; in this case, we multiply the second-to-last layer by 5 and the last layer by 10, keeping the other layers the same before concatenating. To reduce dimensionality, we also apply Single Value Decomposition (SVD) to ﬁx dimensionality to 300 while preserving information from the most important features. We refer to these embeddings as SVD Meta ResNet. The second approach uses a method known as 1ToN (Yin and Sch¨utze, 2016); this method looks to learn set of meta- 1911 K Nearest Neighbour (K=5) Ridge Regression Neural Network (h=1200) Hit@T Accuracy Top 1 Top 5 Top 10 Top 20 Top 1 Top 5 Top 10 Top 20 Top 1 Top 5 Top 10 Top 20 Unimodal Vector Representations ResNet 1.95 29.3 50.0 69.1 5.80 29.1 44.8 61.9 3.23 42.0 60.06 75.5 SVD Meta ResNet 2.04 28.3 50.7 68.6 4.92 43.2 61.0 75.3 2.79 45.1 62.4 76.5 1ToN Meta ResNet 2.76 29.3 53.4 71.8 5.92 41.4 58.4 74.6 2.82 44.1 61.4 76.0 GloVe 1.76 27.3 53.8 70.6 7.18 38.9 56.6 71.1 5.14 44.0 62.0 76.7 Multimodal Vector Representations ResNet + GloVe 2.38 31.7 59.0 76.7 8.87 43.6 60.6 75.5 6.74 50.8 69.5 82.6 SVD Meta ResNet + GloVe 2.32 31.6 58.8 76.4 8.88 43.8 60.8 75.6 6.24 51.9 71.1 83.0 1ToN Meta ResNet + GloVe 2.60 32.3 60.3 78.8 8.18 48.1 66.1 80.6 5.08 52.6 70.9 83.7 Table 2: Results for zero-shot cross-modal mapping task using several predictive models. 5.1 Convolutional Meta-Embeddings The Hit@K tells us the percentage of test features which appear in the top K neighbours with the ground truth representations. Table 2: Results for zero-shot cross-modal mapping task using several predictive models. The Hit@K tells us the percentage of test features which appear in the top K neighbours with the ground truth representations. embeddings using a neural network. For each word, we have a meta-embedding vector, for which the network predicts the associated word embedding for each of our vector space models using several linear layers. A network which combines the information from N embeddings will contain N linear layers which map the meta-embedding into the original constituent word embedding spaces. Suppose we have N distributional models {W 1, W 2, . . . W N}, with equal vocabulary V , and vector lengths (ai)N i=1 ⊂N. We deﬁne the 1ToN neural network with an embeddings matrix E ∈R\|V \|×k for some size k ∈R, with N linear projections of weights Mi ∈Rk×ai and corresponding biases bi, 1 ≤i ≤N. For each word w ∈V , let wi ∈W i be it’s associated word vector for each 1 ≤i ≤N, with meta embeddings E(w). We want to minimize the following loss function, for our neural network parameterized by θ: embeddings using a neural network. For each word, we have a meta-embedding vector, for which the network predicts the associated word embedding for each of our vector space models using several linear layers. A network which combines the information from N embeddings will contain N linear layers which map the meta-embedding into the original constituent word embedding spaces. Suppose we have N distributional models {W 1, W 2, . . . W N}, with equal vocabulary V , and vector lengths (ai)N i=1 ⊂N. We deﬁne the 1ToN neural network with an embeddings matrix E ∈R\|V \|×k for some size k ∈R, with N linear projections of weights Mi ∈Rk×ai and corresponding biases bi, 1 ≤i ≤N. For each word w ∈V , let wi ∈W i be it’s associated word vector for each 1 ≤i ≤N, with meta embeddings E(w). We want to minimize the following loss function, for our neural network parameterized by θ: L(θ) = N X j=1 βj(\| ˆwj −wj\|2 + λ\|Mj\|2) (1) (1) ˆwj = MT j E(w) + bj (2) (2) where [β1, β2 . . . 6 Experiments To evaluate these two meta-embedding models, we repeat the decoding experiment with the SVD and a 1ToN meta-embeddings both of size 300 built using the feature maps from all of the ResNet convolutional layers. We compare the meta-embeddings with embeddings constructed from the penultimate layer of ResNet (i.e. the traditional approach) to see how well they decode each property type. 5.1 Convolutional Meta-Embeddings βN] are the scaler weightings for each component embedding, though we set these values to one. We instead up-weight the embeddings from the last two convolutional layers which we multiply by 5 and 10 respectively. We call this the 1ToN Meta ResNet embedding. where [β1, β2 . . . βN] are the scaler weightings for each component embedding, though we set these values to one. We instead up-weight the embeddings from the last two convolutional layers which we multiply by 5 and 10 respectively. We call this the 1ToN Meta ResNet embedding. 6.1 Property Decodability The results are displayed in Table 4. We see that both meta-embedding approaches, ensembling over the convolutional layers of ResNet, are better representations for decoding human property knowledge than the traditional approach of using the penultimate layer of ResNet alone. We see that there is no real change in how decodable Encyclopaedic or Functional properties are in the meta embeddings, which is to expected, as this is where text-based word embeddings have been shown to perform strongest. Further- more, Taxonomic and Visual Perceptual properties are more decodable since certain layers more strongly encode different types of visual information depending on their location in the DCNN. Surprisingly, Other Perceptual information, such as olfactory or taste-based features are also more decodable in the meta-embeddings. Overall, Taxonomic, Visual Perceptual and Other Perceptual have the most signiﬁcant improvement when using all layers, with the overall F1 score increasing by 5 on average for these three categories, compared with using the penultimate layer of ResNet alone. 1912 (a) Car - Predictions from ResNet convolution layer 3 (b) Fruit - Predictions from ResNet convolution layer 15 (c) Wampimuk - Predictions from ResNet convolution layer 27 (d) Guitar - Predictions from ResNet convolution layer 35 Figure 3: Top predictions from the zero-shot cross-modal mapping task for 4 example images. Property predictions for each image are from different convolutional layers of ResNet. Each colour represents a feature category: Taxonomic , Visual Perceptual , Functional and Other Perceptual . (b) Fruit - Predictions from ResNet convolution layer 15 (a) Car - Predictions from ResNet convolution layer 3 (b) Fruit - Predictions from ResNet convolution layer 15 (a) Car - Predictions from ResNet convolution layer 3 y (c) Wampimuk - Predictions from ResNet convolution layer 27 (d) Guitar - Predictions from ResNet convolution layer 35 (d) Guitar - Predictions from ResNet convolution layer 35 (c) Wampimuk - Predictions from ResNet convolution layer 27 Figure 3: Top predictions from the zero-shot cross-modal mapping task for 4 example images. Property predictions for each image are from different convolutional layers of ResNet. Each colour represents a feature category: Taxonomic , Visual Perceptual , Functional and Other Perceptual . Figure 3: Top predictions from the zero-shot cross-modal mapping task for 4 example images. Property predictions for each image are from different convolutional layers of ResNet. Each colour represents a feature category: Taxonomic , Visual Perceptual , Functional and Other Perceptual . 6.2 Cross-Modal Embedding-to-Property Mapping While the sparse property vectors obtained from norming studies are useful in cognitive science due to their interpretability, as a lexical resource they are very limited in size, due to being created manually. This has driven recent work aiming to learn zero-shot cross-modal mappings between a pretrained se- mantic embedding space and these property vectors, so that property-norm information can be generated automatically from word embeddings (Derby et al., 2019; Fagarasan et al., 2015). Furthermore, we can extend our analysis of the convolutional layers from global to local interpretations based on some sample images (See appendix B). Here we evaluate how accurately our models predict semantic properties for unseen concept words in a zero-shot set-up using several regression models. We predict the property vector for each test concept and ﬁnd the top T nearest neighbours of the predicted vector, to determine whether the concept word for the ground-truth vector is retrieved within that set, which we refer to as a hit. We perform repeated 10-fold cross-validation on the concepts due to the small number of train- ing samples and average the number of hits across all test folds at each T, for T ∈[1, 5, 10, 20] in our evaluations. We perform 5 repeats of each cross-validation. To learn a cross-modal mapping, we report the results for three different models. A k-nearest-neighbours model with k = 5, ridge regression and a neural network with one hidden layer. The neural network had a hidden layer of size 1200, Relu ac- tivations and used the Adam optimiser. We include a neural network as previous work has shown that they give the strongest performance on this zero-shot cross-modal mapping task (Li and Summers-Stay, 2019). The loss function we use is based on the cosine similarity function from Lazaridou et al. (2014). 6.2 Cross-Modal Embedding-to-Property Mapping As the number of models being ensembled increases, information can get lost when concatenating to high dimensionality (Neill and Bollegala, 2018), but with the 1ToN method the network effectively retains the important information from the ensembled component embeddings because of the learning objective. and Moens, 2018). Hence, we perform a hyperparameter search using a small grid of values with 5-fold cross validation to determine the best set of training parameters. To avoid over-ﬁtting, we determine crossvalidation performance using Mean Average Precision (MAP). To illustrate this cross-modal map- pling approach, examples of zero-shot property predictions for held-out images are presented in Figure 3. We also combined the image embeddings with text embeddings to create multimodal distributional models that have been shown to give better performance on cross-modal mapping (Bulat et al., 2016). For the text embeddings, we use Spacy’s GloVe vectors (Pennington et al., 2014), from the large English language model. To build the text+image multimodal models, we concatenate the L2-normalized vectors from the GloVe embeddings with each of our image-based embeddings, which gives us three multimodal models in total. The results are presented in Table 2. We see that in all cases, the meta-embeddings out- perform the embeddings from the penultimate layer of ResNet, and in particular, the 1ToN embeddings show the best performance. As the number of models being ensembled increases, information can get lost when concatenating to high dimensionality (Neill and Bollegala, 2018), but with the 1ToN method the network effectively retains the important information from the ensembled component embeddings because of the learning objective. 6.3 Semantic Similarity Task A common benchmark to evaluate distributional semantic models is to directly compare word similarity scores with human annotator similarity ratings for word pairs. We utilize MEN (Bruni et al., 2012) and SimLex999 (Hill et al., 2015), for which we have 104 and 48 word pair ratings respectively. In this ﬁnal evaluation of the models, we use cosine similarity to score word-pair similarity and then use Spearman ρ to measure the correlation between embedding word similarities and the human annotator ratings. As we can see in Table 3, the results again show the same pattern, with the meta embeddings outperforming the penultimate ResNet layer for both the unimodal and multimodal (text+image) embeddings. 6.2 Cross-Modal Embedding-to-Property Mapping For each ground truth property norm representation y ∈G, with corresponding predicted vector ˆy from the network parameterised by θ, the loss function is L(θ) = X y∈G 1 2(1 −cos(y, ˆy)) (3) (3) Training neural networks on such a small set of data points for zero-shot cross-modal mapping can be difﬁcult, as several problems arise such as “hubness” (Radovanovi´c et al., 2010), “pollution” (Lazari- dou et al., 2015a) and neighbourhood structures resembling the input space more than the output (Collell Training neural networks on such a small set of data points for zero-shot cross-modal mapping can be difﬁcult, as several problems arise such as “hubness” (Radovanovi´c et al., 2010), “pollution” (Lazari- dou et al., 2015a) and neighbourhood structures resembling the input space more than the output (Collell 1913 Model MEN (104) SimLex999 (48) Unimodal Vector Representations ResNet 0.540 0.324 SVD Meta ResNet 0.555 0.360 1ToN Meta ResNet 0.683 0.32 GloVe 0.804 0.210 Multimodal Vector Representations ResNet + GloVe 0.829 0.322 SVD Meta ResNet + GloVe 0.829 0.316 1ToN Meta ResNet + GloVe 0.846 0.491 Table 3: Spearman ρ correlation with MEN and SimLex999 human similarity benchmarks. MEN (104) SimLex999 (48) Table 3: Spearman ρ correlation with MEN and SimLex999 human similarity benchmarks. able 3: Spearman ρ correlation with MEN and SimLex999 human similarity benchmarks. and Moens, 2018). Hence, we perform a hyperparameter search using a small grid of values with 5-fold cross validation to determine the best set of training parameters. To avoid over-ﬁtting, we determine crossvalidation performance using Mean Average Precision (MAP). To illustrate this cross-modal map- pling approach, examples of zero-shot property predictions for held-out images are presented in Figure 3. We also combined the image embeddings with text embeddings to create multimodal distributional models that have been shown to give better performance on cross-modal mapping (Bulat et al., 2016). For the text embeddings, we use Spacy’s GloVe vectors (Pennington et al., 2014), from the large English language model. To build the text+image multimodal models, we concatenate the L2-normalized vectors from the GloVe embeddings with each of our image-based embeddings, which gives us three multimodal models in total. The results are presented in Table 2. We see that in all cases, the meta-embeddings out- perform the embeddings from the penultimate layer of ResNet, and in particular, the 1ToN embeddings show the best performance. References Piotr Bojanowski, Edouard Grave, Armand Joulin, and Tomas Mikolov. 2017. Enriching word vectors with subword information. Transactions of the Association for Computational Linguistics, 5:135–146. Danushka Bollegala and Cong Bao. 2018. Learning word meta-embeddings by autoencoding. In Proceedings of the 27th International Conference on Computational Linguistics, pages 1650–1661. Rose Bruffaerts, Simon De Deyne, Karen Meersmans, Antonietta Gabriella Liuzzi, Gert Storms, and Rik Vanden- berghe. 2019. 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We reveal that, whilst the widely accepted approach for extracting visual semantic representations, using the penultimate layer of DCNNs, yields strong representations of conceptual meaning, they overlook key information generated by the neural network. Instead, we develop meta-embeddings that encompass all the salient feature information encoded in the represen- tations produced at all layers of several DCNNs. These new vector space models are not only closer representations of human conceptual knowledge, but also can be used to build multimodal semantic models that improve performance on a zero-shot cross-modal mapping task and give better ﬁt to human semantic similarity benchmarks. 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By performing a property decoding task on these embeddings, we could then infer what semantic knowledge the model captures at particular layers of the network. Such an approach reﬂects a global interpretation of what information the network captures at each convolutional layer, and is not based on any particular sample we gave to the network. Thankfully, cross-modal map- ping provides us with a simple method for interpreting local instances from our visual data. Mapping Images to Semantic Primitives. When we learn a cross-modal mapping from a distribu- tional feature space onto the conceptual feature space, the model must learn to map common features related to a particular concept onto some plausible semantic properties. Because of this, we can use our trained cross-modal map to predict semantic properties for other instances of the concept, since it has been trained to map common feature onto some associated conceptual knowledge. For example, if the model learns to map features it associates as is-red based on images from some concept such as ROSE, then a new image of a ROSE should still produce features in the convolutional layers that the cross-modal map similarly identiﬁes as is-red. Furthermore, images of other concepts that also have the property is-red, such as STRAWBERRY, should be accurately inferred from the model. For our analysis, we train a ridge regression to map from the convolution layer embeddings of ResNet onto the conceptual space. After training, we apply the appropriate image preprocessing to the sample images that we wish to analyse and extract features across all convolutional layers. Since we frame the task as a regression problem, each predicted conceptual mapping should not only predict the correct properties for a concept, but also the strength of the production frequency for each concept. Production frequencies are count-based statistics that reﬂect the number of times human annotators express that property as true for a particular concept. For our analysis, we predict the conceptual representation for each image and take the highest valued dimensions which correspond to some conceptual property. References How transferable are features in deep neural networks? In Advances in neural information processing systems, pages 3320–3328. Matthew D Zeiler and Rob Fergus. 2014. Visualizing and understanding convolutional networks. In European conference on computer vision, pages 818–833. Springer. 1917 Appendix A. Supplementary Figure 4. Model Encyclo. Functional Taxonomic Visual Perceptual Other Perceptual Overall AlexNet Representations AlexNet 20.86 21.91 36.90 32.00 27.07 27.92 SVD Meta AlexNet 27.68 27.81 56.65 42.09 37.62 37.63 1ToN Meta AlexNet 28.40 27.16 54.84 41.67 34.78 37.00 VGG Representations VGG 25.90 28.74 49.80 39.29 33.34 35.31 SVD Meta VGG 32.51 34.14 64.27 48.26 42.72 43.71 1ToN Meta VGG 33.16 34.93 64.48 48.71 44.47 44.34 ResNet Representations ResNet 33.09 35.97 61.56 46.84 42.83 44.05 SVD Meta ResNet 32.68 35.92 68.12 50.19 48.22 45.80 1ToN Meta ResNet 34.44 37.45 66.77 51.28 48.25 46.74 DenseNet Representations DenseNet 16.66 18.02 31.71 24.40 23.07 22.34 SVD Meta DenseNet 28.44 30.35 59.16 43.10 37.88 39.09 1ToN Meta DenseNet 31.10 32.61 59.86 45.64 41.13 41.42 Table 4: Average cross-validation F1 scores ×100 for each model for each of the ﬁve property classes. We have included the results of all four DCNN’s used in the work. Encyclo. Functional Taxonomic Visual Perceptual Other Perceptual Overall Table 4: Average cross-validation F1 scores ×100 for each model for each of the ﬁve property classes. We have included the results of all four DCNN’s used in the work. Appendix B. Local Interpretation. As there are a large number of layers, we focuses on features at particular intervals of the network, in this 1918 case, layers 3, 15, 27 and 35 of ResNet. (a) Predictions from ResNet convolution layer 3 (b) Predictions from ResNet convolution layer 15 (c) Predictions from ResNet convolution layer 27 (d) Predictions from ResNet convolution layer 35 Figure 4: Top property predictions based on the image of a car with features extracted from ResNet. Each colour represents a feature category: Taxonomic , Visual Perceptual , Functional and Encyclopaedic . (a) Predictions from ResNet convolution layer 3 (b) Predictions from ResNet convolution layer 15 (b) Predictions from ResNet convolution layer 15 (d) Predictions from ResNet convolution layer 35 (a) Predictions from ResNet convolution layer 3 (c) Predictions from ResNet convolution layer 27 (c) Predictions from ResNet convolution layer 27 (d) Predictions from ResNet convolution layer 35 Figure 4: Top property predictions based on the image of a car with features extracted from ResNet. Each colour represents a feature category: Taxonomic , Visual Perceptual , Functional and Encyclopaedic . Concept: Car. We ﬁrst chose an image of the concept CAR (displayed in Figure 4). As we can see the lower layers are dominated by visual perceptual properties, with more high-level properties eventually emerging in the upper layers of the network. Furthermore, the lower layers of the network tend to focus on shape, colour and form across the entire image. We can observe this in the fact that the cross-modal map detects properties such as made-of-wood, is-green and has-leaves, a consequence of the model detecting the trees in the background. Notice also that the dimensions of the object become more precise as we move to the middle layers, with features such as is-small and is-circular-round appearing at the start, while ‘is-big-large appears in the later layers. Not only do these upper layers predict more complex notions about the concept such as made-a-metal or is-expensive, but the attention of these features tend to be solely related to the central object in the image, in this case, a car. Concept: Guitar. Next, we chose an image of the concept GUITAR (displayed in Figure 5), which is another one of the concepts in our lexicon, though is not directly taken from the training data. Appendix B. Local Interpretation. We see that the model detects some visual properties in the lower layers, which it assumes is related to another high-level concept, an animal. We can see this from the top prediction being is-an-animal and other features related to animals such as has-a-tail and has-fur-hair. It is not surprising that there is a high degree of association between certain properties in the norming study, since many related to particular taxonomies, in this case, is-an-animal. Nevertheless, as we move through the network the trajectory of the prediction quickly becomes more related to a guitar, though is-an-animal is still predicted in the top ten features. As we can see, these models generalise quite well to other images and can still decode complex features related to conceptual categories. Concept: Fruit. Next, we chose an image of the concept FRUIT (displayed in Figure 6), which is neither taken from the training data or the lexicon, but instead consists of many concepts from the data such as APPLE, BANANA and KIWI. Here we want to analyse how our cross-modal model copes with multiple instances of the concepts. Here, we see that the model can quickly detect visual properties such 1919 (a) Predictions from ResNet convolution layer 3 (b) Predictions from ResNet convolution layer 15 (c) Predictions from ResNet convolution layer 27 (d) Predictions from ResNet convolution layer 35 Figure 5: Top property predictions based on the image of a guitar with features extracted from ResNet. Each colour represents a feature category: Taxonomic , Visual Perceptual , Functional and Other Perceptual (b) Predictions from ResNet convolution layer 15 (d) Predictions from ResNet convolution layer 35 (a) Predictions from ResNet convolution layer 3 (b) Predictions from ResNet convolution layer 15 (a) Predictions from ResNet convolution layer 3 (b) Predictions from ResNet convolution layer 15 (d) Predictions from ResNet convolution layer 35 (a) Predictions from ResNet convolution layer 3 (c) Predictions from ResNet convolution layer 27 (d) Predictions from ResNet convolution layer 35 (c) Predictions from ResNet convolution layer 27 Figure 5: Top property predictions based on the image of a guitar with features extracted from ResNet. Each colour represents a feature category: Taxonomic , Visual Perceptual , Functional and Other Perceptual . Other Perceptual . as is-yellow, is-red and is-small, though other high-level properties emerge such as is-eaten-edible and is-a-fruit. Again, is-an-animal emerges as a property, which may be due to bias in the model towards high occurring properties. as is-yellow, is-red and is-small, though other high-level properties emerge such as is-eaten-edible and is-a-fruit. Again, is-an-animal emerges as a property, which may be due to bias in the model towards high occurring properties. Concept: Wampimuk. Finally, we chose an imagined concept, known as a WAMPIMUK (displayed in Figure 7). A Wampimuk is a ﬁctitious concept proposed by Lazaridou et al. (2014), to convey how context can shape our perception of a concept, even if we have never heard of it before. Humans are capable of building complete semantic representations for concepts, even when the information is frag- mented (Kivisaari et al., 2019b). Hence, a sentence like ”We found a cute, hairy wampimuk sleeping behind the tree” can communicate a lot of information about what a wampimuk might be, in this case, a small furry animal. The authors create a potential image of such an animal that does not exist, yet we can extract properties about the concept just as well. Hence, we also examine the convolutional layers of the network when given such a creature, to determine whether reasonable semantic properties can be captured by our cross-modal model. We see that the network produces features that the cross-modal map detects as salient aspects of the image such as is-small and has-fur-hair. Furthermore, the model can detect conceptual knowledge related to this imaginary creature based on the context of this information. For example, in the lower layers, has-a-tail is predicted by the cross-modal map, even though there is no evidence of this in the picture, yet it would make sense for a small creature. As we move to the ﬁnal layer of the network, we can even see complex taxonomies emerge, such as is-a-mammal that is quite plausible. 1920 (a) Predictions from ResNet convolution layer 3 (b) Predictions from ResNet convolution layer 15 (c) Predictions from ResNet convolution layer 27 (d) Predictions from ResNet convolution layer 35 Figure 6: Top property predictions based on the image of fruit with features extracted from ResNet. Other Perceptual . Each colour represents a feature category: Taxonomic , Visual Perceptual , Functional and O h l (b) Predictions from ResNet convolution layer 15 (d) Predictions from ResNet convolution layer 35 (b) Predictions from ResNet convolution layer 15 (a) Predictions from ResNet convolution layer 3 (b) Predictions from ResNet convolution layer 15 (a) Predictions from ResNet convolution layer 3 (b) Predictions from ResNet convolution layer 15 (d) Predictions from ResNet convolution layer 35 convolution layer 3 (c) Predictions from ResNet convolution layer 27 (d) Predictions from ResNet convolution layer 35 (c) Predictions from ResNet convolution layer 27 (c) Predictions from ResNet convolution layer 27 Figure 6: Top property predictions based on the image of fruit with features extracted from ResNet. Each colour represents a feature category: Taxonomic , Visual Perceptual , Functional and Figure 6: Top property predictions based on the image of fruit with features extracted from ResNet. Each colour represents a feature category: Taxonomic , Visual Perceptual , Functional and Other Perceptual . (a) Predictions from ResNet convolution layer 3 (b) Predictions from ResNet convolution layer 15 (c) Predictions from ResNet convolution layer 27 (d) Predictions from ResNet convolution layer 35 Figure 7: Top property predictions based on the image of a wampimuk with features extracted from ResNet. Each colour represents a feature category: Taxonomic , Visual Perceptual , Functional and Other Perceptual . (a) Predictions from ResNet convolution layer 3 (b) Predictions from ResNet convolution layer 15 (b) Predictions from ResNet convolution layer 15 (a) Predictions from ResNet convolution layer 3 (a) Predictions from ResNet convolution layer 3 (c) Predictions from ResNet convolution layer 27 (b) Predictions from ResNet convolution layer 15 (d) Predictions from ResNet convolution layer 35 (d) Predictions from ResNet convolution layer 35 (c) Predictions from ResNet convolution layer 27 Figure 7: Top property predictions based on the image of a wampimuk with features extracted from ResNet. Each colour represents a feature category: Taxonomic , Visual Perceptual , Functional and Other Perceptual Figure 7: Top property predictions based on the image of a wampimuk with features extracted from ResNet. Each colour represents a feature category: Taxonomic , Visual Perceptual , Functional and Other Perceptual . 1921
https://openalex.org/W4225585585	https://www.researchsquare.com/article/rs-208666/latest.pdf	English	null	Mechanosensation by endothelial PIEZO1 is required for leukocyte diapedesis	Blood	2,022	cc-by	15,347	Stefan Offermanns (  stefan.offermanns@mpi-bn.mpg.de ) Stefan Offermanns (  stefan.offermanns@mpi-bn.mpg.de ) g p // g/ ShengPeng Wang Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Yanta District, Xi'an, China https://orcid.org/0000-0002-9030-1821 Yue Shi Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center Tanja Moeller Max Planck Institute of Molecular Biomedicine, Muenster, Germany Rebekka Stegmeyer Max Planck Institute of Molecular Biomedicine, Muenster, Germany Boris Strilic Max Planck Institute for Heart and Lung Research Liran Xu Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Zuyi Yuan Xian Jiaotong University Nina Wettschureck Department of Pharmacology, Max Planck Institute for Heart and Lung Research, Bad Nauheim, 61231, Germany Dietmar Vestweber Max Planck Institute for Molecular Biomedicine https://orcid.org/0000-0002-3517-732X Biological Sciences - Article Keywords: leukocyte diapedesis, in§ammation Posted Date: February 12th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-208666/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License ShengPeng Wang Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Yanta District, Xi'an, China https://orcid.org/0000-0002-9030-1821 Yue Shi Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center Tanja Moeller Max Planck Institute of Molecular Biomedicine, Muenster, Germany Rebekka Stegmeyer Max Planck Institute of Molecular Biomedicine, Muenster, Germany Boris Strilic Max Planck Institute for Heart and Lung Research Liran Xu Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Zuyi Yuan Xian Jiaotong University Nina Wettschureck Department of Pharmacology, Max Planck Institute for Heart and Lung Research, Bad Nauheim, 61231 Germany Dietmar Vestweber Max Planck Institute for Molecular Biomedicine https://orcid.org/0000-0002-3517-732X Biological Sciences - Article Posted Date: February 12th, 2021 Mechanosensation by endothelial PIEZO1 1 is required for leukocyte diapedesis 2 3 4 ShengPeng Wang1,2,, Yue Shi1,2, Tanja Möller3, Rebekka I. Stegmeyer3, 5 Boris Strilic2, Liran Xu1, Zuyi Yuan1, Nina Wettschureck2,4,5, 6 Dietmar Vestweber3, Stefan Offermanns2,4,5, 7 8 1 Department of Cardiology, First Affiliated Hospital, Cardiovascular Research 9 Center, School of Basic Medical Sciences, Xi'an Jiaotong University, No.277 West 10 Yanta Road, Yanta District, Xi’an, China 11 2 Max Planck Institute for Heart and Lung Research, Department of Pharmacology, 12 Ludwigstr. 43, 61231 Bad Nauheim, Germany 13 3 Department of Vascular Cell Biology, Max Planck Institute of Molecular 14 Biomedicine, Muenster, Germany 15 4 Center for Molecular Medicine, Goethe University Frankfurt, Theodor-Stern-Kai 7, 16 60590 Frankfurt, Germany 17 5 Cardiopulmonary Institute, 61231 Bad Nauheim, Germany 18 19 20 Mechanosensation by endothelial PIEZO1 1 is required for leukocyte diapedesis 2 3 4 ShengPeng Wang1,2,, Yue Shi1,2, Tanja Möller3, Rebekka I. Stegmeyer3, 5 Boris Strilic2, Liran Xu1, Zuyi Yuan1, Nina Wettschureck2,4,5, 6 Dietmar Vestweber3, Stefan Offermanns2,4,5, 7 8 1 Department of Cardiology, First Affiliated Hospital, Cardiovascular Research 9 Center, School of Basic Medical Sciences, Xi'an Jiaotong University, No.277 West 10 Yanta Road, Yanta District, Xi’an, China 11 2 Max Planck Institute for Heart and Lung Research, Department of Pharmacology, 12 Ludwigstr. 43, 61231 Bad Nauheim, Germany 13 3 Department of Vascular Cell Biology, Max Planck Institute of Molecular 14 Biomedicine, Muenster, Germany 15 4 Center for Molecular Medicine, Goethe University Frankfurt, Theodor-Stern-Kai 7, 16 60590 Frankfurt, Germany 17 5 Cardiopulmonary Institute, 61231 Bad Nauheim, Germany 18 19 20 21 22 23 24 25 26 27 28 29 30 31 * Correspondence: S.P.W. (shengpeng.wang@xjtu.edu.cn) and 32 S.O. (stefan.offermanns@mpi-bn.mpg.de) 33 Summary 1 The extravasation of leukocytes is a critical step during inflammation which requires 2 the localized opening of the endothelial barrier1-3. This process is initiated by the 3 close interaction of leukocytes with various adhesion molecules such as intercellular 4 adhesion molecule-1 (ICAM-1) on the surface of endothelial cells4-6. It is still unclear 5 how these initial processes induce downstream signaling events resulting in the 6 opening of inter-endothelial junctions to allow leukocyte diapedesis. Here we show 7 that mechanical forces induced by leukocyte-induced clustering of ICAM-1 and fluid 8 shear stress exerted by the flowing blood synergistically activate the 9 mechanosensitive cation channel PIEZO1 in endothelial cells. In human and mouse 10 endothelial cells exposed to low flow, PIEZO1 mediates leukocyte-induced increases 11 in [Ca2+]i and activation of downstream signaling events including phosphorylation of 12 SRC, PYK2 and myosin light chain (MLC) leading to endothelial barrier opening. 13 Mice with endothelium-specific loss of Piezo1 show decreased leukocyte 14 extravasation in different inflammation models. We found that actin polymerization 15 and actomyosin contraction induced by ICAM-1 clustering synergistically with fluid 16 shear stress increase endothelial plasma membrane tension to activate PIEZO1. Our 17 data reveal a mechanism by which leukocytes and the hemodynamic 18 microenvironment synergize to mechanically activate endothelial PIEZO1 and 19 subsequent downstream signaling to initiate leukocyte diapedesis. 20 The extravasation of leukocytes is a critical step during inflammation which requires 2 the localized opening of the endothelial barrier1-3. This process is initiated by the 3 close interaction of leukocytes with various adhesion molecules such as intercellular 4 adhesion molecule-1 (ICAM-1) on the surface of endothelial cells4-6. It is still unclear 5 how these initial processes induce downstream signaling events resulting in the 6 opening of inter-endothelial junctions to allow leukocyte diapedesis. Here we show 7 that mechanical forces induced by leukocyte-induced clustering of ICAM-1 and fluid 8 shear stress exerted by the flowing blood synergistically activate the 9 mechanosensitive cation channel PIEZO1 in endothelial cells. In human and mouse 10 endothelial cells exposed to low flow, PIEZO1 mediates leukocyte-induced increases 11 in [Ca2+]i and activation of downstream signaling events including phosphorylation of 12 SRC, PYK2 and myosin light chain (MLC) leading to endothelial barrier opening. 13 Mice with endothelium-specific loss of Piezo1 show decreased leukocyte 14 extravasation in different inflammation models. Mechanosensation by endothelial PIEZO1 is required for leukocyte diapedesis 2 Main Text 1 The endothelial cell layer is a tight barrier for cells in the circulation. However, during 2 inflammation leukocytes are able to transmigrate the endothelium and to extravasate 3 in the perivascular space, a process which involves a well-coordinated cascade of 4 events. This includes initial leukocyte capture and rolling, firm adhesion, crawling, 5 which are then followed by breaching of the endothelial barrier and the 6 extravasation7,8. The molecular mechanisms that control and mediate the initial 7 interactions between leukocytes and endothelial cells are well-characterized and 8 involve interactions between endothelial selectins and glycoproteins of leukocytes 9 during capture and rolling steps, whereas arrest, firm adhesion and crawling are 10 mediated mainly by integrins on leukocytes which bind to endothelial intercellular and 11 vascular cell adhesion molecules (ICAM-1 and VCAM-1) and induce their 12 clustering5,8,9. How these initial processes are linked to the opening of the endothelial 13 barrier, which requires the remodeling of endothelial adherens junction as well as 14 endothelial cell contraction2,3,6,10,11 is, however, poorly understood. 15 The endothelial cell layer is a tight barrier for cells in the circulation. However, during 2 inflammation leukocytes are able to transmigrate the endothelium and to extravasate 3 in the perivascular space, a process which involves a well-coordinated cascade of 4 events. This includes initial leukocyte capture and rolling, firm adhesion, crawling, 5 which are then followed by breaching of the endothelial barrier and the 6 extravasation7,8. The molecular mechanisms that control and mediate the initial 7 interactions between leukocytes and endothelial cells are well-characterized and 8 involve interactions between endothelial selectins and glycoproteins of leukocytes 9 during capture and rolling steps, whereas arrest, firm adhesion and crawling are 10 mediated mainly by integrins on leukocytes which bind to endothelial intercellular and 11 vascular cell adhesion molecules (ICAM-1 and VCAM-1) and induce their 12 clustering5,8,9. How these initial processes are linked to the opening of the endothelial 13 barrier, which requires the remodeling of endothelial adherens junction as well as 14 endothelial cell contraction2,3,6,10,11 is, however, poorly understood. 15 Opening of endothelial junctions and endothelial cell contraction during 16 leukocyte transmigration requires activation of endothelial signaling pathways, and 17 several studies have shown that leukocytes induce an increase in the cytosolic Ca2+ 18 concentration in endothelial cells12-16. This calcium signal is not necessary for 19 leukocyte adhesion but is required to induce transendothelial migration12-14. Summary 1 We found that actin polymerization 15 and actomyosin contraction induced by ICAM-1 clustering synergistically with fluid 16 shear stress increase endothelial plasma membrane tension to activate PIEZO1. Our 17 data reveal a mechanism by which leukocytes and the hemodynamic 18 microenvironment synergize to mechanically activate endothelial PIEZO1 and 19 subsequent downstream signaling to initiate leukocyte diapedesis. 20 3 Main Text 1 ICAM-1 20 has been shown to be involved in lymphocyte-induced Ca2+ transients in endothelial 21 cells14, and more recently, the transient receptor potential (TRP) channel C6 (TRPC6) 22 was shown to be involved in endothelial calcium transients induced by neutrophils 23 and for their transendothelial migration17, but how leukocytes induce endothelial Ca2+ 24 transients is still unclear. 25 4 The Piezo proteins PIEZO1 and PIEZO2 are mechanically activated cation 1 channels that form homotrimeric complexes18-20, which are sufficient to mediate 2 mechanically induced currents18. PIEZO1 has been shown to be gated directly by 3 changes in membrane tension21,22 and to mediate multiple cellular functions including 4 endothelial flow sensing23,24. 5 Endothelial PIEZO1 is critically involved in leukocyte extravasation in vivo 1 y y To study leukocyte extravasation in vivo, we injected TNFα into the peritoneal cavity 2 and determined the number of CD11b+/Ly6G+ myeloid cells in the peritoneal cavity 6 3 hours later. While TNFα induced a significant influx of cells into the peritoneal cavity 4 of wild-type mice compared to untreated controls, the effect of TNFα was strongly 5 reduced in EC-Piezo1-KO mice (Fig. 1d). We then studied the role of endothelial 6 PIEZO1 in a model of acute dermatitis of the ear by applying croton oil to the ear 7 surface. Six hours later, when analyzing postcapillary venules characterized by a 8 diameter of 20-30 µm, the primary site of leukocyte extravasation, we found that the 9 majority of neutrophils had completed extravasation and were found in the 10 perivascular space in wild-type mice, whereas about 25-30 % of the leukocytes were 11 found in the lumen of vessels (Fig. 1e-g). However, in EC-Piezo1-KO mice, a 12 significantly reduced portion of leukocytes had completed extravasation, and the 13 majority, about 70 % of cells, showed arrest at the luminal surface of the endothelium 14 (Fig. 1e,g), suggesting that they adhered to the endothelium but were not able to 15 initiate the process of endothelial transmigration. Also intravital microscopy of the 16 cremaster of EC-Piezo1-KO mice revealed a reduced extravasation of neutrophils 17 compared to wild-type animals after intrascrotal injection of IL-1β (Fig. 1h). 18 Hemodynamic parameters were similar in both mouse types, and there was no 19 significant different in leukocyte rolling and adhesion within venules (Extended Data 20 Fig. 2a-e). Also basal extravasation of Evans blue and extravasation after 21 subcutaneous injection of histamine or VEGF were indistinguishable between wild- 22 type and EC-Piezo1-KO mice (Fig. 1i), indicating that vascular permeability was 23 unchanged. Expression of genes encoding proteins involved in endothelial functions 24 was not changed in endothelial cells from EC-Piezo1-KO mice (Extended Data Fig. 25 To study leukocyte extravasation in vivo, we injected TNFα into the peritoneal cavity 2 and determined the number of CD11b+/Ly6G+ myeloid cells in the peritoneal cavity 6 3 hours later. While TNFα induced a significant influx of cells into the peritoneal cavity 4 of wild-type mice compared to untreated controls, the effect of TNFα was strongly 5 reduced in EC-Piezo1-KO mice (Fig. 1d). PIEZO1 is required for leukocyte transendothelial migration in vitro 7 In a screen to identify endothelial transmembrane proteins involved in the 8 transendothelial migration of leukocytes, we identified the mechanosensitive cation 9 channel PIEZO1 (Fig. 1a). The siRNA-mediated knock-down of PIEZO1 in human 10 umbilical venous endothelial cells (HUVECs) or in the mouse brain endothelial cell 11 line bEnd.3 strongly reduced endothelial transmigration of polymorphonuclear 12 leukocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) (Fig. 1b, 13 Extended Data Fig. 1a,b). Similarly, PMN transmigration through mouse lung 14 endothelial cells (MLECs) from mice with endothelium-specific loss of Piezo1 (EC- 15 Piezo1-KO) was strongly reduced compared to wild-type MLECs (Fig. 1c). Basal 16 expression of VE-cadherin and PECAM-1 as well as TNFα-induced expression of 17 ICAM-1 was not affected by loss of PIEZO1 (Extended Data Fig. 1c). Both, PMN 18 transmigration of human and murine endothelial cells could be stimulated by Yoda1, 19 an activator of PIEZO1, and this effect was not seen after knock-down of PIEZO1 in 20 endothelial cells (Fig. 1c, Extended Data Fig. 1b,d,e). PMN rolling on and adhesion to 21 endothelial cells was not affected by loss of endothelial Piezo1 expression (Fig. 1b, 22 Extended Data Fig. 1a), and endothelial barrier function analyzed by measuring the 23 electrical impedance of the endothelial cell layer in vitro or by determining the 24 permeability of the endothelial layer for FITC-labelled dextran in vivo was not affected 25 by loss of PIEZO1 (Extended Data Fig. 1f,g). 26 5 5 Endothelial PIEZO1 is critically involved in leukocyte extravasation in vivo 1 We then studied the role of endothelial 6 PIEZO1 in a model of acute dermatitis of the ear by applying croton oil to the ear 7 surface. Six hours later, when analyzing postcapillary venules characterized by a 8 diameter of 20-30 µm, the primary site of leukocyte extravasation, we found that the 9 majority of neutrophils had completed extravasation and were found in the 10 perivascular space in wild-type mice, whereas about 25-30 % of the leukocytes were 11 found in the lumen of vessels (Fig. 1e-g). However, in EC-Piezo1-KO mice, a 12 significantly reduced portion of leukocytes had completed extravasation, and the 13 majority, about 70 % of cells, showed arrest at the luminal surface of the endothelium 14 (Fig. 1e,g), suggesting that they adhered to the endothelium but were not able to 15 initiate the process of endothelial transmigration. Also intravital microscopy of the 16 cremaster of EC-Piezo1-KO mice revealed a reduced extravasation of neutrophils 17 compared to wild-type animals after intrascrotal injection of IL-1β (Fig. 1h). 18 Hemodynamic parameters were similar in both mouse types, and there was no 19 significant different in leukocyte rolling and adhesion within venules (Extended Data 20 Fig. 2a-e). Also basal extravasation of Evans blue and extravasation after 21 subcutaneous injection of histamine or VEGF were indistinguishable between wild- 22 type and EC-Piezo1-KO mice (Fig. 1i), indicating that vascular permeability was 23 unchanged. Expression of genes encoding proteins involved in endothelial functions 24 6 Leukocytes and low flow synergistically induce downstream signaling via 1 PIEZO1 2 Leukocytes and low flow synergistically induce downstream signaling via 1 PIEZO1 2 Since increases in [Ca2+]i are involved in the initiation of leukocyte transendothelial 3 migration and since leukocyte diapedesis occurs in the presence of low flow in vivo, 4 we studied leukocyte-induced increases in endothelial cytosolic Ca2+ in the absence 5 and presence of flow at a low shear rate (1.2 dynes/cm2). In control HUVECs loaded 6 with Fluo4, low flow or addition of human neutrophils alone had only a small effect on 7 the cytosolic [Ca2+] (Fig. 2a and b). However, when given together, endothelial 8 cytosolic Ca2+ concentration strongly increased (Fig. 2a and b). Leukocyte-dependent 9 increases in endothelial [Ca2+]i were rarely seen during rolling or initial arrest of 10 leukocytes but during crawling and also during the transmigration phase (Extended 11 Data Fig. 3a). After knock-down of endothelial PIEZO1, calcium transients induced by 12 leukocytes in the presence of low flow were strongly reduced or absent (Fig. 2c and 13 d). 14 We then tested the potential involvement of PIEZO1 in the induction of 15 downstream signaling events mediating leukocyte-induced opening of endothelial 16 junctions. Again, low flow alone or addition of PMNs had hardly any effect on the 17 phosphorylation of PYK2, SRC and the myosin light chain (MLC) in endothelial cells. 18 However, application of both flow and PMNs synergistically induced endothelial 19 PYK2, SRC and MLC phosphorylation, and this effect was strongly reduced after 20 knock-down of PIEZO1 (Fig. 2e,f). The effect of PMNs and flow was mimicked by 21 application of Yoda1, and this effect was blocked after knock-down of PIEZO1 (Fig. 22 2g,h). Inhibition of endothelial PYK2 or SRC by PF431396 or PP2, respectively, 23 reduced basal transmigration and blocked Yoda1-induced increases in PMN 24 transmigration (Fig. 2i). These data strongly indicate that PMNs and low flow 25 synergistically induce downstream signaling events through endothelial PIEZO1 26 7 resulting in the opening of endothelial junctions and leukocyte transmigration. 1 Consistent with this, we also observed synergism in the ability of flow and PMNs to 2 induce internalization of VE-cadherin, an effect strongly inhibited after siRNA- 3 mediated suppression of PIEZO1 expression (Fig. 2j). 4 resulting in the opening of endothelial junctions and leukocyte transmigration. 1 Consistent with this, we also observed synergism in the ability of flow and PMNs to 2 induce internalization of VE-cadherin, an effect strongly inhibited after siRNA- 3 mediated suppression of PIEZO1 expression (Fig. 2j). 4 Endothelial PIEZO1 is activated by flow-induced ICAM-1 clustering 6 Since engagement of endothelial ICAM-1 by leukocyte β2 integrins is essential for 7 induction of increases in [Ca2+]i and diapedesis, we suppressed expression of 8 endothelial ICAM-1 and found that this strongly inhibited PMN-induced Ca2+ 9 transients as well as PYK2, SRC and MLC phosphorylation (Extended Fig. 3b-e). 10 Clustering of ICAM-1 using beads coated with anti-ICAM-1 antibodies mimicked the 11 effect of PMNs and induced Ca2+ transients as well as phosphorylation of PYK2, 12 SRC and MLC synergistically with low flow (Fig. 3a,b, Extended Data Fig. 3f,g). The 13 effects of ICAM-1 clustering were inhibited after knock-down of PIEZO1 and ICAM-1 14 (Fig. 3a-d). To test the direct effect of ICAM-1 clustering on PIEZO1-dependent 15 signaling, we cross-linked bound anti-ICAM-1 antibodies. As shown in Fig. 3e-h, 16 clustering of ICAM-1 induced by antibody cross-linking mimicked the effect of PMNs 17 and of anti-ICAM-1 beads and induced Ca2+ transients as well as phosphorylation of 18 PYK2, SRC and MLC synergistically with application of low flow in a PIEZO1- 19 dependent manner. This strongly indicates that clustering and activation of ICAM-1 20 by leukocytes in the presence of flow results in PIEZO1-mediated downstream 21 signaling leading to the opening of endothelial junctions. 22 ICAM-1 clustering and flow synergistically increase membrane tension 24 To analyze how ICAM-1 clustering leads to PIEZO1 activation, we determined 25 membrane tension using the fluorescent lipid tension sensor, FliptR. We found that 26 8 clustering of ICAM-1 leads to a small increase in endothelial membrane tension (Fig. 1 4a and b). Low flow, which by itself had no significant effect on endothelial membrane 2 tension, when given together with ICAM-1 clustering agents, resulted in a very strong 3 increase in plasma membrane tension (Fig. 4a and b). This indicates that low flow 4 and ICAM-1 clustering synergistically increase endothelial membrane tension. Since 5 ICAM-1 clustering has been shown to induce localized actin polymerization and 6 myosin activity resulting in a localized reorganization of the cortical cytoskeleton25, 7 we analyzed the effect of cytochalasin D and blebbistatin on membrane tension and 8 on phosphorylation of PYK2, SRC and MLC induced by ICAM-1 clustering. Both 9 agents blocked ICAM-1-dependent changes in membrane tension and downstream 10 signalling (Fig. 4c,d, Extended Data Fig. 4a,b). We then tested whether increased 11 membrane tension and downstream signaling induced by ICAM-1 clustering involves 12 the actin adapter proteins α-actinin-4 and cortactin which have been shown to be 13 recruited after clustering of ICAM-1 and to be required for ICAM-1-mediated 14 endothelial actin filament branching as well as for ICAM-1-dependent 15 transendothelial migration of neutrophils26-28. As shown in Fig. 4e,f and Extended 16 Data Fig. 4c-e, siRNA-mediated knock-down of the RNAs encoding α-actinin-4 and 17 cortactin (ACTN4 and CTTN, respectively) blocked the effect of ICAM-1 clustering on 18 membrane tension and downstream signaling. 19 Discussion 21 These include exposure to fluid shear stress, mechanical 2 indentation of the cell surface, compression of the cell membrane or forces generated 3 at the cell-cell or cell-matrix interface19,38. Our data show that low-level fluid shear 4 stress as well as interaction of leukocytes with the endothelial surface act in an 5 synergistic manner to activate endothelial PIEZO1 and to initiate leukocyte 6 transendothelial migration. In postcapillary venules, the place where leukocyte 7 extravasation mainly takes place, the shear stress exerted by the flowing blood is 8 relatively low at about 1-2 dynes/cm2 39,40, a shear rate hardly able to induce PIEZO1 9 mediated signaling24. Consistent with this, we saw only very small increases in [Ca2+]i 10 and no significant increase in the phosphorylation of PYK2, SRC or MLC in response 11 to fluid shear stress of 1.2 dynes/cm2. Similarly, when ICAM-1 clustering was induced 12 in TNFα-pretreated endothelial cells by PMNs or anti-ICAM-1 antibodies, only small 13 increases in [Ca2+]i and phosphorylation of PYK2, SRC and MLC could be observed, 14 which were further reduced after suppression of PIEZO1 expression. However, when 15 endothelial ICAM-1 clustering was induced while exposing cells to low flow, 16 downstream signaling was strongly activated in a PIEZO1-dependent manner. This 17 raised the question as to how ICAM-1 clustering promotes PIEZO1 activation. Both 18 ICAM-1 clustering and adhesion of leukocytes to endothelial cells have been shown 19 to induce stiffening of the endothelial surface and to induce traction stress25,41-44. 20 These endothelial responses are due to increased actin polymerization and 21 actomyosin contractility of the cortical cytoskeleton which lead to increased cortical 22 tension45,46 and require recruitment of the actin adapter proteins α-actinin-4 and 23 cortactin25,26. Since the plasma membrane and the underlying cortical cytoskeleton 24 are closely interconnected45,46, changes in the actomyosin cortical tension directly 25 Various mechanical stimuli acting on cellular membranes have been shown to 1 be able to activate PIEZO1. These include exposure to fluid shear stress, mechanical 2 indentation of the cell surface, compression of the cell membrane or forces generated 3 at the cell-cell or cell-matrix interface19,38. Our data show that low-level fluid shear 4 stress as well as interaction of leukocytes with the endothelial surface act in an 5 synergistic manner to activate endothelial PIEZO1 and to initiate leukocyte 6 transendothelial migration. Discussion 21 We here report that the mechanosensitive cation channel PIEZO1 plays a critical role 22 in transendothelial migration of leukocytes in vitro and in vivo by integrating 23 coincident mechanical signals induced by low levels of fluid shear stress and by 24 leukocyte-dependent clustering of ICAM-1. PIEZO1 thereby mediates an increase in 25 [Ca2+]i which leads to localized opening of the endothelial barrier (Fig. 4g). Recent 26 9 9 data reported that TRPC6 is critically involved in leukocyte-induced increases in 1 endothelial [Ca2+]i during leukocyte transendothelial migration17. In in vitro 2 experiments, we were not able to observe a contribution of TRPC6 in leukocyte- 3 induced calcium transients, but it could well be that both PIEZO1 and TRPC6 operate 4 in parallel under in vivo conditions or that PIEZO1 is involved in the initiation of 5 leukocyte extravasation whereas TRPC6 mediates increases in [Ca2+]i mainly at later 6 stages of diapedesis. However, our study considerably differed from the study 7 reported by Weber et al. in that we investigated the role of PIEZO1 in the presence of 8 physiological flow conditions and therefore also addressed whether the local 9 hemodynamic environment of the adhering and transmigrating leukocyte has an 10 effect on leukocyte-induced downstream signaling and transmigration. 11 ICAM-1 is a central endothelial adhesion receptor that functions as a ligand for 12 β2 integrins on leukocytes and promotes leukocyte spreading, migration and 13 transmigration29,30. Engagement of ICAM-1 leads to clustering of ICAM-1 molecules 14 and cytoskeletal changes such as actin polymerization, MLC phosphorylation and 15 actomyosin contractility, which promote junctional opening16,25,30,31. ICAM-1 also 16 promotes increase in [Ca2+]i levels12,32, which has been shown to lead to activation of 17 SRC via protein kinase C14, and ICAM-1-mediated activation of SRC and PYK2 has 18 been shown to be required for VE-cadherin-dependent leukocyte transendothelial 19 migration33. This involves direct phosphorylation of VE-cadherin33-35 as well as 20 indirect regulation of VE-cadherin through VE-PTP36 or by phosphorylation of β- 21 catenin37. How ICAM-1 clustering induces activation of these downstream signaling 22 events resulting in junctional opening and transendothelial migration was unclear. 23 Our data indicate that downstream signaling through ICAM-1 requires co-activation of 24 PIEZO1 by fluid shear stress and ICAM-1-induced reorganization of the cortical 25 cytoskleleton. 26 10 Various mechanical stimuli acting on cellular membranes have been shown to 1 be able to activate PIEZO1. Discussion 21 In postcapillary venules, the place where leukocyte 7 extravasation mainly takes place, the shear stress exerted by the flowing blood is 8 relatively low at about 1-2 dynes/cm2 39,40, a shear rate hardly able to induce PIEZO1 9 mediated signaling24. Consistent with this, we saw only very small increases in [Ca2+]i 10 and no significant increase in the phosphorylation of PYK2, SRC or MLC in response 11 to fluid shear stress of 1.2 dynes/cm2. Similarly, when ICAM-1 clustering was induced 12 in TNFα-pretreated endothelial cells by PMNs or anti-ICAM-1 antibodies, only small 13 increases in [Ca2+]i and phosphorylation of PYK2, SRC and MLC could be observed, 14 which were further reduced after suppression of PIEZO1 expression. However, when 15 endothelial ICAM-1 clustering was induced while exposing cells to low flow, 16 downstream signaling was strongly activated in a PIEZO1-dependent manner. This 17 raised the question as to how ICAM-1 clustering promotes PIEZO1 activation. Both 18 ICAM-1 clustering and adhesion of leukocytes to endothelial cells have been shown 19 to induce stiffening of the endothelial surface and to induce traction stress25,41-44. 20 These endothelial responses are due to increased actin polymerization and 21 actomyosin contractility of the cortical cytoskeleton which lead to increased cortical 22 tension45,46 and require recruitment of the actin adapter proteins α-actinin-4 and 23 cortactin25,26. Since the plasma membrane and the underlying cortical cytoskeleton 24 are closely interconnected45,46, changes in the actomyosin cortical tension directly 25 affect plasma membrane tension46 and therefore are likely to regulate PIEZO1 26 1 11 activity. Consistent with this, we found that inhibition of actin polymerization and 1 myosin activity as well as siRNA-mediated knock-down of α-actinin-4 and cortactin 2 blocked ICAM-1-mediated increases in membrane tension as well as PIEZO1- 3 dependent downstream signaling required for leukocyte transendothelial migration. 4 Recent data indicate that changes in plasma membrane tension are restricted 5 to subcellular domains of endothelial cells as local increases in membrane tension 6 lead only to local activation of mechanosensitive ion channels such as PIEZO147. 7 The finding that leukocyte-induced endothelial downstream signaling and diapedesis 8 require PIEZO1 and flow is consistent with earlier observations, which showed that 9 fluid shear stress promotes transendothelial leukocyte migration48-50. Our data 10 identify a novel synergism of local hemodynamic forces and initial endothelial 11 leukocyte adhesion to induce plasma membrane tension and endothelial signaling 12 events which promote leukocyte extravasation. Discussion 21 The discovery of a novel 13 mechanosensing and mechanosignalling process required for the initial phase of 14 leukocyte diapedesis may also lead to new anti-inflammatory therapeutic approaches. 15 16 12 Acknowledgements 1 The authors wish to thank Svea Hümmer for secretarial help, Yin Hao (Instrument 2 Analysis Center of Xi’an Jiaotong University) for assistance with fluorescence-lifetime 3 imaging microscopy and Shuya Liu, Martina Finkbeiner, Ulrike Krüger and Claudia 4 Ullmann for technical help. This work was supported by the Collaborative Research 5 Centre 834 of the German Research Foundation (S.O.), the Collaborative Research 6 Center 1348 of the German Research Foundation (D.V.) and the National Natural 7 Science Foundation of China (grant #81870220, S.P.W), Shaanxi Natural Science 8 Fund for Distinguished Young Scholars of China (S2020-JC-JQ-0239, S.P.W). 9 10 Author contributions 11 S.P.W. initiated and designed the study, performed experiments, analyzed data and 12 wrote the manuscript; Y.S. performed in vitro experiments; T.M. and R.I.S. performed 13 in vivo experiments; B.S. helped with in vitro and in vivo experiments; L.X. and Z.Y. 14 helped with in vitro experiments; N.W. supervised part of the study and discussed 15 data; D.V. supervised part of the in vivo experiments and analyzed and discussed 16 data; S.O. designed and supervised the study, discussed data and wrote the 17 manuscript. All authors commented on the manuscript. 18 Author information 20 The authors declare no competing financial interests. Correspondence and request 21 for materials should be addressed to S.O. (stefan.offermanns@mpi-bn.mpg.de) or 22 S.P.W. (shengpeng.wang@xjtu.edu.cn). 23 13 References 1 et al. TRPC6 is the endothelial calcium channel that 1 leukocyte transendothelial migration during the inflammatory respons 2 Med 212, 1883-1899, doi:10.1084/jem.20150353 (2015). 3 18 Coste, B. et al. Piezo proteins are pore-forming subunits of me 4 activated channels. Nature 483, 176-181, doi:10.1038/nature10812 (201 5 19 Murthy, S. E., Dubin, A. E. & Patapoutian, A. Piezos thrive under 6 mechanically activated ion channels in health and disease. Nature 7 Molecular cell biology 18, 771-783, doi:10.1038/nrm.2017.92 (2017). 8 20 Zhao, Q. et al. Structure and mechanogating mechanism of the Piezo1 9 Nature 554, 487-492, doi:10.1038/nature25743 (2018). 10 21 Lewis, A. H. & Grandl, J. 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Shear stress affects 9 migration behavior of polymorphonuclear cells arrested on endothelium. Cell 10 Immunol 203, 39-46, doi:10.1006/cimm.2000.1671 (2000). 11 12 17 Figures and figure legends 1 Figures and figure legends 1 Figures and figure legends 1 g g g 2 3 Figure 1. PIEZO1 mediates leukocyte transendothelial migration in vitro and in vivo. 4 3 3 3 Figure 1. PIEZO1 mediates leukocyte transendothelial migration in vitro and in vivo. 4 (a) HUVECs pretreated with 10 ng/ml TNFα were transfected with 360 siRNAs pools 5 against RNAs encoding transmembrane proteins expressed in endothelial cells and 6 were then exposed to THP-1 monocytic cells for 3 hours. Shown is the ratio of THP-1 7 cells which transmigrated the HUVEC monolayer transfected with a particular siRNA 8 pool and with control siRNA. The plot shows the ranked average ratios of three 9 independent experiments. (b) HUVECs were transfected with control (siCtrl) or 10 PIEZO1-specific siRNA (siPIEZO1), and rolling, adhesion and transmigration of 11 human PMNs applied together with flow (1.2 dynes/cm2) were analyzed (n=8 per 12 group). Cells treated with control siRNA were set as 100%. (c) Mouse lung 13 18 endothelial cells (MLECs) were isolated from wild-type (WT) and EC-Piezo1-KO mice 1 and transmigration of mouse PMNs was determined after pretreament without or with 2 1 μM Yoda1 for 15 min (n=5). (d) Wild-type (WT) and endothelium-specific PIEZO1 3 deficient mice (EC-Piezo1-KO) were injected intraperitoneally with PBS or 500 ng of 4 TNFα, and the number of peritoneal CD11b+;Ly6G+ neutrophils was determined by 5 flow cytometry (n=4 mice (-TNFα); n=5 mice (+TNFα)). (e-g) Wild-type (WT) and EC- 6 Piezo1-KO mice were treated with croton oil on one ear. 6 h later, animals were killed 7 and ears were immunostained as whole mounts with antibodies against PECAM-1 8 (blue, endothelium), collagen-IV (red, basement membrane) and MRP14 (green, 9 neutrophil). Arrows indicate neutrophils. Scale bar: 10 μm. (e) Representative images 10 of stained ears. (f) Schematic drawing illustrating the criteria to delineate the 5 11 positions in which leukocyte are found during extravasation. (g) Distribution pattern of 12 neutrophil positions relative to the endothelium and basement membrane (n=16 mice 13 (WT); n=14 mice (EC-Piezo1-KO), 3-5 vessels were analyzed per animal). (h) WT 14 and EC-Piezo1-KO mice were analyzed by intravital microscopy of cremaster 15 venules 4 hours after injection of 50 ng IL-1β for extravasated leukocytes (n=9 mice 16 per group; 4-10 measurements per animal). Figures and figure legends 1 (i) Evans blue extravasation was 17 assessed after subcutaneous injection of 20 µl of PBS without or with 100 μM of 18 histamine or 100 ng/ml of VEGF (n=8 mice (PBS and histamine); n=4 mice (VEGF)). 19 Shown are mean values ± s.e.m.; P ≤ 0.05; P ≤ 0.01; *P ≤ 0.001 (unpaired two- 20 tailed t-test). 21 19 Figure 2. Leukocytes and flow synergistically induce PIEZO1 activation to stimulate endothelial downstream signaling. Figure 2. Leukocytes and flow synergistically induce PIEZO1 activation to stimulate 2 endothelial downstream signaling. 3 (a-d) Untransfected HUVECs (a,b) or HUVECs transfected with control (siCtrl) or 4 PIEZO1-specific siRNA (siPIEZO1) (c,d) were preactivated with TNFα, loaded with 5 Fluo-4 and were then exposed to PMNs alone, to low flow (1.2 dynes/cm2) alone or 6 to both (a,b) or to PMNs and low flow together (c,d). [Ca2+]i was determined as 7 fluorescence intensity (RFU, relative fluorescence units) (a, c). b and d show the area 8 under curve (AUC) of the [Ca2+]i-trace from 6 independent experiments (a.u., 9 arbitrary units). (e-h) Immunoblot analysis of total and phosphorylated PYK2, SRC 10 and MLC in lysates of TNFα-activated HUVECs transfected with control siRNA (siCtrl) 11 or siRNA directed against PIEZO1 and incubated without or with human PMNs in the 12 (a-d) Untransfected HUVECs (a,b) or HUVECs transfected with control (siCtrl) or 4 PIEZO1-specific siRNA (siPIEZO1) (c,d) were preactivated with TNFα, loaded with 5 Fluo-4 and were then exposed to PMNs alone, to low flow (1.2 dynes/cm2) alone or 6 to both (a,b) or to PMNs and low flow together (c,d). [Ca2+]i was determined as 7 fluorescence intensity (RFU, relative fluorescence units) (a, c). b and d show the area 8 under curve (AUC) of the [Ca2+]i-trace from 6 independent experiments (a.u., 9 arbitrary units). (e-h) Immunoblot analysis of total and phosphorylated PYK2, SRC 10 and MLC in lysates of TNFα-activated HUVECs transfected with control siRNA (siCtrl) 11 or siRNA directed against PIEZO1 and incubated without or with human PMNs in the 12 20 absence or presence of low flow (1.2 dynes/cm2) (e) or without or with 5 μM Yoda1 1 (g). Immunoblot analysis of PIEZO1 and GAPDH served as controls. Bar diagrams 2 (f,h) show the densitometric analysis of 3 independent experiments. Figures and figure legends 1 (i) 3 Transmigration of human PMNs across TNFα-activated HUVECs preincubated for 30 4 min with the PYK2 and SRC inhibitors PF431396 (10 μM) and PP2 (10 μM), 5 respectively (n=5 independent experiments). (j) HUVECs transfected with control 6 (siCtrl) or PIEZO1-specific siRNA (siPIEZO1) were preactivated with TNFα and were 7 then exposed to PMNs alone, to low flow (1.2 dynes/cm2) alone or to both. After 15 8 minutes VE-cadherin internalization was determined as described in the Methods 9 (n=4). Shown are mean values ± s.e.m.; P ≤ 0.05; *P ≤ 0.01(unpaired two-tailed t- 10 test). 11 absence or presence of low flow (1.2 dynes/cm2) (e) or without or with 5 μM Yoda1 1 (g). Immunoblot analysis of PIEZO1 and GAPDH served as controls. Bar diagrams 2 (f,h) show the densitometric analysis of 3 independent experiments. (i) 3 Transmigration of human PMNs across TNFα-activated HUVECs preincubated for 30 4 min with the PYK2 and SRC inhibitors PF431396 (10 μM) and PP2 (10 μM), 5 respectively (n=5 independent experiments). (j) HUVECs transfected with control 6 (siCtrl) or PIEZO1-specific siRNA (siPIEZO1) were preactivated with TNFα and were 7 then exposed to PMNs alone, to low flow (1.2 dynes/cm2) alone or to both. After 15 8 minutes VE-cadherin internalization was determined as described in the Methods 9 (n=4). Shown are mean values ± s.e.m.; P ≤ 0.05; *P ≤ 0.01(unpaired two-tailed t- 10 test). 11 21 Figure 3. Endothelial PIEZO1 activation by leukocytes involves ICAM-1 activation and flow. 1 Figure 3. Endothelial PIEZO1 activation by leukocytes involves ICAM-1 activation 2 and flow. 3 (a-h) TNFα-activated HUVECs transfected with control siRNA (siCtrl) or siRNA 4 directed against ICAM-1 or PIEZO1 were exposed to low flow alone, anti-ICAM-1 5 antibody beads (ICAM-1 beads) alone or both (a-d) or to low flow alone, anti-ICAM-1 6 clustering antibodies (ICAM-1 XL) or both (e-h), and immunoblot analysis of total and 7 phosphorylated PYK2, SRC and MLC was performed. Immunoblot analysis of 8 GAPDH served as controls. Bar diagrams (b,f) show the densitometric analysis of 3 9 independent experiments. Alternatively, the free [Ca2+]i was determined after loading 10 of cells with Fluo4 (c,g). Bar diagrams (d,h) show the area under the curve (AUC) of 11 the [Ca2+]i-trace from 3 independent experiments (a.u., arbitrary units). Shown are 12 mean values ± s.e.m.; P ≤ 0.05; *P ≤ 0.01 (unpaired two-tailed t-test). 13 22 Figure 4. Figures and figure legends 1 Flow and ICAM-1 clustering synergistically increase endothelial membrane tension. (a b) Fluorescence lifetime τ1 images of FliptR in TNFα-activated HUVECs kept Figure 4. Flow and ICAM-1 clustering synergistically increase endothelial membrane 3 tension. 4 (a,b) Fluorescence lifetime τ1 images of FliptR in TNFα-activated HUVECs kept 5 under static conditions or in the presence of low flow (1.2 dynes/cm2) or exposed to 6 anti-ICAM-1 antibody beads or to anti-ICAM-1-crosslinking antibodies (ICAM-1 XL) 7 without or together with low flow. The color bar corresponds to lifetime in 8 nanoseconds (ns). Bar length: 15 µm. Corresponding lifetime mean values indicating 9 membrane tension are shown in the bar diagram (b; n = 40 measurements from 5 10 (a,b) Fluorescence lifetime τ1 images of FliptR in TNFα-activated HUVECs kept 5 under static conditions or in the presence of low flow (1.2 dynes/cm2) or exposed to 6 anti-ICAM-1 antibody beads or to anti-ICAM-1-crosslinking antibodies (ICAM-1 XL) 7 without or together with low flow. The color bar corresponds to lifetime in 8 nanoseconds (ns). Bar length: 15 µm. Corresponding lifetime mean values indicating 9 membrane tension are shown in the bar diagram (b; n = 40 measurements from 5 10 23 independent experiments). (c-f) HUVECs were preincubated without or with 10 µM 1 cytochalasin D (CytoD) or 30 µM blebbistatin (Bleb) (c,d) or were transfected with 2 control siRNA (siCtrl) or siRNA directed against the RNA encoding α-actinin-4 3 (siACTN4) or cortactin (siCTTN) (e,f) and were exposed to low flow alone, anti-ICAM- 4 1 clustering antibodies (ICAM-1 XL) alone or both, and membrane tension was 5 determined using FliptR (c,e; n = 20 measurements from 3 independent experiments) 6 or immunoblot analysis of total and phosphorylated PYK2, SRC and MLC was 7 performed (d,f). Bar diagrams show lifetime mean values (c,e). (g) Schematic 8 representation showing how fluid shear stress exerted by the flowing blood and 9 leukocyte-induced ICAM-1 clustering synergistically activate PIEZO1 to induce 10 downstream signaling events resulting in opening of the endothelial barrier. Shown 11 are mean values ± s.e.m.; P ≤ 0.05; P ≤ 0.01; P ≤ 0.001 (unpaired two-tailed t- 12 test). 13 24 2 3 Ext. Data Figure 1. PIEZO1 mediates leukocyte transendothelial migration in vitro. 4 (a-g) The indicated endothelial cells were transfected with control (siCtrl) or PIEZO1- 5 specific siRNA (siPIEZO1). Figures and figure legends 1 (a) Rolling, adhesion and transmigration of mouse PMNs 6 (n=8 per group) applied together with flow (1.2 dynes/cm2) to a bEnd.3 cell 7 monolayer. Cells treated with control siRNA were set as 100%. (b,d,e) 8 Transmigration of human PBMCs (b) (n=4 per group), human PMNs (d) (n=6 per 9 group) or mouse PMNs (e) (n=6 per group) across HUVECs (b,d) or bEnd.3 cells (e) 10 pre-treated without or with 1 μM Yoda1 for 15 min. (c) HUVECs were transfected with 11 control siRNA or siRNA directed against PIEZO1 and were incubated with 10 ng/ml 12 TNFα for 15 h. Cells were then lysed and the indicated proteins were analyzed by 13 immunoblotting using the indicated antibodies. (f) HUVEC barrier integrity was 14 assessed using an electric cell-substrate impedance sensing (ECIS) system in the 15 absence or presence of 1 μM Yoda1 (n=8 per group). (g) Paracellular permeability of 16 the endothelial monolayer cultured in transwell plates was determined using 40 kDa 17 FITC-dextran (n=5 per group; a.u., arbitrary units). Shown are mean values ± s.e.m.; 18 P ≤ 0.05; P ≤ 0.01; P ≤ 0.001 (unpaired two-tailed t-test). 19 2 3 Ext. Data Figure 1. PIEZO1 mediates leukocyte transendothelial migration in vitro 4 2 2 3 Ext. Data Figure 1. PIEZO1 mediates leukocyte transendothelial migration in vitro. 4 3 Data Figure 1. PIEZO1 mediates leukocyte transendothelial migration in vitro. Ext. Data Figure 1. PIEZO1 mediates leukocyte transendothelial migration in vitro. (a-g) The indicated endothelial cells were transfected with control (siCtrl) or PIEZO1- 5 specific siRNA (siPIEZO1). (a) Rolling, adhesion and transmigration of mouse PMNs 6 (n=8 per group) applied together with flow (1.2 dynes/cm2) to a bEnd.3 cell 7 monolayer. Cells treated with control siRNA were set as 100%. (b,d,e) 8 Transmigration of human PBMCs (b) (n=4 per group), human PMNs (d) (n=6 per 9 group) or mouse PMNs (e) (n=6 per group) across HUVECs (b,d) or bEnd.3 cells (e) 10 pre-treated without or with 1 μM Yoda1 for 15 min. (c) HUVECs were transfected with 11 control siRNA or siRNA directed against PIEZO1 and were incubated with 10 ng/ml 12 TNFα for 15 h. Cells were then lysed and the indicated proteins were analyzed by 13 immunoblotting using the indicated antibodies. (f) HUVEC barrier integrity was 14 assessed using an electric cell-substrate impedance sensing (ECIS) system in the 15 absence or presence of 1 μM Yoda1 (n=8 per group). Figures and figure legends 1 (g) Paracellular permeability of 16 the endothelial monolayer cultured in transwell plates was determined using 40 kDa 17 FITC-dextran (n=5 per group; a.u., arbitrary units). Shown are mean values ± s.e.m.; 18 P ≤ 0.05; P ≤ 0.01; P ≤ 0.001 (unpaired two-tailed t-test). 19 25 Ext. Data Fig. 2. Effect of endothelial PIEZO1 knock-out on hemodynamic parameters, leukocyte rolling and adhesion as well as on expression of various genes. Ext. Data Fig. 2. Effect of endothelial PIEZO1 knock-out on hemodynamic 2 parameters, leukocyte rolling and adhesion as well as on expression of various 3 genes. 4 Ext. Data Fig. 2. Effect of endothelial PIEZO1 knock-out on hemodynamic 2 parameters, leukocyte rolling and adhesion as well as on expression of various 3 genes. 4 (a-e) WT and EC-Piezo1-KO mice were analyzed by intravital microscopy of 5 Cremaster venules 4 hours after injection of 50 ng IL1β for the number of adhering 6 PMNs (a), rolling PMNs per second per mm of vessel length (b), rolling flux fraction 7 (c), Newtonian wall shear rates (d) and wall shear rates (e) (n=9 animals per group). 8 (f,g) Lung endothelial cells were isolated from wild-type (WT) and EC-Piezo1-KO 9 mice and analyzed by RNA-sequencing (f) or by immunoblotting (g). Shown are 10 mean values ± s.e.m.; n.s., not significant (unpaired two-tailed t-test). 11 26 1 Ext. Data Fig. 3. Increases in [Ca2+]i and downstream signaling mediated by PIEZO1 2 and ICAM-1. 3 Ext. Data Fig. 3. Increases in [Ca2+]i and downstream signaling mediated by PIEZO1 2 and ICAM-1. 3 (a) HUVECs loaded with Fluo4 were exposed to human neutrophils together with flow 4 (1.2 dynes/cm2), and intracellular free [Ca2+]i was measured during different phases 5 of PMN-endothelial cell interaction (n=6 per group) (RFU, relative fluorescence units). 6 (b-e) TNFα-activated HUVECs transfected with control siRNA (siCtrl) or siRNA 7 directed against ICAM-1 were exposed to low flow alone, human PMNs alone or both, 8 and immunoblot analysis of total and phosphorylated PYK2, SRC and MLC was 9 performed. Immunoblot analysis of GAPDH served as control. The bar diagram (c) 10 shows the densitometric analysis of 3 independent experiments. Alternatively, the 11 free [Ca2+]i was determined after loading of cells with Fluo4 (d,e). The bar diagram (e) 12 shows the area under the curve (AUC) of the [Ca2+]i-trace from 3 independent 13 experiments (a.u., arbitrary units). diagrams show the statistical analysis of 3 independently performed immunoblot 1 experiments. (d,e) Analysis of knock-down efficiency in HUVECs. HUVECs were 2 transfected with control siRNA (siCtrl) or siRNA directed against ACTN4 (d) or CTTN 3 (e). Knock-down efficiency was analyzed by qRT-PCR (n=3). Shown are mean 4 values ± s.e.m.; P ≤ 0.05; *P ≤ 0.01 (unpaired two-tailed t-test). 5 Cell culture and cell isolation 2 Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza and 3 cultured with EGM-2 (Lonza) supplemented with 5% fetal bovine serum (FBS) 4 (Lonza). Only confluent cells at passage ≤4 were used in experiments. THP-1 5 monocyte cells were obtained from Sigma (Cat. No. 88081201) and were cultured in 6 RPMI 1641 medium (Invitrogen) supplemented with 2 mM glutamine and 10% FBS. 7 The bEnd.3 cell line was obtained from ATCC and cells were cultured in DMEM 8 medium (Invitrogen) supplemented with 10% FBS. Primary mouse lung endothelial 9 cells (MLECs) were isolated using CD31-labelled dynabead (Miltenyi Biotec, Cat. No. 10 130-097-418) and were further purified by FACS using anti-CD144-PE antibodies 11 (BD Biosciences, Cat. No. 562243) as described previously51,52. Human 12 polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells 13 (PBMC) were isolated from peripheral blood by density gradient centrifugation using 14 Histopaque 1077 and 1119 (Sigma-Aldrich) and were resuspended in Hanks 15 balanced salt solution (HBSS) with 20 mM HEPES (pH 7.4) and 0.5% human serum 16 albumin (Sigma-Aldrich)53,54. Mouse PMNs from murine femora and tibiae were 17 isolated with an EasySep Mouse Neutrophil Enrichment Kit (STEMCELL 18 Technologies, Canada) according to manufacturer’s instructions. The PMNs were 19 resuspended in Hanks balanced salt solution (HBSS) containing 20 mM HEPES (pH 20 7.4) and 0.5% fetal calf serum and were immediately used for transmigration assays. 21 22 Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza and 3 cultured with EGM-2 (Lonza) supplemented with 5% fetal bovine serum (FBS) 4 (Lonza). Only confluent cells at passage ≤4 were used in experiments. THP-1 5 monocyte cells were obtained from Sigma (Cat. No. 88081201) and were cultured in 6 RPMI 1641 medium (Invitrogen) supplemented with 2 mM glutamine and 10% FBS. 7 The bEnd.3 cell line was obtained from ATCC and cells were cultured in DMEM 8 medium (Invitrogen) supplemented with 10% FBS. Primary mouse lung endothelial 9 cells (MLECs) were isolated using CD31-labelled dynabead (Miltenyi Biotec, Cat. No. 10 130-097-418) and were further purified by FACS using anti-CD144-PE antibodies 11 (BD Biosciences, Cat. No. 562243) as described previously51,52. Cell culture and cell isolation 2 Human 12 polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells 13 (PBMC) were isolated from peripheral blood by density gradient centrifugation using 14 Histopaque 1077 and 1119 (Sigma-Aldrich) and were resuspended in Hanks 15 balanced salt solution (HBSS) with 20 mM HEPES (pH 7.4) and 0.5% human serum 16 albumin (Sigma-Aldrich)53,54. Mouse PMNs from murine femora and tibiae were 17 isolated with an EasySep Mouse Neutrophil Enrichment Kit (STEMCELL 18 Technologies, Canada) according to manufacturer’s instructions. The PMNs were 19 resuspended in Hanks balanced salt solution (HBSS) containing 20 mM HEPES (pH 20 7.4) and 0.5% fetal calf serum and were immediately used for transmigration assays. 21 22 Figures and figure legends 1 (f,g) HUVECs were loaded with Fluo4 and 14 exposed to low flow (1.2 dynes/cm2) alone, anti-ICAM-1 antibody-linked beads 15 (beads) or both flow and beads, and free [Ca2+]i was determined (RFU, relative 16 fluorescence units). The bar diagram shows the statistical evaluation of the area 17 under the curve (AUC) (n=3) (a.u., arbitrary units). Shown are mean values ± s.e.m.; 18 P ≤ 0.05; *P ≤ 0.01 (unpaired two-tailed t-test). 19 27 27 1 2 Ext. Data Fig. 4. Flow and ICAM-1 clustering synergistically induce downstream 3 signalling. 4 (a-c) HUVECs were preincubated without or with 10 µM cytochalasin D (CytoD) or 30 5 1 2 Ext. Data Fig. 4. Flow and ICAM-1 clustering synergistically induce downstream 3 Ext. Data Fig. 4. Flow and ICAM-1 clustering synergistically induce downstream 3 signalling. 4 (a-c) HUVECs were preincubated without or with 10 µM cytochalasin D (CytoD) or 30 5 µM blebbistatin (Bleb) (a,b) or were transfected with control siRNA (siCtrl) or siRNA 6 directed against the RNA encoding α-actinin-4 (siACTN4) or cortactin (siCTTN) (c) 7 and were exposed to low flow alone, anti-ICAM-1 clustering antibodies (ICAM-1 XL) 8 alone or both and membrane tension was determined using FliptR (a), or immunoblot 9 analysis of total and phosphorylated PYK2, SRC and MLC was performed (b,c). Bar 10 28 diagrams show the statistical analysis of 3 independently performed immunoblot 1 experiments. (d,e) Analysis of knock-down efficiency in HUVECs. HUVECs were 2 transfected with control siRNA (siCtrl) or siRNA directed against ACTN4 (d) or CTTN 3 (e). Knock-down efficiency was analyzed by qRT-PCR (n=3). Shown are mean 4 values ± s.e.m.; P ≤ 0.05; *P ≤ 0.01 (unpaired two-tailed t-test). 5 diagrams show the statistical analysis of 3 independently performed immunoblot 1 experiments. (d,e) Analysis of knock-down efficiency in HUVECs. HUVECs were 2 transfected with control siRNA (siCtrl) or siRNA directed against ACTN4 (d) or CTTN 3 (e). Knock-down efficiency was analyzed by qRT-PCR (n=3). Shown are mean 4 values ± s.e.m.; P ≤ 0.05; *P ≤ 0.01 (unpaired two-tailed t-test). 5 diagrams show the statistical analysis of 3 independently performed immunoblot 1 experiments. (d,e) Analysis of knock-down efficiency in HUVECs. HUVECs were 2 transfected with control siRNA (siCtrl) or siRNA directed against ACTN4 (d) or CTTN 3 (e). Knock-down efficiency was analyzed by qRT-PCR (n=3). Shown are mean 4 values ± s.e.m.; P ≤ 0.05; *P ≤ 0.01 (unpaired two-tailed t-test). 5 29 siRNA-mediated knockdown 23 Cells were transfected with siRNAs using Opti-MEM and Lipofectamine RNAiMAX 24 (Invitrogen) as described previously55. For transfection of cells in ibidi flow chambers, 25 20 pmoles of siRNA were mixed gently with RNAiMAX in 20 μl Opti-MEM (Thermo 26 30 Fisher) and incubated for 30 minutes at room temperature. The mixture was then 1 added to 100 μl of cell culture medium, and cells in the flow chamber (ibidi µ-slide I) 2 were covered by the medium. The medium was changed after 6 hours. 18 hours later, 3 the siRNA transfection was repeated, and experiments were performed 48 hours 4 later. For transfection of cells in 96-transwell plates (Corning), 2.5 pmoles siRNA in 5 10 μl were prepared and added to 50 μl cell culture medium. siRNAs against PIEZO1 6 and ICAM1 were from Sigma-Aldrich, and siRNAs against ACTN4 and CTTN were 7 from Genepharma. The targeted sequences of siRNAs directed against RNAs 8 encoding PIEZO1, ICAM-1, α-actinin-4 and cortactin were: PIEZO1 (human), 5′- 9 CCAAGTACTGGATCTATGT-3′, 5′-GCAAGTTCGTGCGCGGATT-3′, and 5′- 10 AGAAGAAGATCGTCAAGTA-3′; ICAM-1 (human), 5′-CAGCGGAAGATCAAGAAAT- 11 3′, 5′-CCGAGCTCAAGTGTCTAAA-3′, 5′-CAACCAATGTGCTATTCAA-3′; α-actinin-4 12 (human) 5′-CCACATCAGCTGGAAGGATGGTC-3′, 5′- 13 Fisher) and incubated for 30 minutes at room temperature. The mixture was then 1 added to 100 μl of cell culture medium, and cells in the flow chamber (ibidi µ-slide I) 2 were covered by the medium. The medium was changed after 6 hours. 18 hours later, 3 the siRNA transfection was repeated, and experiments were performed 48 hours 4 later. For transfection of cells in 96-transwell plates (Corning), 2.5 pmoles siRNA in 5 10 μl were prepared and added to 50 μl cell culture medium. siRNAs against PIEZO1 6 and ICAM1 were from Sigma-Aldrich, and siRNAs against ACTN4 and CTTN were 7 from Genepharma. The targeted sequences of siRNAs directed against RNAs 8 encoding PIEZO1, ICAM-1, α-actinin-4 and cortactin were: PIEZO1 (human), 5′- 9 CCAAGTACTGGATCTATGT-3′, 5′-GCAAGTTCGTGCGCGGATT-3′, and 5′- 10 AGAAGAAGATCGTCAAGTA-3′; ICAM-1 (human), 5′-CAGCGGAAGATCAAGAAAT- 11 3′, 5′-CCGAGCTCAAGTGTCTAAA-3′, 5′-CAACCAATGTGCTATTCAA-3′; α-actinin-4 12 (human) 5′-CCACATCAGCTGGAAGGATGGTC-3′, 5′- 13 GCAGCAGCGCAAGACCTTC-3′; cortactin (human) 5′-CCAGGAGCATATCAACATA- 14 3′; 5′-GCAACTTATTGTATCTGAA-3. 15 SiRNAs used for the screen were pools of siRNAs of a customized siRNA 16 library (Sigma) directed against 360 genes encoding transmembrane proteins 17 enriched in HUVECs. Only siRNAs resulting in suppression of expression levels to 18 less than 25% of control levels as determined by quantitative RT-PCR were used. 19 20 In vitro transmigration assay 21 HUVECs or MLECs were seeded at 1.5 × 104 cells in 100 μl and were cultured on 22 collagen-coated 96-transwell plates with polyester membranes of 8 μm pore size 23 (Corning) until reaching confluency and were then incubated with 10 ng/ml 24 recombinant TNFα (PeproTech, Cat. No. 300-01A) for 16 hours prior to the assay. 25 For transmigration experiments, the medium of the upper compartment was removed 26 31 and 8 × 103 calcein-AM-labelled PMNs were added in 50 μl of HBSS alone or in the 1 presence of the indicated substances. 30 min later, transmigrated PMNs on the lower 2 side of the filter were imaged (Olympus IX81 or Zeiss Axio Observer Z1) and 3 quantified with ImageJ. The screen to identify potential transmembrane proteins that 4 mediate trans-endothelial migration of THP-1 cells was performed in a 96-well format. 5 The ratio of transmigration for each condition was defined as transmigration of THP-1 6 cells after transfection of HUVECs with an individual siRNA pool divided by the 7 transmigration after transfection with control siRNA. For transmigration assay under 8 flow, 1.5 x 104 HUVECs were cultured per channel in a fibronectin-coated ibidi μ- 9 Slide I using a parallel-plate flow chamber and stimulated with 10 ng/ml recombinant 10 TNFα (PeproTech) 16 hours prior to the assay. The flow chamber was perfused with 11 HBSS at a constant shear flow (1.2 dyne/cm2) using a computer controlled air 12 pressure pump (ibidi) for 15 min. PMNs were subsequently injected into the perfusion 13 medium and the transmigration process was recorded for 30 min at 0.5 frames/s 14 using a IX81 (Olympus) microscope at 37°C in the presence of 5% CO2. Percentage 15 of rolling, adhering, crawling and transmigrated PMNs were manually quantified using 16 ImageJ as described previously56. Rolling cells were defined as those that move 17 more than 1 cell diameter within 10 s, while adhering cells were those that moved 18 less than 1 cell diameter within 5 s. Cells transmigrating the endothelial monolayer 19 were directly visualized and crawling was defined as the period between adhesion 20 Determination of [Ca2+]i. 7 For the determination of the intracellular Ca2+ concentration, endothelial cells were 8 loaded with 5 μM Ca2+-sensitive dye Fluo-4 AM (Molecular Probes, Cat. No. F14201) 9 in HBSS supplemented with 20 mM HEPES for 30 min at 37 °C and were then 10 washed with HBSS 3 times at room temperature. Live-cell images were acquired with 11 an IX81 microscope (Olympus) at a frequency of 1 Hz. Fluorescence intensity was 12 measured using a FlexStation 3 (Molecular Devices). 13 Immunoblot analysis 23 Cells were lysed in cell lysis buffer (Cell Signaling, Cat. No. 9803) containing 1% 24 triton X-100 or in RIPA buffer (Cell Signaling, Cat. No. 9806) supplemented with 25 protease and phosphatase inhibitors (Cell Signaling, Cat. No. 5872). Lysates were 26 32 centrifuged at 10,000 x g at 4°C for 10 minutes. Supernatants were then subjected to 1 SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed 2 with primary and HRP-conjugated secondary antibodies (Cell Signaling Technology, 3 Cat. No. 8884 and 7076, respectively) and were developed using the ECL detection 4 system (Pierce). 5 6 Transendothelial electrical resistance measurement 15 Endothelial barrier function was assessed in real-time by continuously recording 16 change in trans-endothelial electrical resistance using the ECIS ZTheta system 17 (Applied BioPhysics) as described previously57. In brief, 40,000 HUVECs were 18 seeded per well of a 96W1E + PET electrode plate coated with 1% gelatin (Applied 19 BioPhysics). HUVEC barrier integrity was analyzed after 48 h when cells had formed 20 a confluent monolayer. 21 FITC-dextran permeability assay 23 1.5 x 104 HUVECs were seeded per well of a collagen-coated transwell plate (3 μm 24 pore size, Corning) and were cultured with daily medium changes until reaching 25 confluency. For knockdown experiments, 8000 cells were transfected using 26 33 Lipofectamine RNAiMAX with the indicated siRNAs. For permeability assay, the 1 medium of the upper insert was removed and replaced with medium containing 250 2 µg/ml fluorescein isothiocyanate (FITC)-conjugated dextran (relative molecular mass 3 40 kDa; Molecular Probes). The permeability was determined by passage of FITC- 4 dextran through the endothelial monolayer into the lower chamber using FlexStation- 5 3 (Molecular Devices). 6 Lipofectamine RNAiMAX with the indicated siRNAs. For permeability assay, the 1 medium of the upper insert was removed and replaced with medium containing 250 2 µg/ml fluorescein isothiocyanate (FITC)-conjugated dextran (relative molecular mass 3 40 kDa; Molecular Probes). The permeability was determined by passage of FITC- 4 dextran through the endothelial monolayer into the lower chamber using FlexStation- 5 3 (Molecular Devices). 6 ICAM-1 clustering 1 For antibody-mediated clustering of ICAM-1, sheep-anti mouse IgG-coupled 2 dynabeads (Invitrogen, Cat. No. M280) were coated with mouse anti-human ICAM-1 3 antibody (R&D Systems, Cat. No. BBIG-I1) or IgG1 control (R&D Systems, Cat. No. 4 MAB002) overnight at 4 °C according to the manufacture’s protocol. To induce 5 clustering, 1.5 x 106 antibody-coated beads were added to a TNFα-stimulated 6 HUVEC monolayer cultured in a 6-well dish and incubated for 15 min. For some 7 experiments, antibody-coated beads were injected into the ibidi perfusion system 8 containing HUVECs to induce ICAM-1 clustering on HUVECs under physiological 9 flow conditions. Alternatively, ICAM-1 was ligated with 15 μg/ml mouse monoclonal 10 antibodies (R&D Systems, Cat. No. BBIG-I1) for 30 min, followed by washing and 11 ICAM-1 cross-linking with 50 μg/ml mouse secondary antibody (R&D Systems, Cat. 12 No. AF007) for 20 min at 37°C. Live cell imaging of membrane tension and 13 intracellular Ca2+ during ICAM-1 clustering were performed using a Leica SP8 or an 14 Olympus IX81 microscope (see above). For immunoblot analysis, beads were 15 isolated using a magnetic holder (Miltenyi Biotec), and cells were lysed with RIPA 16 buffer as described above. 17 Determination of cell membrane tension 8 Membrane tension was determined as previously described58,59. Briefly, endothelial 9 cells cultured in ibidi flow chambers or collagen-coated 8-well glass chambers (Nunc) 10 were stimulated with TNFα overnight and incubated with 1 μM of the membrane 11 tension probe Flipper-TR® (Tebu-bio, Cat. No. SC020) for 30 min at 37 °C. Cells 12 were then washed 3 times with HBSS and subjected to flow (1.2 dynes/cm2) alone, 13 PMNs alone, or flow together with PMNs, anti-ICAM-1 beads or ICAM-1-clustering 14 antibodies (see below) and imaged with a Leica-SP8 FLIM microscope. Excitation 15 was performed using a pulsed 488 nm laser (Laser kit WLL2+pulse picker, Leica 16 Microsystems) operating at 80 MHz, and the emission signal was collected from 549 17 to 651 nm with acousto-optical beam splitter (AOBS) using a gated hybrid (HyD SMD) 18 detectors and a TimeHarp 300 TCSPC Module and Picosecond Event Timer 19 (PicoQuant). SymPhoTime 64 software (PicoQuant) was then used to fit fluorescence 20 decay data. To extract lifetime information, the photon histograms from membrane 21 regions were fitted with a double exponential, and 2 fluorescence emission decay 22 times (τ1 and τ2) are extracted. The longest lifetime with the higher fit amplitude τ1 is 23 used to report membrane tension 59. 24 34 VE-cadherin internalization assay 19 The endocytosis of VE-cadherin was assayed as described60. HUVECs were grown 20 to confluency on collagen-coated eight-well glass chamber (Nunc) or fibronectin- 21 coated flow chambers (ibidi, µ-slide I). Cells were stimulated with 10 ng/ml TNFα 22 before they were incubated with 150 μM chloroquine (Sigma, Cat. No. C6628). The 23 antibody against VE-cadherin (Becton Dickinson, Cat. No. 555661) was dialyzed into 24 the cell culture medium and incubated with cells for 1h at 4 °C. Free antibody was 25 removed by rinsing cells in cold EGM-2 medium. Cells were switched back to 37°C 26 35 35 and were incubated for 15 min with 1 × 104 freshly isolated PMNs per chamber 1 without or with flow (1.2 dynes/cm2). PMNs were then removed by rinsing of cells 2 three times with PBS. To remove cell surface-bound antibody while retaining the 3 internalized antibody, cells were washed with PBS (pH 2.7) containing 25 mM glycine 4 and 5% BSA for 15 min. Cells were then fixed with 4% paraformaldehyde and 5 processed for permeabilization and immunofluorescence staining with secondary 6 antibodies Alexa Fluor 488–goat anti-mouse (Invitrogen, Cat. No. A28175), and DNA 7 was stained with DAPI (Invitrogen, Cat. No. D1306). Fluorescence signals were 8 detected with a confocal laser-scanning microscope (Leica SP8 or Olympus C2). 9 10 Mice 11 All mice were backcrossed onto a C57BL/6N background at least 10 times, and 12 experiments were performed with littermates as controls. Male and female animals at 13 an age of 8-12 weeks were used unless stated otherwise. Mice were housed under a 14 12-hour light-dark cycle with free access to food and water and under specific 15 pathogen-free conditions. The generation of inducible endothelium-specific PIEZO1- 16 deficient mice (Tek-CreERT2;Piezo1fl/fl [EC-Piezo1-KO]) was described previously 55. 17 To induce recombination, animals were injected on 5 consecutive days with 1 mg/d 18 tamoxifen (Sigma) dissolved in corn oil, and 10-14 days later experiments were 19 started. All animals which served as controls for tamoxifen-induced endothelial 20 specific animals were treated with the same amount of tamoxifen under the same 21 conditions. All procedures of animal care and use in this study were approved by the 22 local animal welfare authorities and committees (Regierungspräsidium Darmstadt, 23 Germany and Ethical Committee of Xi’an Jiaotong University, China). VE-cadherin internalization assay 19 24 and were incubated for 15 min with 1 × 104 freshly isolated PMNs per chamber 1 without or with flow (1.2 dynes/cm2). PMNs were then removed by rinsing of cells 2 three times with PBS. To remove cell surface-bound antibody while retaining the 3 internalized antibody, cells were washed with PBS (pH 2.7) containing 25 mM glycine 4 and 5% BSA for 15 min. Cells were then fixed with 4% paraformaldehyde and 5 processed for permeabilization and immunofluorescence staining with secondary 6 antibodies Alexa Fluor 488–goat anti-mouse (Invitrogen, Cat. No. A28175), and DNA 7 was stained with DAPI (Invitrogen, Cat. No. D1306). Fluorescence signals were 8 detected with a confocal laser-scanning microscope (Leica SP8 or Olympus C2). 9 10 All mice were backcrossed onto a C57BL/6N background at least 10 times, and 12 experiments were performed with littermates as controls. Male and female animals at 13 an age of 8-12 weeks were used unless stated otherwise. Mice were housed under a 14 12-hour light-dark cycle with free access to food and water and under specific 15 pathogen-free conditions. The generation of inducible endothelium-specific PIEZO1- 16 deficient mice (Tek-CreERT2;Piezo1fl/fl [EC-Piezo1-KO]) was described previously 55. 17 To induce recombination, animals were injected on 5 consecutive days with 1 mg/d 18 tamoxifen (Sigma) dissolved in corn oil, and 10-14 days later experiments were 19 started. All animals which served as controls for tamoxifen-induced endothelial 20 specific animals were treated with the same amount of tamoxifen under the same 21 conditions. All procedures of animal care and use in this study were approved by the 22 local animal welfare authorities and committees (Regierungspräsidium Darmstadt, 23 Germany and Ethical Committee of Xi’an Jiaotong University, China). 24 36 TNFα-induced peritonitis and flow cytometry 11 Wild-type or EC-Piezo1-KO mice were injected intraperitoneally with 100 µl PBS 12 containing no or 500 ng TNFα prewarmed to 37 °C. After 60 min, animals were killed 13 and cells in the peritoneal cavity were collected by flushing with 5 ml ice-cold PBS. 14 Peritoneal cells were filtered using a 70-μm strainer and analyzed by flow cytometry 15 (BD FACS Canto II). The following antibodies were used: FITC conjugated anti- 16 mouse CD11b (BioLegend, Cat. No. 101205) and APC-conjugated anti-mouse Ly6G 17 (BioLegend, Cat. No. 127614). 18 Intravital microscopy 1 Mice were anesthetized with an intraperitoneal injection of 125 mg/kg ketamine 2 hydrochloride (Zoetis) and 12.5 mg/kg xylazine (Bayer). Cremaster muscle was 3 prepared, and intravital microscopy was carried out as previously described 61. 4 Postcapillary venules with a diameter of 20 to 40 µm were chosen for recording using 5 an intravital upright microscope (Zeiss Axio Examiner Z.1) with a 20x W.Plan 6 Apochromat 1.0 numerical aperture saline immersion objective (Zeiss). Inflammation 7 was induced 4 hours before the experiment by intrascrotal injection of interleukin-1β 8 (IL-1β). 9 In vivo vascular permeability assay 20 A modified Miles assay to determine vascular permeability in the skin was performed 21 as described62. Mice were anaesthetized by isoflurane and shaved 2 days before the 22 assay. Animals were then intraveneously injected with 150 µl of 1% Evans blue 23 solution, and 15 min later 20 µl of VEGF (100 ng/ml, PreproTech) or histamine (100 24 μM, Sigma-Aldrich) in PBS was injected intradermally at the shaved back skin of the 25 mice. 20 µl of PBS was injected as control. 30 min later mice were killed by CO2, 26 37 perfused with PBS, and the area around the injection site was dissected. The 1 extravascular dye was then extracted by incubation with formamide at 56°C for 2 2 days. The degree of vascular leakage was determined by measuring 3 spectrophotometrically at 620 nm the ratio of Evans blue light absorption in test 4 samples and control samples. 5 Ear dermatitis induced by croton oil 7 The method of croton oil-induced dermatitis was perfomed as previously described 8 17,63,64. Briefly, wild-type or EC-Piezo1-KO mice were treated on the inner surface of 9 the right ear with croton oil (Sigma-Aldrich, 2% v/v in a 4:1 mixture of acetone and 10 olive oil). The left ear was treated with vehicle solution as control. Mice were killed 6 11 h later, and both ears were harvested and fixed overnight in 4% paraformaldehyde at 12 4°C in PBS. Ears were then permeabilized with 0.5% Triton X-100, 5% BSA in PBS 13 and incubated in blocking solution (0.5% Triton X-100, 5% BSA in PBS) for 24 h at 14 4°C. Thereafter samples were incubated with anti-PECAM-1 (BD, clone MEC 13.3, 15 Cat. No. 550274), anti-MRP-14 (R&D Systems, Cat. No. AF2065), or anti-collagen IV 16 (Bio-Rad, Cat. No. 2150-1470) overnight at 4°C. Alexa Fluor 488 donkey anti-goat, 17 Alexa Fluor 594 goat anti-rabbit and Alexa Fluor 647 chicken anti-rat (Thermo Fisher 18 Scientific, Cat. No. A11055, A11037 and A21472, respectively) secondary antibodies 19 were used. Tissue was mounted in fluorescent mounting medium (Polysciences, Cat. 20 No. 18606-5), Z-stack projection were acquired using a Leica SP5 or SP8 confocal 21 microscope and image analysis was performed with Imaris (Bitplane) and ImageJ. 22 Other reagents and antibodies 24 Yoda1 (Cat. No. 5586) was from Tocris Bioscience. Cytochalasin D (Cat. No. 25 C8273), Blebbistatin (Cat. No. B0560) and PF431396 (Cat. No. PZ0185) were from 26 Yoda1 (Cat. No. 5586) was from Tocris Bioscience. Cytochalasin D (Cat. No. 25 C8273), Blebbistatin (Cat. No. B0560) and PF431396 (Cat. No. PZ0185) were from 26 38 Sigma. PP2 (Cat. No. 529576) was from Merck Chemicals. Anti-PIEZO1-antibody 1 was from Proteintech (Cat. No. 15939-1-AP). Anti-GAPDH (Cat. No. #5174), anti- 2 PYK2 (Cat. No. #3292), anti-p-PYK2(Tyr402, Cat. No. #3291), anti-SRC (Cat. No. 3 #2109), anti-p-SRC (Tyr416, Cat. No. #6943), anti-MLC (Cat. No. #3672) and anti-p- 4 MLC (Ser19, Cat. No. #3675) were from Cell Signaling Technology. Anti-endomucin- 5 antibody was from Santa Cruz (Cat. No. sc-65495). 6 Statistical analysis 7 Trial experiments or experiments done previously were used to determine sample 8 size with adequate statistical power. Samples were excluded in cases where 9 RNA/cDNA quality or tissue quality after processing was poor (below commonly 10 accepted standards). Data are presented as means ± SEM. Comparisons between 2 11 groups were performed with unpaired 2-tailed Student’s t test, and multiple group 12 comparisons at different time points were performed by 2-way ANOVA followed by 13 Bonferroni’s post hoc test. P ≤ 0.05 was considered to be statistically significant. 14 Data availability 16 All data are available from the corresponding authors upon request. 17 18 19 References (Methods) 20 51 Wang, S. et al. P2Y₂ and Gq/G₁₁ control blood pressure by mediating endothelial 21 mechanotransduction. J Clin Invest 125, 3077-3086 (2015). 22 52 Kaur, H. et al. Single-cell profiling reveals heterogeneity and functional patterning 23 of GPCR expression in the vascular system. Nat Commun 8, 15700, 24 doi:10.1038/ncomms15700 (2017). 25 53 Frye, M. et al. Interfering with VE-PTP stabilizes endothelial junctions in vivo via 26 Tie-2 in the absence of VE-cadherin. J Exp Med 212, 2267-2287, 27 doi:10 1084/jem 20150718 (2015) 28 All data are available from the corresponding authors upon request. 17 18 19 References (Methods) 20 References (Methods) 20 51 Wang, S. et al. P2Y₂ and Gq/G₁₁ control blood pressure by mediating endothelial 21 mechanotransduction. J Clin Invest 125, 3077-3086 (2015). 22 52 Kaur, H. et al. Single-cell profiling reveals heterogeneity and functional patterning 23 of GPCR expression in the vascular system. Nat Commun 8, 15700, 24 doi:10.1038/ncomms15700 (2017). 25 53 Frye, M. et al. Interfering with VE-PTP stabilizes endothelial junctions in vivo via 26 Tie-2 in the absence of VE-cadherin. J Exp Med 212, 2267-2287, 27 doi:10.1084/jem.20150718 (2015). 28 54 Braun, L. J. et al. Platelets docking to VWF prevent leaks during leukocyte 29 extravasation by stimulating Tie-2. Blood 136, 627-639, 30 doi:10.1182/blood.2019003442 (2020). 31 51 Wang, S. et al. P2Y₂ and Gq/G₁₁ control blood pressure by mediating endothelial 21 mechanotransduction. J Clin Invest 125, 3077-3086 (2015). 22 51 Wang, S. et al. P2Y₂ and Gq/G₁₁ control blood pressure by mediating endothelial 21 mechanotransduction. J Clin Invest 125, 3077-3086 (2015). 22 52 Kaur, H. et al. Single-cell profiling reveals heterogeneity and functional patterning 23 of GPCR expression in the vascular system. Nat Commun 8, 15700, 24 doi:10.1038/ncomms15700 (2017). 25 53 Frye, M. et al. Interfering with VE-PTP stabilizes endothelial junctions in vivo via 26 Tie 2 in the absence of VE cadherin J Exp Med 212 2267 2287 27 52 Kaur, H. et al. Single-cell profiling reveals heterogeneity and functional patterning 23 of GPCR expression in the vascular system. Nat Commun 8, 15700, 24 doi:10.1038/ncomms15700 (2017). 25 53 Frye, M. et al. Interfering with VE-PTP stabilizes endothelial junctions in vivo via 26 Tie-2 in the absence of VE-cadherin. J Exp Med 212, 2267-2287, 27 doi:10.1084/jem.20150718 (2015). 28 53 Frye, M. et al. Interfering with VE-PTP stabilizes endothelial junctions in vivo via 26 Tie-2 in the absence of VE-cadherin. J Exp Med 212, 2267-2287, 27 doi:10.1084/jem.20150718 (2015). 28 54 Braun, L. J. et al. Platelets docking to VWF prevent leaks during leukocyte 29 extravasation by stimulating Tie-2. Blood 136, 627-639, 30 doi:10.1182/blood.2019003442 (2020). 31 39 55 Wang, S. et al. Endothelial cation channel PIEZO1 controls blood pressure by 1 mediating flow-induced ATP release. J Clin Invest 126, 4527-4536, 2 doi:10.1172/JCI87343 (2016). 3 56 Heemskerk, N. et al. F-actin-rich contractile endothelial pores prevent vascular 4 leakage during leukocyte diapedesis through local RhoA signalling. Nat Commun 5 7, 10493, doi:10.1038/ncomms10493 (2016). 6 57 Stanicek, L. et al. Long non-coding RNA LASSIE regulates shear stress sensing 7 and endothelial barrier function. References (Methods) 20 Commun Biol 3, 265, doi:10.1038/s42003-020- 8 0987-0 (2020). 9 58 Wang, S. et al. Adipocyte Piezo1 mediates obesogenic adipogenesis through the 10 FGF1/FGFR1 signaling pathway in mice. Nat Commun 11, 2303, 11 doi:10.1038/s41467-020-16026-w (2020). 12 59 Colom, A. et al. A fluorescent membrane tension probe. Nat Chem 10, 1118- 13 1125, doi:10.1038/s41557-018-0127-3 (2018). 14 60 Wessel, F. et al. Leukocyte extravasation and vascular permeability are each 15 controlled in vivo by different tyrosine residues of VE-cadherin. Nat Immunol 15, 16 223-230, doi:10.1038/ni.2824 (2014). 17 61 Artz, A., Butz, S. & Vestweber, D. GDF-15 inhibits integrin activation and mouse 18 neutrophil recruitment through the ALK-5/TGF-betaRII heterodimer. Blood 128, 19 529-541, doi:10.1182/blood-2016-01-696617 (2016). 20 62 Mikelis, C. M. et al. RhoA and ROCK mediate histamine-induced vascular 21 leakage and anaphylactic shock. Nat Commun 6, 6725, 22 doi:10.1038/ncomms7725 (2015). 23 63 Tubaro, A., Dri, P., Delbello, G., Zilli, C. & Della Loggia, R. The croton oil ear test 24 revisited. Agents Actions 17, 347-349, doi:10.1007/BF01982641 (1986). 25 64 Schenkel, A. R., Chew, T. W. & Muller, W. A. Platelet endothelial cell adhesion 26 molecule deficiency or blockade significantly reduces leukocyte emigration in a 27 majority of mouse strains. J Immunol 173, 6403-6408, 28 doi:10.4049/jimmunol.173.10.6403 (2004). 29 30 Figures Figures Figures Figure 1 PIEZO1 mediates leukocyte transendothelial migration in vitro and in vivo. (a) HUVECs pretreated with ng/ml TNFα were transfected with 360 siRNAs pools against RNAs encoding transmembrane protein expressed in endothelial cells and were then exposed to THP-1 monocytic cells for 3 hours. Shown is t ratio of THP-1 cells which transmigrated the HUVEC monolayer transfected with a particular siRNA po Figure 1 PIEZO1 mediates leukocyte transendothelial migration in vitro and in vivo. (a) HUVECs pretreated with 10 ng/ml TNFα were transfected with 360 siRNAs pools against RNAs encoding transmembrane proteins expressed in endothelial cells and were then exposed to THP-1 monocytic cells for 3 hours. Shown is the ratio of THP-1 cells which transmigrated the HUVEC monolayer transfected with a particular siRNA pool and with control siRNA. The plot shows the ranked average ratios of three independent experiments. (b) HUVECs were transfected with control (siCtrl) or PIEZO1-speci¦c siRNA (siPIEZO1), and rolling, adhesion and transmigration of human PMNs applied together with §ow (1.2 dynes/cm2) were analyzed (n=8 per group). Cells treated with control siRNA were set as 100%. References (Methods) 20 (c) Mouse lung 13 endothelial cells (MLECs) were isolated from wild-type (WT) and EC-Piezo1-KO mice and transmigration of mouse PMNs was determined after pretreament without or with 1 μM Yoda1 for 15 min (n=5). (d) Wild-type (WT) and endothelium-speci¦c PIEZO1 de¦cient mice (EC-Piezo1-KO) were injected intraperitoneally with PBS or 500 ng ofTNFα, and the number of peritoneal CD11b+;Ly6G+ neutrophils was determined by§ow cytometry (n=4 mice (-TNFα); n=5 mice (+TNFα)). (e-g) Wild-type (WT) and EC- Piezo1-KO mice were treated with croton oil on one ear. 6 h later, animals were killed and ears were immunostained as whole mounts with antibodies against PECAM-1(blue, endothelium), collagen-IV (red, basement membrane) and MRP14 (green, neutrophil). Arrows indicate neutrophils. Scale bar: 10 μm. (e) Representative images of stained ears. (f) Schematic drawing illustrating the criteria to delineate the 5 positions in which leukocyte are found during extravasation. (g) Distribution pattern of neutrophil positions relative to the endothelium and basement membrane (n=16 mice (WT); n=14 mice (EC-Piezo1-KO), 3-5 vessels were analyzed per animal). (h) WT and EC-Piezo1-KO mice were analyzed by intravital microscopy of cremaster venules 4 hours after injection of 50 ng IL-1β for extravasated leukocytes (n=9 mice per group; 4-10 measurements per animal). (i) Evans blue extravasation was assessed after subcutaneous injection of 20 μl of PBS without or with 100 μM of histamine or 100 ng/ml of VEGF (n=8 mice (PBS and histamine); n=4 mice (VEGF)). Shown are mean values ± s.e.m.; P ≤ 0.05; P ≤ 0.01; P ≤ 0.001 (unpaired two- tailed t-test). Figure 2 Figure 2 Leukocytes and §ow synergistically induce PIEZO1 activation to stimulate endothelial downstream signaling.(a-d) Untransfected HUVECs (a,b) or HUVECs transfected with control (siCtrl) or PIEZO1-spec siRNA (siPIEZO1) (c,d) were preactivated with TNFα, loaded with Fluo-4 and were then exposed to PM alone, to low §ow (1.2 dynes/cm2) alone or to both (a,b) or to PMNs and low §ow together (c,d). [Ca2+ was determined as §uorescence intensity (RFU, relative §uorescence units) (a, c). b and d show the are under curve (AUC) of the [Ca2+]i-trace from 6 independent experiments (a.u., arbitrary units). (e-h) Immunoblot analysis of total and phosphorylated PYK2, SRC and MLC in lysates of TNFα-activated Figure 2 Figure 2 Leukocytes and §ow synergistically induce PIEZO1 activation to stimulate endothelial downstream signaling.(a-d) Untransfected HUVECs (a,b) or HUVECs transfected with control (siCtrl) or PIEZO1-speci¦c siRNA (siPIEZO1) (c,d) were preactivated with TNFα, loaded with Fluo-4 and were then exposed to PMNs alone, to low §ow (1.2 dynes/cm2) alone or to both (a,b) or to PMNs and low §ow together (c,d). [Ca2+]i was determined as §uorescence intensity (RFU, relative §uorescence units) (a, c). b and d show the area under curve (AUC) of the [Ca2+]i-trace from 6 independent experiments (a.u., arbitrary units). (e-h) Immunoblot analysis of total and phosphorylated PYK2, SRC and MLC in lysates of TNFα-activated HUVECs transfected with control siRNA (siCtrl) or siRNA directed against PIEZO1 and incubated without or with human PMNs in the absence or presence of low §ow (1.2 dynes/cm2) (e) or without or with 5 μM Yoda1 (g). Immunoblot analysis of PIEZO1 and GAPDH served as controls. Bar diagrams (f,h) show the densitometric analysis of independent experiments. (i) Transmigration of human PMNs across TNFα- activated HUVECs preincubated for 30 min with the PYK2 and SRC inhibitors PF431396 (10 μM) and PP2 (10 μM), respectively (n=5 independent experiments). (j) HUVECs transfected with control (siCtrl) or PIEZO1-speci¦c siRNA (siPIEZO1) were preactivated with TNFα and were then exposed to PMNs alone, to low §ow (1.2 dynes/cm2) alone or to both. After 15 minutes VE-cadherin internalization was determined as described in the Methods (n=4). Shown are mean values ± s.e.m.; P ≤ 0.05; *P ≤ 0.01(unpaired two- tailed t- test). ) igure 3 ndothelial PIEZO1 activation by leukocytes involves ICAM-1 activation and §ow. (a-h) TNFα-activated UVECs transfected with control siRNA (siCtrl) or siRNA directed against ICAM-1 or PIEZO1 were exposed o low §ow alone, anti-ICAM-1 antibody beads (ICAM-1 beads) alone or both (a-d) or to low §ow alone, nti-ICAM-1 clustering antibodies (ICAM-1 XL) or both (e-h), and immunoblot analysis of total and Figure 3 Endothelial PIEZO1 activation by leukocytes involves ICAM-1 activation and §ow. (a-h) TNFα-activated HUVECs transfected with control siRNA (siCtrl) or siRNA directed against ICAM-1 or PIEZO1 were exposed to low §ow alone, anti-ICAM-1 antibody beads (ICAM-1 beads) alone or both (a-d) or to low §ow alone, anti-ICAM-1 clustering antibodies (ICAM-1 XL) or both (e-h), and immunoblot analysis of total and phosphorylated PYK2, SRC and MLC was performed. Immunoblot analysis of GAPDH served as controls. Bar diagrams (b,f) show the densitometric analysis of 3 independent experiments. Figure 2 Alternatively, the free [Ca2+]i was determined after loading of cells with Fluo4 (c,g). Bar diagrams (d,h) show the area under the curve (AUC) of the [Ca2+]i-trace from 3 independent experiments (a.u., arbitrary units). Shown are mean values ± s.e.m.; P ≤ 0.05; *P ≤ 0.01 (unpaired two-tailed t-test). curve (AUC) of the [Ca2+]i-trace from 3 independent experiments (a.u., arbitrary units). Shown are values ± s.e.m.; P ≤ 0.05; *P ≤ 0.01 (unpaired two-tailed t-test). Figure 4 Figure 4 Figure 4 Flow and ICAM-1 clustering synergistically increase endothelial membrane tension. (a,b) Fluorescence lifetime τ1 images of FliptR in TNFα-activated HUVECs kept under static conditions or in the presence of low §ow (1.2 dynes/cm2) or exposed to anti-ICAM-1 antibody beads or to anti-ICAM-1-crosslinking antibodies (ICAM-1 XL) without or together with low §ow. The color bar corresponds to lifetime in nanoseconds (ns). Bar length: 15 μm. Corresponding lifetime mean values indicating membrane tension are shown in the bar diagram (b; n = 40 measurements from 5 independent experiments). (c-f) HUVECs were preincubated without or with 10 μM 1 cytochalasin D (CytoD) or 30 μM blebbistatin (Bleb) (c,d) or were transfected with control siRNA (siCtrl) or siRNA directed against the RNA encoding α-actinin-4 3 (siACTN4) or cortactin (siCTTN) (e,f) and were exposed to low §ow alone, anti-ICAM-4 1 clustering antibodies (ICAM-1 XL) alone or both, and membrane tension was determined using FliptR (c,e; n = 20 measurements from independent experiments) or immunoblot analysis of total and phosphorylated PYK2, SRC and MLC was performed (d,f). Bar diagrams show lifetime mean values (c,e). (g) Schematic representation showing how §uid shear stress exerted by the §owing blood and leukocyte-induced ICAM-1 clustering synergistically activate PIEZO1 to induce downstream signaling events resulting in opening of the endothelial barrier. Shown are mean values ± s.e.m.; P ≤ 0.05; P ≤ 0.01; *P ≤ 0.001 (unpaired two- tailed t- test). Wangetaleditorialpolicychecklist.pdf Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download.
https://openalex.org/W2198019162	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0143826&type=printable	English	null	Genome Wide Methylome Alterations in Lung Cancer	PloS one	2,015	cc-by	12,791	RESEARCH ARTICLE OPEN ACCESS OPEN ACCESS Citation: Mullapudi N, Ye B, Suzuki M, Fazzari M, Han W, Shi MK, et al. (2015) Genome Wide Methylome Alterations in Lung Cancer. PLoS ONE 10(12): e0143826. doi:10.1371/journal.pone.0143826 Genome Wide Methylome Alterations in Lung Cancer Nandita Mullapudi1☯, Bin Ye2☯, Masako Suzuki3, Melissa Fazzari3, Weiguo Han1, Miao K. Shi1, Gaby Marquardt1, Juan Lin4, Tao Wang5, Steven Keller6, Changcheng Zhu7, Joseph D. Locker7¤, Simon D. Spivack1,3,5* 1 Department of Medicine/Pulmonary, Albert Einstein College of Medicine, Bronx, New York, United States of America, 2 Department of Bioinformatics, Albert Einstein College of Medicine, Bronx, New York, United States of America, 3 Department of Genetics, Albert Einstein College of Medicine, Bronx, New York, United States of America, 4 Department of Epidemiology & Population Health, Division of Biostatistics, Albert Einstein College of Medicine, Bronx, New York, United States of America, 5 Department of Epidemiology & Population Health, Albert Einstein College of Medicine, Bronx, New York, United States of America, 6 Department of Cardiovascular &Thoracic Surgery, Montefiore Medical Center, Bronx, New York, United States of America, 7 Department of Pathology, Montefiore Medical Center, Bronx, New York, United States of America a1111 ☯These authors contributed equally to this work. ¤ Current address: Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America * simon.spivack@einstein.yu.edu ¤ Current address: Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Abstract Citation: Mullapudi N, Ye B, Suzuki M, Fazzari M, Han W, Shi MK, et al. (2015) Genome Wide Methylome Alterations in Lung Cancer. PLoS ONE 10(12): e0143826. doi:10.1371/journal.pone.0143826 Citation: Mullapudi N, Ye B, Suzuki M, Fazzari M, Han W, Shi MK, et al. (2015) Genome Wide Methylome Alterations in Lung Cancer. PLoS ONE 10(12): e0143826. doi:10.1371/journal.pone.0143826 Aberrant cytosine 5-methylation underlies many deregulated elements of cancer. Among paired non-small cell lung cancers (NSCLC), we sought to profile DNA 5-methyl-cytosine features which may underlie genome-wide deregulation. In one of the more dense interroga- tions of the methylome, we sampled 1.2 million CpG sites from twenty-four NSCLC tumor (T)–non-tumor (NT) pairs using a methylation-sensitive restriction enzyme- based HELP- microarray assay. We found 225,350 differentially methylated (DM) sites in adenocarcino- mas versus adjacent non-tumor tissue that vary in frequency across genomic compartment, particularly notable in gene bodies (GB; p<2.2E-16). Further, when DM was coupled to differ- ential transcriptome (DE) in the same samples, 37,056 differential loci in adenocarcinoma emerged. Approximately 90% of the DM-DE relationships were non-canonical; for example, promoter DM associated with DE in the same direction. Of the canonical changes noted, pro- moter (PR) DM loci with reciprocal changes in expression in adenocarcinomas included HBEGF, AGER, PTPRM, DPT, CST1, MELK; DM GB loci with concordant changes in expres- sion included FOXM1, FERMT1, SLC7A5, and FAP genes. IPA analyses showed adenocar- cinoma-specific promoter DMxDE overlay identified familiar lung cancer nodes [tP53, Akt] as well as less familiar nodes [HBEGF, NQO1, GRK5, VWF, HPGD, CDH5, CTNNAL1, Editor: Osman El-Maarri, University of Bonn, Institut of Experimental Hematology and Transfusion Medicine, GERMANY Editor: Osman El-Maarri, University of Bonn, Institut of Experimental Hematology and Transfusion Medicine, GERMANY Received: August 18, 2015 Accepted: November 10, 2015 Published: December 18, 2015 Copyright: © 2015 Mullapudi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2015 Mullapudi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Relevant data can be found on the GEO repository-GSE315520, GSE 31552. PTPN13, DACH1, SMAD6, LAMA3, AR]. Introduction Lung cancer is responsible for the highest number of cancer-related deaths in the United States [1]. Cancer is characterized by genome-wide changes in CpG methylation, including a general- ized genome-wide hypomethylation (loss of methylation) including at oncogenes, and recipro- cal hypermethylation at particular loci (increased methylation), including tumor suppressor gene promoters [2,3]. Recent studies have shown that the functional consequence of 5-methyl- ation of cytosine is dependent on the genomic context and specific sequence in which it occurs [4,5]. Methylation of CG residues within CG islands (CGI) in gene promoters is associated with gene silencing. However, methylation of CGI within gene bodies is found to be associated with tissue-specific expression and gene activation in cancer genomes [6–8]. Panels of well-known candidate tumor suppressor genes have been examined in clinical lung cancer specimens to characterize promoter-methylation [9,10], yielding concise methyla- tion signatures [11] as well as to distinguish the different histological sub-types [12]. Methyla- tion changes occur early during the development of lung cancer [13] and thus can be used as predictive markers to detect potential malignancies [14,15]. Thus, the identification of discrim- inatory methylation marks can be further developed into diagnostic assays to aid in risk assess- ment and diagnostics. DNA methylation can be measured by targeted methods such as bisulfite sequencing (tBGS) [16], methylation-specific PCR (MSP) [17], and mass spectrometry-based methods (Epityper1) [18]. Each platform assays locus-specific methylation at higher resolution, wherein a defined panel of genes can be assessed for the methylation status of a select number of CpG residues within them. However these methods depend on prior knowledge of specific epigenomic loci to design the assay. Among discovery methods to detect methylation patterns at a genome-wide scale, one approach is to employ methylation-sensitive and resistant isoschizmer restriction enzymes (HELP, RLM, others). Other approaches include chromatin immunoprecipitation with methylated DNA- binding antibodies (MBD, MeDIP, others), or bisulfite sequencing of a reduced component of the genome (RRBGS, others) [19]. Each of these methods has its own biases and by necessity of scale, samples only a small subset of the human methylome. Whole genome bisulfite sequencing [20] is designed to densely query the entire methylome at single base-specific resolution. However cur- rently this method is too costly and analytically intensive to perform on large sample sizes. Recent studies have assessed genome-wide methylation in lung cancer to discover tumor specific methylation signatures of cancer genomes [13,21,22]. Abstract The unique findings from this study include the dis- covery of numerous candidate The unique findings from this study include the discovery of numerous candidate methylation sites in both PR and GB regions not previously identified in NSCLC, and many non-canonical relationships to gene expression. These DNA methylation features could potentially be developed as risk or diagnostic biomarkers, or as candidate tar- gets for newer methylation locus-targeted preventive or therapeutic agents. Funding: This work was supported by NCI 1RC1 CA145422-01 (SDS); 1K24-CA139054-01 (SDS); Stony-Wold Herbert Foundation (NM); 1R01- CA180126-01 (SDS Co-PI); P30 CA013330 (Albert Einstein Cancer Center core grant). Competing Interests: The authors have declared that no competing interests exist. 1 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 Methylome Alterations in Lung Cancer PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 Genome-wide survey of differentially methylated loci in lung tumor versus non-tumor Among 24 NSCLC lung resection donors (S1 Table), using the HELP-microarray assay we identified 452,754 HpaII fragments significantly differentially methylated (DM; p<0.05, FDR- adjusted) in tumor versus adjacent non-tumor (Table 1). Of these DM sites, 57% were found in coding regions (comprising 38% of those CCGG sites represented on the array). (Fig 1) Another 39% of these were found in intergenic regions (48% of those sites represented on the array) and were mostly hypomethylated in tumors. Approximately 7% were found in promoter regions (26% of those sites on the array). Gene promoters (PR) and gene bodies (GB) showed both hyper- and hypo-methylation. (Table 1). Promoter hypomethylation exceeded hyper- methylation in number (Table 1). Based on a permutation test conducted using random sam- pling within compartments (PR/GB/IG) we found that DM loci are significantly over- represented in gene body regions (p< 2.2e-16). The magnitude of differential methylation (delta) varied by compartment and direction of change. Moderate/large degrees of DM hypermethylation in PR and GB (delta>1; PR = 74%, GB = 63%) were more common than small degrees (1<delta<0.5; PR = 24%, GB = 33%) of hypermethylation changes in these compartments (Fig 2). The magnitude of moderate/large hypermethylation changes were distinct from that of hypomethylation changes, where the moderate/large distribution by genomic region was PR = 12%, GB = 14%, IG = 17%. Within tumor promoters, CG islands (CGI) and CG shores (CGS) were more often hypermethylated than hypomethylated (Fig 2B). Overall distribution of DM loci varied by PR genomic location (CGI, CGS, other) among all NSCLC histologies (ChiSquare p = 2.2E-16) and among adeno- carcinoma-only (ChiSquare1.9E-4). There was substantial DM outside of CGI and CGS. Individual cancer genes identified by differential methylation The top 25 differentially methylated loci within promoter regions and gene bodies are listed (S2 Table). In brief, for all histologies combined DM was observed in many promoters (S2A Table) [hypermethylation in C7orf54, DARS, SPTAN1, DOM3Z, PCNX, CTNNAL1, others; hypomethylation in NQO1, SIRP1B, UNC5CL, NFIA, CST1, others] and in gene bodies [hyper- methylation in NOL10, ARHGEF12, UST, RGS3, MBNL2, others; hypomethylation FBXL7, RYR2, NTRK3, ADAMTS12, PARK2, others]. For adenocarcinoma specifically, DM was observed in promoters [hypermethylated RASL12; SPTAN1, mir-26a, hypomethylated NQO1, SIRPB1, NF1A] and gene bodies [hypermethylated AKAP13, ANK family, PRKCE, ROS1; hypo- methylated FAM171A1, PARK2, BCAS3, RHOJ] and many others. Introduction Selamat et al [23] used the Illu- mina Infinium HumanMethylation27k platform to characterize genome-wide methylation of ~27,000 CpG sites in 59 matched T/NT lung adenocarcinoma samples, and coupled that to transcriptome arrays. Comprehensive molecular profiling of 230 patients (Adenocarcinoma) and 178 patients (Squamous Cell Carcinomas) by TCGA [24,25] made use of an expanded ver- sion of the same platform, HM450k, which interrogates more than 480,000 CpG sites, across CpG islands and shores in the human genome. We hypothesized that an unbiased genome-wide tumor vs non-tumor search for differen- tially methylated loci will lead to the identification of novel and known loci deregulated in lung cancer. Additionally, investigating the same specimens for differential gene expression would allow identification of higher impact DM loci, by virtue of potential impact on expression. To test these hypotheses, we used the HELP assay [26] to assay the CpG methylation of 24 pairs of tumor (T) and adjacent non-tumor (NT) human samples. This assay queries 1.2 million CCGG motif-defined fragments across the genome by restriction enzymes HpaII (methylation sensitive) and MspI (methylation resistant) to isolate differentially methylated fragments of the genome. These fragments are then adapter-ligated and amplified and labeled, following which 2 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 Methylome Alterations in Lung Cancer they are co-hybridized to a high density microarray. Methylation is detected at ends of enzyme-generated fragments (CCGG sites) and measured as a ratio of MspI-generated frag- ments to HpaII-generated fragments. Reasoning that methylation-deregulated genes might be more apparent if cognate/proximate gene expression is altered, we further examined the associ- ation of differentially methylated (DM) regions with differentially expressed (DE) genes, using mRNA expression data from the same paired T and NT surgical resection samples. Methylome Alterations in Lung Cancer es. 452754 loci are significantly differentially methylated (DM) between T and NT based on an FDR < 0.05. Major- vs NT Table 1. Genomic distribution of DM sites. 452754 loci are significantly differentially methylated (DM) between T and NT based on an FDR < 0.05. Major- ity of the DM loci are hypomethylated in T vs NT. Genomic compartment # loci on array Differentially methylated loci FDR p< 0.05 (% of loci represented) Hypomethylated in tumors Hypermethylated in tumors Promoters 151568 32037 (26%) 68% 31% Gene Body regions 551628 248721 (38%) 74% 25% Intergenic regions 540473 171996 (48%) 93% 6% doi:10 1371/journal pone 0143826 t001 Several loci within the same gene show DM, resulting in the recurrence of gene names in the heat maps. Those multiple loci within a gene (e.g. PR: TMEM88, GIMAP6, RUSC2, others; GB: CDH13, CACNA203, NOMO3, others) tended to be concordant, albeit imperfectly, with the direction of DM (hyper- vs hypomethylation). CpG methylation validation The methylation states of three representative DM CCGG loci chosen on the basis of DM magni- tude (one in the promoter of DARS and two in the promoter of RGS3) were quantitatively deter- mined by the high resolution Sequenom MassARRAY1 method, and compared with the results from the HELP microarray-based assay using Spearman rank order correlation software [27]. The correlation (rho) was 0.72 (p = 0.0006), indicating that the results of HELP assay significantly correlated with the reference results of the Sequenom MassARRAY1 reference assay (S1 Fig) Heat maps of top 50 most differentially methylated loci within the subset of adenocarcinomas (Fig 3A and 3B) The top 50 most DM loci (FDR adjusted p<0.05, ranked by magnitude of delta), reflect separa- tion of T and NT in most of the paired samples, except in the samples 603T, 653T and 542T. 3 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 Identification of discriminatory classifiers The average accuracy for top 100 or top 25 DM loci tumor versus non-tumor classification models, all NSCLC histologies in aggregate, was 87% and 90% respectively. On the Fig 1. The genome compartment represented on the HELP Nimblegen microarray and statistically significant DM loci. (A) Approximately 91% of the 1.2 million loci represented on the HELP microarray are located in gene body (GB) and intergenic (IG) regions, with a small minority (9%) of the loci located within promoters (PR). (B) Statistical significance (Y-axis) vs. delta (X-axis) (magnitude) of DM. Delta (X-axis) indicates the difference in methylation between tumor (T) vs non-tumor (NT) at a given locus. Loci hypermethylated in T relative to NT have delta < 0. P-value (Y-axis) is calculated based on Benjamini Hochberg adjusted FDR. At FDR p < 0.05, 433,505 loci across all genomic compartments are found to be differentially methylated in T vs NT. Red dots indicate statistically significant DM loci. doi:10.1371/journal.pone.0143826.g001 Fig 1. The genome compartment represented on the HELP Nimblegen microarray and statistically significant DM loci. (A) Approximately 91% of the 1.2 million loci represented on the HELP microarray are located in gene body (GB) and intergenic (IG) regions, with a small minority (9%) of the loci located within promoters (PR). (B) Statistical significance (Y-axis) vs. delta (X-axis) (magnitude) of DM. Delta (X-axis) indicates the difference in methylation between tumor (T) vs non-tumor (NT) at a given locus. Loci hypermethylated in T relative to NT have delta < 0. P-value (Y-axis) is calculated based on Benjamini Hochberg adjusted FDR. At FDR p < 0.05, 433,505 loci across all genomic compartments are found to be differentially methylated in T vs NT. Red dots indicate statistically significant DM loci. doi:10.1371/journal.pone.0143826.g001 4 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 Methylome Alterations in Lung Cancer Fig 2. Magnitude and Direction of differential methylation and its distribution across genomic compartments. (A) All NSCLC histologies DM was classified as negligible, small or moderate based on the absolute value. (1< abs delta <2 is Moderate/Large; 1< abs delta <0.5 is Small; 0.5< abs delta <0 is Negligible). DM loci with FDR p<0.05 based on paired T-test were considered for this analysis. Majority of hypermethylation in tumors is observed to be of moderate/large magnitude in promoters and gene bodies, while in the intergenic regions, small changes are most frequent. Identification of discriminatory classifiers The majority of hypomethylation is observed to be of small magnitude in all the three compartments. A significant fraction of hypomethylation changes are of negligible magnitude yet statistically significant. (B) Direction of DM and the distribution within promoters categorized based on location within CG-islands and CG-shores. Within the category of DM promoter loci, hypermethylation is more frequent in tumors as compared to hypomethylation for those loci within CG-islands and CG-shores. Overall DM differences do vary by PR genomic location (CGI, CGS, other); all NSCLC histologies were ChiSquare p = 2.2E-16; adenocarcinoma-only histology ChiSquare p = 1.9E-4. doi:10.1371/journal.pone.0143826.g002 adenocarcinoma data subset, the average accuracy for top 100 and top 25 DM loci was 80% and 79% respectivelyIn general, the classification models tend to be more specific than sensi- tive. (S7 Table). Two loci (LOXL4 and LINC00841) were repeatedly selected within the top DM loci during the classification process. Identification of discriminatory classifiers The majority of hypomethylation is observed to be of small magnitude in all the three compartments. A significant fraction of hypomethylation changes are of negligible magnitude yet statistically significant. (B) Direction of DM and the distribution within promoters categorized based on location within CG-islands and CG-shores. Within the category of DM promoter loci, hypermethylation is more frequent in tumors as compared to hypomethylation for those loci within CG-islands and CG-shores. Overall DM differences do vary by PR genomic location (CGI, CGS, other); all NSCLC histologies were ChiSquare p = 2.2E-16; adenocarcinoma-only histology ChiSquare p = 1.9E-4. d i 10 1371/j l 0143826 002 Fig 2. Magnitude and Direction of differential methylation and its distribution across genomic Fig 2. Magnitude and Direction of differential methylation and its distribution across genomic compartments. (A) All NSCLC histologies DM was classified as negligible, small or moderate based on the absolute value. (1< abs delta <2 is Moderate/Large; 1< abs delta <0.5 is Small; 0.5< abs delta <0 is Negligible). DM loci with FDR p<0.05 based on paired T-test were considered for this analysis. Majority of hypermethylation in tumors is observed to be of moderate/large magnitude in promoters and gene bodies, while in the intergenic regions, small changes are most frequent. The majority of hypomethylation is observed to be of small magnitude in all the three compartments. A significant fraction of hypomethylation changes are of negligible magnitude yet statistically significant. (B) Direction of DM and the distribution within promoters categorized based on location within CG-islands and CG-shores. Within the category of DM promoter loci, hypermethylation is more frequent in tumors as compared to hypomethylation for those loci within CG-islands and CG-shores. Overall DM differences do vary by PR genomic location (CGI, CGS, other); all NSCLC histologies were ChiSquare p = 2.2E-16; adenocarcinoma-only histology ChiSquare p = 1.9E-4. Fig 2. Magnitude and Direction of differential methylation and its distribution across genomic compartments. (A) All NSCLC histologies DM was classified as negligible, small or moderate based on the absolute value. (1< abs delta <2 is Moderate/Large; 1< abs delta <0.5 is Small; 0.5< abs delta <0 is Negligible). DM loci with FDR p<0.05 based on paired T-test were considered for this analysis. Majority of hypermethylation in tumors is observed to be of moderate/large magnitude in promoters and gene bodies, while in the intergenic regions, small changes are most frequent. PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 Methylation x Expression Merge DNA loci were integrated with previously generated mRNA transcriptome microarray data among the 21 T-NT pairs where both datasets were available. (S2 Fig). This analysis yielded n = 433,666 DM loci in all compartments (Table 2). For example, pooling all histologies, we identified n = 75 loci that showed hypermethylation in PR regions and concurrent down-regu- lation of mRNA expression by microarray. There were n = 219 loci within GB regions that showed concurrent hypermethylation and up-regulation of expression. The promoter-specific subcompartment distribution (CGI, CGS, other) of canonical DMxGE relationships (e.g. promoter hypermethylation: gene down-regulation) versus non- canonical relationships (e.g. promoter hypermethylation: gene up-regulation) is displayed in Fig 4. Overall, within PR regions, CGI patterns tended to follow canonical DM:GE patterns (first bar of each of the leftmost two bargraph triplets within a panel) somewhat less frequently than the other promoter compartments. Notable is that non-canonical DM:DE relationships were approximately equal in overall frequency to canonical relationships, as assessed by this analysis. For all 21 pairs (all NSCLC histologies, Fig 4A), overall distribution of DMxDE 5 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 Methylome Alterations in Lung Cancer Fig 3. Heat Map of the Top 50 DM Loci within Adenocarcinomas. (A) Promoter regions; and (B) Gene body regions. Several genes show differential methylation (DM) at more than one locus and appear multiple times in the heatmap. Blue = Non Tumor, Red = Tumor. nomas (A) Promoter regions; and (B) Gene body regions Several genes show differential ( ) Fig 3. Heat Map of the Top 50 DM Loci within Adenocarcinomas. (A) Promoter regions; and (B) Gene body regions. Several genes show differential methylation (DM) at more than one locus and appear multiple times in the heatmap. Blue = Non Tumor, Red = Tumor. Fig 3. Heat Map of the Top 50 DM Loci within Adenocarcinomas. (A) Promoter regions; and (B) Gene body regions. Several genes show differential methylation (DM) at more than one locus and appear multiple times in the heatmap. Blue = Non Tumor, Red = Tumor. doi:10.1371/journal.pone.0143826.g003 differences do vary by PR genomic location (CGI, CGS, other), ChiSquare p = 3.32E-4. Simi- larly, within the set of adenocarcinomas (Fig 4B), overall DMxDE differences do vary by PR genomic location (CGI, CGS, other), ChiSquare p = 1.10E-7. The majority of PR DM loci are associated with hypermethylation when the DM loci are within CG islands. Methylation x Expression Merge This effect is Table 2. Merge of Differential Methylation and Differential Expression (All Histologies– 21 pairs). All Histologies (21 pairs). Assay Total # loci FDR p < 0.05 HELP 1135549 433666 Gene Expression 18208 7957 Regions Correlation # of loci Promoter Hypermethylated and Downregulated 75 Hypomethylated and Upregulated 38 other 3113 Genebody Hypermethylated and Upregulated 219 Hypomethylated and Downregulated 3753 Other 71542 Total 78740 doi:10.1371/journal.pone.0143826.t002 Table 2. Merge of Differential Methylation and Differential Expression (All Histologies– 21 pairs). All Histologies (21 pairs). 6 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 Methylome Alterations in Lung Cancer Fig 4. Methylation vs Expression in Promoter regions. Analysis of DM loci within promoter regions and their overlap with differential gene expression. (Left panel A) All 21 pairs (all NSCLC histologies), overall differences do vary by PR genomic location (CGI, CGS, other), ChiSquare p = 3.32E-4. (Right panel B) Within the set of adenocarcinomas overall differences do vary by PR genomic location (CGI, CGS, other), ChiSquare p = 1.10E-7. Majority of DM promoter loci are associated with hypermethylation when the DM loci are within CG islands. This effect is more pronounced among adenocarcinomas, where the DM loci in CG islands are mostly associated with downregulation of the gene. KEY: “M” = methylation, “E” = expression. Upward arrow indicates increase and downward arrow indicates decrease. doi:10.1371/journal.pone.0143826.g004 Fig 4. Methylation vs Expression in Promoter regions. Analysis of DM loci within promoter regions and their overlap with differential gene expression. (Left panel A) All 21 pairs (all NSCLC histologies), overall differences do vary by PR genomic location (CGI, CGS, other), ChiSquare p = 3.32E-4. (Right panel B) Within the set of adenocarcinomas overall differences do vary by PR genomic location (CGI, CGS, other), ChiSquare p = 1.10E-7. Majority of DM promoter loci are associated with hypermethylation when the DM loci are within CG islands. This effect is more pronounced among adenocarcinomas, where the DM loci in CG islands are mostly associated with downregulation of the gene. KEY: “M” = methylation, “E” = expression. Upward arrow indicates increase and downward arrow indicates decrease. doi:10.1371/journal.pone.0143826.g004 doi:10.1371/journal.pone.0143826.g004 notable among the adenocarcinomas subset as well, where the DM hypermethylated loci in CG islands are mostly associated with downregulation of the gene. Methylation x Expression Merge The number of loci obtained from expression-methylation overlay is displayed in 3D coor- dinates in panel A (S3 Fig). Genomic coordinates were also displayed by circos plots; an exam- ple of chromosome 3 is displayed in panel B (S3 Fig). These panels denote the overall patterns of gene body and promoter methylation and accompanying gene expression changes. The overlap between DM and GE for GB was more frequent than PR (in part due to relative over- representation of the GB versus PR regions on the HELP microarray (Table 1). The canonical pattern most often seen in GB was hypomethylation and down-regulation (S3 Fig). overlap between DM and GE for GB was more frequent than PR (in part due to relative over- representation of the GB versus PR regions on the HELP microarray (Table 1). The canonical pattern most often seen in GB was hypomethylation and down-regulation (S3 Fig). Examples of the quantitative relationship of DM to GE in promoters and gene bodies are displayed in selected scatter plots (S4 Fig). Most genes that were qualitatively canonical for the DM⬄GE relationship showed an ambiguous quantitative relationship (not displayed); those genes that are selected for display do exemplify a canonical relationship. The top eight differentially expressed (DE) genes associated with promoter DM loci (S3 Table: FILIP, HBEF, TMEM88, VWR, CASP12, NQO1, CST, XAGE1D) underwent a quantita- tive verification of microarray-based GE expression changes, using qRT-PCR scaled to GAPDH internal housekeeper. S5 Fig displays these results, showing general concordance of direction of GE between the two platforms (microarray and qRT-PCR; r2 = 0.9367815, p< 0.0007), albeit with a compressed dynamic range of the microarray data, as is typical in the lit- erature [28]. Individual genes revealed by DM x GE Merge All NSCLC histologies: Overlay of DM x DE overlay (S3 Table) yielded additional genomic DM loci with canonical expression patterns (e.g. PR hypermethylation:mRNA downregulated and PR hypomethylation:mRNA upregulated; GB hypermethylated:mRNA upregulated and 7 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 Methylome Alterations in Lung Cancer hypomethylated:mRNA downregulated) [PR n = 113; GB n = 3972] (Table 2). Notable hyper- methylated PR loci with reciprocal decreased expression GE were HBEGF, DPT, AGER, SPARCL1, PTPRM, ARHGEF6, TMEM88, SEMA6A. Those PR hypomethylated loci with increased GE were NQO1, CST1, TNS4, FUT2, MELK, FAM83A, MMP9, and SLCO1B3. Those GB loci with concordant methylation and expression included: hypermethylated/increased GE: FERMT1, SLC7A5, FAP, KRT15, ETV4, TFAP2a TPX2, FOXM1; hypomethylated/decreased GE: AGBL1, RHOJ, LDB2, GHR, ITGA8, ABCB1, SEMA5A, GPM6A. Within the category of adenocarcinomas alone, we merged the results of DM with DE, and discovered several loci with differential methylation in promoters or gene bodies and cognate gene expression changes. Hypermethylated PR loci with GE downregulation include RPL23AP32, CTNNAL1, HBEGF, TMEM88 and CASP12. Loci showing PR hypomethylation and upregulation include NQO1, CST1, XAGE1D, IGKC and AIM2. GB loci showing hyper- methylation and upregulation include FAP, NLN, TPX2, and KIF26B and others. GB loci show- ing hypomethylation and downregulation include AGBL1, RHOJ, LDB2, GHR, ITGA8 and others (S3 Table) Methylation-Expression relationship in CG-islands and CG-shores We queried the association between DM and DE for DM loci located within promoter CG islands (CGI) and CG shores (CGS; defined as 2 kb upstream of a CG island; Fig 4; S5 Table). We observed only a small percentage of loci (3–11%) that exhibited DM within CGI or CGS associated with the expected change in gene-expression of the nearest gene. These include genes such as TMEM88, S1P1R, FZD4, GIPC2, DNAJB4, ADAMTS1 (hypermethylated in CGI and CGS and downregulated) and BUB1 (hypomethylated and upregulated). PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 Current vs Former smokers The sample set of 24 subjects consisted of 11 former smokers and 10 current smokers. We investigated the presence of differentially methylated loci based on smoking status. At adjusted p<0.05 level, no loci were found to be DM between current and former smokers. Methylome Alterations in Lung Cancer significant tumor suppressor gene, known to be mutated or deleted in different cancers. ZEB1, GB hypomethylated, is also highlighted as a central node in this cancer-related network. It encodes a zinc finger transcription factor (also known as TCF8) which is known to be an inducer of epithelial-mesenchymal transition in NSCLC [29]. Within the category of adenocarcinomas specifically (16 pairs S6B Fig shows the cancer- related gene network identified from the most significant (adjusted p<0.05) DM loci (within GB). A central hub of this network is the gene AR (androgen receptor) which is found to be GB hypomethylated in tumors. AR is a transcription factor activated by the steroid hormone androgen. It plays an important role in cell-growth, proliferation, cell-death and invasion. Because of an apparent centrality in this particular DM network, we further explored the DM methylation pattern of AR as it relates to gender. We observed that the two relevant DM frag- ments (within GB) were notable for hypermethylation in GB in males compared to females in NT tissue uniquely (t-test FDR, fragment 1 = 0.041, fragment 2 = 3.76E-5). Supervillin (SVIL), is also a gene at a hub of this network (S6B Fig), and is GB hypomethylated in tumors. SVIL is a peripheral membrane protein that regulates cell motility, spreading and is known to enhance cell survival by interacting with the tumour suppressor gene p53 and its downstream targets [30]. Adenocarcinoma-specific promoter DM x DE overlay did highlight familiar (SMAD6, tP53, CTNNB1, NQO1) as well as unfamiliar lung cancer IPA nodes (S6C Fig). The cancer-related network derived from this analysis consisted of a single hypomethylated gene promoter (NQO1) at the node of a cluster interacting with TP53, HSP70 and NPM1. Several hypermethy- lated gene promoters including HBEGF, SMAD6, PTPN13, CDH5 and SFTPC were found at the periphery of the network. This network is comprised of several genes that are not identified as DM from our study, but do form a part of the network by virtue of their previously pub- lished interactions with other DM loci, as depicted in open/white shapes. PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 Pathway analyses A tabular summary of IPA analyses is offered in S6 Table. All DM loci (Bejamini-Hochberg adj p < 0.05) corresponding to eight categories (based on genomic compartment, histology and with/without gene expression merge) were separately analyzed using IPA, to identify gene net- works enriched within the sets of DM loci. In all the eight cases, “Cancer” was the top disease associated with the input data set, although the constituent genes were different. Canonical pathways from Ingenuity’s knowledge base that were found to be enriched within the gene sets with adj. p < 0.05 are reported. Three of the networks (All NSCLC histologies, DM only, GB; Adenocarcinomas DM only, GB; and Adenocarcinomas DM+DE both, PR) among the eight categories were found to have a statistically significant association with a canonical pathway with adj. p < 0.05, and are further outlined below. IPA Cancer-related Networks depictions. Networks analysis of those significant net- works tabulated in S6 Table are summarized in S6 Fig. The displays show that several genes that play an important role in cancer-related pathways are differentially methylated in T rela- tive to NT in various categories, and highlights some genes that are not known to do so. For example, pooling all NSCLC histologies, S6A Fig shows the cancer-related network derived from IPA of all DM loci (adj. p<0.05) within GB across all the 24 T/NT pairs. Genes such as EZH2, CDH1, CDKN2A and DNMT3A/3B are found at central points in this network. EZH2 (hypomethylated) is a member of the Polycomb-group family and plays an important role in cell proliferation, growth, cell cycle progression, transcriptional repression and invasion. DNMT3A/3B (hypermethylated) encodes a DNA methyl-transferase that is purported to carry out de novo methylation and has an important role in transcriptional repressional signaling. CDH1 (hypermethylated) encodes E-Cadherin, a known surface adhesion molecule downregu- lated in cancers. CDKN2A (hypermethylated) is an inhibitor of CDK4 kinase and is a 8 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 Methylome Alterations in Lung Cancer in multiple samples. The regions of the genome assayed by the HELP assay are dependent upon the occurrences of CCGG sites within the genome, and not by any prior functional or compartment-wise classification of the loci. The HELP assay is less promoter biased (GB and IG regions are represented 3.5x times promoter regions), thereby allowing for the discovery of novel events associated with methylation in other genomic regions in tumor samples [36]. However, the HELP assay misses non-CCGG embedded CpG sites as well as those CCGGs that would define a size range outside the target fragment size range (200-2000bp). HELP (unlike Infinium HM arrays) is not focused on detecting contiguous CpGs of pre-defined gene promoters. While the magnitude of the overall DM differences between tumors and nontumor lung tissue at a given locus tended to be small (generally < 2-fold), reassuring is that the valida- tion of pre-selected DM loci compared favorably with the quantitative reference technique (Sequenom MassARRAY1). Upon examining the gene lists of top DM loci discovered from among other published genome-wide studies in lung cancer to those reported in our study, we found that, as expected, the degree of overlap between our studies and others is modest, possibly indicative of the differ- ences in the HELP platform (which detects fragments bounded by individual CpG sites, but not additional fragment-internal CpG sites), and the target regions queried (which in HELP are equally distributed among PR, GB, and IG regions of the genome. Additionally, we note that the extent of overlap across studies that used the same microarray-based methylation(for e.g. Infinium array) ([23,24,35] was also not large. This could stem from various subject and sample heterogeneity factors, and criteria used to rank DM loci. For example, Sandoval et al [35] identified a HOXA7 region amongst the top most variable CpG promoters, whereas the same locus is not reported in the TCGA study [24] that utilizes the same platform. On the other hand, both these studies report differential methylation of the HOXA9 locus. Both of these loci do not figure in the lists of top DM loci from our study. Overall findings from this study include that: there are many individual DM loci/genes, par- ticularly in gene bodies (S2 and S3 Tables); there are many non-canonical DMxDE relation- ships; and genelists and network relationships include both previously recognized and myriad previously unrecognized loci. PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 Discussion We report a methylome comparison survey for a set of NSCLC tumors versus the paired non- tumor tissue in surgical resection samples in order to identify genome-wide methylation signa- tures in lung cancer, and filter them for those germane to gene expression alterations from the same samples. The goal is informing diagnostic biomarker work already underway in the labo- ratory, [31] and target identification for future development in diagnostic and preventive/ther- apeutic trials [32,33]. Using the HELP assay, we were able to query 1.2 million discontinuous CCGG loci (~1% of the methylome) in a manner representative of all three genomic (PR, GB, IG) regions. Using an FDR adjusted p<0.05 as the cutoff, 452,754 loci across all regions and histologies show sta- tistically significant differential methylation in tumors. The distribution of these DM sites was notably more concentrated in gene bodies than in promoter regions, even considering regional representation variations on the detector microarray, as supported by a permutation test. Studies thus far have typically focused on promoter methylation in lung cancer, utilizing promoter-focused custom microarrays [10,15,34] or bead arrays [23,35] and thus often query only for pre-selected genomic regions and loci. This is one of the few studies to date that more agnostically examines genome-wide methylation across all regions of the lung cancer genome 9 / 21 Methylome Alterations in Lung Cancer detected genes with those in the recent literature was apparent in promoters with prior genome surveys [PR: SLC27A6, SIRPB1; GB: CNTNAP5, CDH13]. However, many “hits” in this study with no readily apparent representation in the lung cancer methylome-related literature were found, [e.g., PR: DARS, CLDN18, APIP; GB: ARHGEF12 PRKCE], and are likely worthy of pur- suit. The potential relevance of some of the unique DM genes identified here is described in Table 4i [41–46]. As for the general magnitude of DM in tumors, we observed that hypermethylation changes were generally higher in magnitude across all genomic compartments (PR, GB, IG), predomi- nantly greater than 50% increased, as compared to the magnitude of hypomethylation changes That is, hypermethylation in tumors was predominantly between 1.5–2-fold, albeit notably more common within CGI regions than was hypomethylation. Given that our study employed homogenized, non-microdissected tissue samples by neces- sity of the platforms available at study commencement, the mixed cell populations could obscure changes in individual cell types from being identified. Also, the now-appreciated rele- vance of more local effects of higher resolution patterns of CpG methylation [4,16] within each CCGG-defined fragment could not be assessed with this HELP platform, such that weak or even powerful effects from smaller fragments or motif fine details could not be ascertained at this resolution, and requires high resolution sequencing based follow-up, examples of which are displayed in this report. Notable is that DNA methylation, despite some inter-locus concor- dance observed here (e.g. CDH13, others), is not “linked” to anywhere near the degree of link- age disequilibrium of native germline nucleotide sequence itself, so that inferences of DNA methylation status at even modest (1kb) distances carry much uncertainty [4]. To further characterize the functional effects of PR and GB methylation, we then analyzed differential mRNA expression data generated from the same donated lung resection specimen and examined the overlay with differentially methylated loci. The idea was to use gene expres- sion as a filter for DM changes, to ascertain those DM sites more likely to have functional con- sequence. We used a simple approach to cross-platform integromics. Using a t-test comparison, after correcting for false discovery rate, we identified among 37,056 DM sites of which only 3,216 were canonically related (DM loci within a 2 kb vicinity of a qualitatively dif- ferentially expressed gene, in the expected direction). PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 As previously recognized, the genome is overall more hypo- methylated, but also displays promoter hypermethylation in cancer versus paired non-cancer tissues, as was true from early genome-wide studies of differential methylation in lung cancer [2,21–23]. However, many features clearly differ; for example, we observed no significant over- lap with CIMP—based classifications [24,25,37], this was possibly due to the difference in the assays used to determine DM (ours more comprehensive, and included many GB abd IG regions), as well as the limited sample size, and therefore power, of this study. We uniquely report here the list of genes showing gene-body (GB) methylation alterations in a group of NSCLCs. This finding is of interest as gene body methylation is an understudied phenomenon and the biological effect is not fully understood; gene expression effects from pro- moter methylation alterations are much better understood [3–5,38]. We also found genome- wide hypomethylation in NSCLC tumors is especially pronounced in the intergenic regions, not previously well explored, and representing the largest proportion of the genome overall. Differential methylation may have functional impact based largely on sequence and struc- tural chromatin context. That is, while cytosine 5-methylation may silence genes or activate genes, depending on precise position and pattern in the promoter, in intergenic and intronic regions, it may have as much to do with gene splicing and effects at a distance, as with expres- sion of the most proximate, neighboring genes [39,40]. The lists of DM loci in promoters (PR) and gene bodies (GB) revealed many sites that were different from the typical list of methylation silenced “players” in NSCLC, derived from assays that are often a priori selected, candidate-gene focused. Clearly, overlap of some of these DM- 10 / 21 Methylome Alterations in Lung Cancer Table 3. Merge of Differential Methylation and Differential Expression (Adenocarcinomas only). Assay Total # Loci FDR p < 0.05 HELP 1135549 225350 Gene Expression 18208 6378 Regions Correlation # of loci Promoter Hypermethylated and Downregulated 64 Hypomethylated and Upregulated 16 other 1239 Genebody Hypermethylated and Upregulated 138 Hypomethylated and Downregulated 2998 Other 32601 Total 37056 doi:10.1371/journal.pone.0143826.t003 Table 3. Merge of Differential Methylation and Differential Expression (Adenocarcinomas only). validation to verify that the DMxDE relationship does indeed exist. We undertook technical validation of gene-expression levels of eight genes identified through the DMxDE analysis: NQO1, CST1, XAGE1D (PR: hypomethylated and up-regulated) and FILIP1, HBEGF, validation to verify that the DMxDE relationship does indeed exist. We undertook technical validation of gene-expression levels of eight genes identified through the DMxDE analysis: NQO1, CST1, XAGE1D (PR: hypomethylated and up-regulated) and FILIP1, HBEGF, TMEM88, VWR and CASP12 (PR: hypermethylated and down-regulated) and confirmed the Table 4. Relevance of some novel DM and DM+DE genes in Lung cancer. Gene name Function Change identiﬁed Ref DARS Aspartyl-tRNA synthetase, a member of a multienzyme complex that mediates the attachment of amino acids to their cognate tRNAs. It is also known to function in inducing cell proliferation in its non-canonical role PR hypermethylated [41] CLDN18 Claudin 18 is an integral membrane protein identiﬁed as an early stage marker of pancreatic cancer. Claudins are involved in the regulation of epithelial-cell barrier function and polarity. CLDN18 was previously identiﬁed as a potential therapy target in NSCLC by virtue of its overexpression PR hypomethylated [42,43] APIP APAF1-interacting protein which functions in the methionine salvage pathway and plays a role in apoptosis. It has been reported to be downregulated in NSCLC at the mRNA and protein level [44](44) and the same study has also shown that other mechanisms in addition to DNA methylation may be involved in its regulation PR hypermethylated [44] PRKCE (Protein Kinase C, epsilon) is a member of the serine-threonine protein kinase C (PKC) family with a role in diverse cellular signaling pathways including cell adhesion, motility, migration, cell cycle functions, cancer cell invasion and apoptosis. PRKCE overexpression has been associated with tumor aggressiveness, malignant transformation and metastases in a variety of cancers including mammary, prostate and lung cancer. GB hypermethylated [45,46] CST1 Cystatin SN) (PR hypomethylated and upregulated) is a cysteine protease inhibitor with a role in inﬂammation and tumorigenesis. doi:10.1371/journal.pone.0143826.t004 The majority of these DMxDE canonical loci were in the GB. Among adenocarcinomas, only a small fraction of these DMxDE loci (8.7%) show the expected canonical association between methylation and gene-expression (PR hyper/hypo-methylation: down/up-regulation, respectively, n = 100; GB hyper/hypo-methyla- tion and up/down-regulation, respectively, n = 3136) (Table 3). This could imply that for the vast majority of statistically significant DM loci within a 2 kb vicinity of differentially expressed genes in tumor, the DMxDE “co-occurrence” is coincidental, with no functional implication. Or alternately, this implies that the DMxDE relationship relies instead on more high resolution detail at single CpG site resolution, rather than estimates of overall fragment methylation [4], as is inherent to the HELP assay. Of course, many competing non-CpG methylation inputs to gene expression are also likely. To further characterize the functional effects of PR and GB methylation, we then analyzed differential mRNA expression data generated from the same donated lung resection specimen and examined the overlay with differentially methylated loci. The idea was to use gene expres- sion as a filter for DM changes, to ascertain those DM sites more likely to have functional con- sequence. We used a simple approach to cross-platform integromics. Using a t-test With the current dataset, the genes so discovered in this functional (DMxDE) subset, where methylation does relate to expression, could be appropriate candidates to prioritize to further understand the functional impact of DNA methylation on gene expression, and its role in lung tumor biology. These merged DMxDE datasets suggest some known or previously reported/ suspected cancer genes [PR: SPARCL1, NQO1, CST1, MELK1, DPT, FAM83A, MMP9; GB: FOXM1, TFAP2, GREM1, ITGA8, GRIA1, SLIT3] as well as many previously unreported genes/loci. The known relevance of some of the above mentioned genes, identified through the DMxDE screen is summarized in Table 4 [47–52]. While provocative in this ‘omics level screen, each of these putative deregulated candidates requires technical as well as biological 11 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 Methylome Alterations in Lung Cancer qualitative (up/down) gene-expression regulation, as assessed by qRT-PCR. The magnitude of fold-changes observed by qRT-PCR were larger than those observed by the genome-wide microarray for most genes; that may be explained by the differences in inherent normalization procedures for the two techniques, as well as the ability for qRT-PCR to span a larger dynamic range of mRNA levels [28]. When examining the gene networks formed from groups of DM genes in various categories using IPA analysis, there were several networks where both known and unknown lung cancer genes/nodes were apparent. For example, the zinc finger transcription factor ZEB1 (TCF8) (GB hypomethylated) was identified within the IPA network generated from GB DM loci, all NSCLC histologies (S6A Fig). While the role of ZEB1 as an inducer of EMT (epithelial-mesen- chymal transition) in NSCLC is well studied [53] and, its regulation by miR-200c has been reported [54], it is yet to be determined if differential gene body methylation observed in this study confers an additional level of gene expression regulation. If indeed GB hypomethylation is found to be tightly associated with ZEB1 expression, it can potentially serve as a biomarker of erlotinib resistance by virtue of its role in EMT [55]. The refined GB, DM set of genes for adenocarcinoma specifically showed enrichment for cancer-related canonical pathways (BH adjusted p-value = 0.0152). The top network in this subset displayed a centrality of the androgen receptor (AR), not generally implicated in lung cancer to date. We noted that AR differed in expression across gender in the non-tumor com- partment, but was not gender-specific in the tumor compartment. SVIL (supervilin) (GB:hypo- methylated) is involved in actin-myosin and cell spreading, a plausible but unexpected finding in lung cancer as well. A new network discovery pattern was also apparent for the DMxDE merged datasets, even if the DM locus in isolation was not readily apparent in the IPA nodes. For example, examining the refined adenocarcinoma only, PR only, DMxDE network for adenocarcinoma displayed the known cancer-related genes [TP53, Akt, NQO1], but also myriad additional nodes [SMARCA4, ITGB1, CTNNB1, Hsp70, AR, others], to date of unknown significance. Similarly, DACH1 (PR hypermethylated in tumor) was one of the IPA-defined nodes identified as a chro- matin-binding protein that associates with other transcription factors to govern gene-expres- sion during development. PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 It was identiﬁed as a target of the Wnt signaling pathway and reported to be overexpressed in endometroid and colorectal cancers. It has also been reported to be overexpressed in NSCLC, and its overexpression is associated with increased risk of recurrence, metastasis and poor survival in NSCLC patients that have undergone surgical resection PR hypomethylated and expression upregulated [47] DPT Dermatopontin is a matricellular protein that accelerates collagen ﬁbrinogenesis and may play an important role in wound healing. It is known to be downregulated in oral and hepatocellular carcinoma (HCC). Silencing of DPT in HCC has been shown to be mediated by DNA methylation. While DPT has been reported to downregulated in the normal bronchial epithelium of smokers relative to non-smokers PR hypermethylated and expression downregulated [48,49] MELK MELK is a serine threonine protein kinase is known to be upregulated in lung, colon, breast, and ovarian cancers. It has been identiﬁed as a promising drug target and a MELK-inhibitor molecule OTSSP167 has been developed and is currently undergoing Phase III trials PR hypomethylated and expression upregulated [50, 51] FOXM1 FOXM1 is a transcription factor that regulates the expression of cell-cycle genes. It is regarded as a proto-oncogene and is upregulated in several cancers including NSCLC. In NSCLC, it is required for K-Ras-mediated tumorigenesis by activating NF-κB and JNK pathways GB hypermethylated and expression upregulated [52] Table 4. Relevance of some novel DM and DM+DE genes in Lung cancer. doi:10.1371/journal.pone.0143826.t004 12 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 HELP assay We used a microarray based version of the HELP assay [26], whereby methylation sensitive (HpaII) versus insensitive (MspI) enzyme pair digests the genome, detecting fragments con- taining paired CCGG sites at the ends of the fragments of 200–2000 bp. To investigate relative ratios of HpaII and MspI digested products from the same sample, a Nimbelgen whole genome high density tiling microarray was used. This array contained 2.3 million probes corresponding to 1.2 million HpaII sites throughout the human genome. For each sample, HpaII and MspI LM-PCR libraries were labeled with Cy5 and Cy3 dyes respec- tively and cohybridized to the Nimbelgen array. Array images generated as cel files were pre- processed and then analyzed. We used custom R-scripts [61] to carry out data preprocessing. Array data were subject to detailed quality control checks (QC) by generating intensity plots. Based on the intensity plots, six pairs were discarded from further analysis due to the presence of non-uniformities and biased intensities. QC-pass array data for 24 pairs were subsequently subjected to normaliza- tion and computation of HpaII to MspI ratios using an R pipeline [61]. Patient recruitment and Sample collection All subjects were enrolled under, and this study was approved by, the Albert Einstein College of Medicine IRB—protocol (#2007–407). All subjects provided fully informed written consent approved by Albert Einstein College of Medicine IRB. This study was comprised of a total of 30 consenting individuals undergoing lung resectional surgery for clinically suspected non-small cell carcinoma. Patient recruitment was conducted as previously described [58–60]. We sur- veyed tumor and adjacent non-tumor tissue from the initial 30 donors drawn from our lung cancer tissue repository. Paired tissue samples were collected in the operating room after lobar resection and immediately snap frozen in liquid isopentane within 15 min of surgical resection; and stored in a −180°C liquid nitrogen tissue bank until analyzed. Sections from these snap fro- zen blocks were examined by a pathologist to confirm tumor presence and composition, to dis- tinguish between adenocarcinomas, squamous cell carcinomas and mixed adenosquamous type. The assigned clinical surgical pathologist confirmed the diagnosis of lung cancer in all cases, per clinical routine, and classified the samples according to the 1999 WHO histologic classifica- tion of lung and pleural tumors, and recent updates [58]. All adenocarcinomas were invasive adenocarcinoma, rather than adenocarcinoma in situ, or minimally invasive adenocarcinomas. Additionally, all selected cases were independently re-reviewed by two pathologists (JL, CZ), blinded to prior histologic diagnosis, clinical, and methylome and transcriptome data. All subjects were enrolled under, and this study was approved by, the Albert Einstein College of Medicine IRB—protocol (#2007–407). All subjects provided fully informed written consent approved by Albert Einstein College of Medicine IRB. This study was comprised of a total of 30 consenting individuals undergoing lung resectional surgery for clinically suspected non-small cell carcinoma. Patient recruitment was conducted as previously described [58–60]. We sur- veyed tumor and adjacent non-tumor tissue from the initial 30 donors drawn from our lung cancer tissue repository. Paired tissue samples were collected in the operating room after lobar resection and immediately snap frozen in liquid isopentane within 15 min of surgical resection; and stored in a −180°C liquid nitrogen tissue bank until analyzed. Sections from these snap fro- zen blocks were examined by a pathologist to confirm tumor presence and composition, to dis- tinguish between adenocarcinomas, squamous cell carcinomas and mixed adenosquamous type. Materials and Methods (Details available in S1 File) (Details available in S1 File) Patient recruitment and Sample collection The assigned clinical surgical pathologist confirmed the diagnosis of lung cancer in all cases, per clinical routine, and classified the samples according to the 1999 WHO histologic classifica- tion of lung and pleural tumors, and recent updates [58]. All adenocarcinomas were invasive adenocarcinoma, rather than adenocarcinoma in situ, or minimally invasive adenocarcinomas. Additionally, all selected cases were independently re-reviewed by two pathologists (JL, CZ), blinded to prior histologic diagnosis, clinical, and methylome and transcriptome data. DACH1 expression has been reported to be reduced in human NSCLC where it was determined to bind tp53 and block lung adenocarcinoma cell growth [56]. Our study was necessarily limited in sample size to accommodate this dense two-platform analysis within available resources, and therefore did not detect DM loci in squamous cell car- cinomas within the statistical threshold applied (adjusted p < 0.05) to any significant degree. This was most likely due to the smaller number of squamous cell carcinoma samples (n = 6) available to us for multiplatform analysis at the time/funding of the study. Similarly, the overall small sample size may have precluded the robust identification of statistically significant changes in current vs former smokers. Another possibility, however, is that tobacco smoke- induced methylation changes are persistent, and incompletely reversed by smoking cessation, in both tumor and non-tumor tissue alike, which is compatible with the epidemiology [57]. We were unable to evaluate EGFR/KRAS somatic mutation data for adenocarcinoma sub- grouping, as most resections accrued before this was routine somatic mutation clinical testing, and post-hoc subject permission was not possible to obtain for many subjects, due to interval subject deaths, and other factors. In summary, a genome-wide query of DNA methylation in lung cancer was performed, showing significant alterations in gene bodies as well as gene promoters and intergenic regions, including many previously unrecognized loci. An initial integromics overlay of genome-wide DNA methylation with gene expression data yielded many hits and coupled DMxDE nodes, 13 / 21 Methylome Alterations in Lung Cancer worthy of further validation. One can envision exploration of those potential targets that vali- date in future observational and experimental studies, for the purposes of risk and diagnostic biomarker development, and for targeted tumor modulation and/or prevention. worthy of further validation. One can envision exploration of those potential targets that vali- date in future observational and experimental studies, for the purposes of risk and diagnostic biomarker development, and for targeted tumor modulation and/or prevention. Pathway Analysis To determine pathways and networks associated with DM loci, we conducted Ingenuity IPA1 analysis. All DM loci (FDR adjusted p< 0.05) as well as DM loci within the vicinity of DE genes were subject to analysis. Fishers t-test was used to assign statistical significance of the association of a given pathway with the set of DM loci. We used multiple-hypothesis corrected p-values to assign significance to the canonical pathways discovered associated with each cate- gory of DM loci. Methylome Alterations in Lung Cancer Significance of distribution of DM loci within the various genomic compartments (PR/GB/ IG) was tested: 433505 loci (same as the number of statistically DM loci (FDR p<0.05)) were first picked at random. 1000 such iterations were performed to assess compartment-wise distri- bution of DM loci. These distributions were compared to the actual distribution of DM loci observed to determine the statistical significance of over-representation of DM loci within GB regions. Identification of discriminatory of classifiers The complete (all NSCLC histologies) data set, as well as the set of adenocarcinomas alone, were split (2/3 and 1/3) into training and test data sets respectively. The top 25 or 100 DM loci were selected from within the training sets, and the process was repeated iteratively 10 times. The success of these DM loci to separate T and NT samples within the test data set was evaluated. Technical validation Statistically significant DM loci were ranked based on the proximity of a locus to other loci showing the same direction of change in methylation, as well as belonging to the same genomic compartment (PR, GB, IG). This strategy helps assess loco-regional methylation consistency across adjacent CCGG-defined fragments. Top ranking promoter loci thus identified (DARS, RGS3) were further evaluated for validation by Sequenom MassARRAY EpiTYPER1 [18]. Methylation-Expression Correlation Paired patient samples were processed with HELP assay or expressionmicroarray using Affy- metrix HuGene 1.0 st chips. HELP assay’s p values were adjusted for multiple testing using FDR method with R package multi-test function p.adjust (method = “fdr”). Significance was defined by FDR adjusted p value < 0.05 for both HELP loci selection and microarray gene selection. We performed paired t tests of tumor vs non-tumor samples for HELP assay and expression microarray data separately. Significant HELP loci were correlated with significantly expressed genes by genome location, if methylation loci were located within 2kb upstream of gene tran- scription starting site, the loci were classified as in promoter region; if loci located within 2 kb downstream of gene transcription starting and upstream/downstream of ending sites, the loci were classified as in gene body region. For promoter-specific analyses, regions within 2 kb upstream of annotated CG islands were classified as CG-shores. Regional validation In the initial DM-only analysis, differences in T vs NT at the fragment locus level were exam- ined using paired t-tests and an additional test to correct for FDR was also applied [62]. Loci were then ranked by their corresponding p-values, and top-ranked loci (FDR p-value < 0.05) considered for subsequent analyses. 14 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 Supporting Information S1 Fig. Validation of selected individual DM sites. Two index genes were used, DARS and RGS3 gene. Left panel A) UCSC genome-browser screen shots for 3 different T/NT pairs; DARS and RGS3 is displayed. Red indicates Tumor, and blue indicates Non-Tumor. PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 15 / 21 Methylome Alterations in Lung Cancer Methylated fragments are represented as quantitative Sequenom MassArray EpiTYPER1 mea- surements shown in a thin vertical bar graph from 0–100% methylation. The CpG locus-spe- cific T-NT differences are subtle. Right panel B) For RGS3 gene, a 495 bp DNA fragment upstream of the transcription start site (Chr9:116,262,214–116,262,708) was amplified for MassARRAY EpiTYPER1 analysis. The methylation state of one CCGG site was quantitatively analyzed from four pairs of tumor and nontumor tissues. For DARS gene, a 308 bp DNA frag- ment upstream the transcription start site (Chr2:136,744,845–136,745,152) was amplified for Sequenom MassARRAY EpiTYPER analysis. The methylation state of two CCGG sites was quantitatively analyzed from four pairs of tumor and nontumor tissues. The methylation degree was calculated by methylated CCGG/methylated +unmethylated CCGG (Methylation ratio by rank, Y-axis). For HELP assay, the methylation degree was indicated by delta value from HpaII vs MspI (delta value by rank, X-axis). Spearman Rank Order Correlation software was used for analysis. The correlation (rho) was 0.72 (p = 0.0006). (PDF) S2 Fig. Strategy for arriving at DM loci associated with DE genes (DMxDE). Statistically sig- nificant DM loci (FDR p<0.05) within promoters and gene bodies and DE genes (FDR p<0.05) were chosen. These DM loci were queried for position within 2 kb of a DE gene. Such loci thus associated with FDR p<0.05 are considered to be associated with differential gene expression, and the direction and location of DM and DE were further analyzed (Tables 2 and 3). (PDF) S3 Fig. Integration of DNA methylation (DM) and gene expression (GE) for 14 lung adeno- carcinomas vs. paired non-tumor samples. (Left panel, A) Methylome data were overlaid on mRNA expression data for gene promoters (left) and gene bodies (right, to demonstrate capac- ity and feasibility. X-axis is the delta readout of the HELP assay; negative (leftward deflection) by convention is for hypermethylated in the test sample tumor, compared to the comparison sample (far-adjacent non-tumor alveolar tissue). Y axis represents the inverse log10 of the false discovery rate (FDR), and z axis is log2 fold change (mRNA levels in tumor:non-tumor). Supporting Information The color of the dots depict “coherent” patterns, where expected biological relationships are mani- fest. For example hypermethylation in a promoter region correlates to decreased expression (green dots), whereas hyper-methylation in a gene body correlates with increased mRNA expression (orange). KEY: Red: gene fold change >2 & delta>0 (T hypomethylated); Green: fold change < -2 & delta < 0 (T hypermethylated); Orange: fold change > 2 & delta < 0; Blue: fold change < -2 & delta > 0. (Right panel B) The circos plot for chromosome 3 is an example of mapping deregulated “hot- spots” to chromosomal coordinates, and as internal check, here highlights several well-known tumor suppressor and other known cancer-related genes (MASP1, WNT7a, TGFBR2, GATA2). Fragments that are hypomethylated are in green (outer circle), HELP tags that are hypermethy- lated are shown in blue (middle circle); and expression microarray genes are shown in yellow for down-regulation, and red for up-regulation (inner circle). The longer purple lines that cut through the chromosome marked the correlated promoter region, while the shorter brown lines mark the gene body regions. S4 Fig. Scatter plots of select genes depicting canonical relationships between methylation and expression. (PR: hypermethylated, downregulated or hypomethylated, upregulated; and, GB: hypermethylated, upregulated or hypomethylated, downregulated). Only a small fraction of genes (8%) identified from the significant DMxDE overlay analyses displayed these 16 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 Methylome Alterations in Lung Cancer S5 Fig. Validation of gene-expression changes by qRT-PCR. Verification was performed in top representative genes that show canonical promoter patterns; PR:hypermethylation and GE downregulation and PR:hypomethylation & GE upregulation. Among DE genes associated with promoter DM loci (S3 Table), these eight genes were selected for qRT-PCR quantitation of gene-expression. All fold changes are depicted for T relative to matched NT; gene-expression values were normalized to GAPDH expression levels. Microarray fold-change values are depicted alongside as a reference. PCR primers and conditions used are described in S4 Table. (PDF) S6 Fig. IPA network analyses. S6A Fig Top IPA network generated from DM loci within gene bodies from all 24 pairs. Previously well-known cancer-related genes such as EZH2, CNR1, SUZ2 (GB hypomethylated), and CDH1, DNMT3A/B, CNR1 (GB hypermethylated) form major nodes in this network. At the periphery of the network several lung cancer—related genes can be detected such as SFRP5, MUC4, PTPRF. PLOS ONE \| DOI:10.1371/journal.pone.0143826 December 18, 2015 References 1. American Cancer Society. Cancer facts & figures. 2014. 1. American Cancer Society. Cancer facts & figures. 2014. 1. American Cancer Society. Cancer facts & figures. 2014. 2. Ehrlich M. DNA methylation in cancer: too much, but also too little. Oncogene. 2002; 21: 5400–13. doi: 10.1038/sj.onc.1205651 PMID: 12154403 2. Ehrlich M. DNA methylation in cancer: too much, but also too little. Oncogene. 2002; 21: 5400–13. doi: 10.1038/sj.onc.1205651 PMID: 12154403 3. Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med. 2003; 349: 2042–2054. doi: 10.1056/NEJMra023075 PMID: 14627790 3. Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med. 2003; 349: 2042–2054. doi: 10.1056/NEJMra023075 PMID: 14627790 4. Han W, Shi M, Spivack SD. 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Anglim PP, Alonzo T a, Laird-Offringa I a. DNA methylation-based biomarkers for early detection of non-small cell lung cancer: an update. Mol Cancer. 2008; 7: 81. doi: 10.1186/1476-4598-7-81 PMID: 18947422 10. Hatada I, Fukasawa M, Kimura M, Morita S, Yamada K, Yoshikawa T, et al. Genome-wide profiling of promoter methylation in human. Oncogene. 2006; 25: 3059–64. doi: 10.1038/sj.onc.1209331 PMID: 16407832 11. Tsou J a, Hagen J a, Carpenter CL, Laird-Offringa I a. DNA methylation analysis: a powerful new tool for lung cancer diagnosis. Oncogene. 2002; 21: 5450–61. doi: 10.1038/sj.onc.1205605 PMID: 12154407 12. Toyooka S, Maruyama R, Toyooka KO, McLerran D, Feng Z, Fukuyama Y, et al. Smoke exposure, his- tologic type and geography-related differences in the methylation profiles of non-small cell lung cancer. Int J Cancer. Author Contributions Conceived and designed the experiments: NM M. Suzuki J. Lin SDS. Performed the experi- ments: NM M. Suzuki WH GM CZ J. Locker. Analyzed the data: NM BY M. Suzuki MF J. Lin TW SDS. Contributed reagents/materials/analysis tools: M. Suzuki MF J. Lin TW SK. Wrote the paper: NM BY WH GM J. Locker SDS. Lab support and RNA studies: M. Shi. Acknowledgments John Greally, MBBS, PhD, for developing and refining the HELP assay, and general guidance; Shahina Maqbool PhD for Epigenomics Core execution of the HELP assay; David Reynolds of the Genomics Core for DNA sequencing and MassARRAY EPITYPER1 guidance. Supporting Information S6B Fig Top Gene network generated from DM loci within gene bodies in Adenocarcinomas alone (16 pairs). AR (GB hypo- methylated in tumors) the androgen receptor gene until now not closely associated with lung cancer forms a central node in this network, and was noted to be more methylated in the GB of normal lung tissue of men than women (not shown here). SVIL (GB hypomethylated) is involved in actin-myosin and cell spreading, and, connects with several other gnes involved in cytoskeletal function including MYO1B (GB hypermethylated), TUBA, TUBB, LMNB etc. S6C Fig Cancer-related gene network generated from DM loci within promoters in the vicinity of DE genes, Adenocarcinomas alone (13 pairs). The cancer-related network derived from this analysis consisted of a single hypomethylated gene promoter (NQO1) at the node of a clus- ter interacting with TP53, HSP70 and NPM1. Several hypermethylated gene promoters includ- ing HBEGF, SMAD6, PTPN13, CDH5 and SFTPC were found at the periphery of the networks. This DMxDE network is comprised of several genes that are not identified as DM from this study, but form a part of the network by virtue of their interactions with other DM loci and are depicted in white shapes. S1 File. Supplementary Materials and Methods. (PDF) S1 Table. Donor Characteristics. (PDF) S2 Table. Top 25 Differentially Methylated Loci. (PDF) S3 Table. Methylation-Expression Overlay. (PDF) S4 Table. Primers used. (PDF) S5 Table. Promoter CGI and CGS distinction in DMxDE analysis. (PDF) S6 Table. Summary of IPA Analyses. (PDF) S1 File. Supplementary Materials and Methods. (PDF) S1 File. Supplementary Materials and Methods. (PDF) S1 Table. Donor Characteristics. (PDF) S2 Table. Top 25 Differentially Methylated Loci. (PDF) S2 Table. Top 25 Differentially Methylated Loci. (PDF) S3 Table. Methylation-Expression Overlay. (PDF) S3 Table. Methylation-Expression Overlay. (PDF) S3 Table. Methylation-Expression Overlay. S4 Table. Primers used. (PDF) S5 Table. Promoter CGI and CGS distinction in DMxDE analysis. (PDF) S6 Table. Summary of IPA Analyses. 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https://openalex.org/W3185975249	https://www.nature.com/articles/s41598-021-94731-2.pdf	English	null	Zooarchaeology through the lens of collagen fingerprinting at Denisova Cave	Scientific reports	2,021	cc-by	9,403	Zooarchaeology through the lens of collagen fingerprinting at Denisova Cave Samantha Brown1,2, Naihui Wang2, Annette Oertle2, Maxim B. Kozlikin3, Michael V. Shunkov3, Anatoly P. Derevianko3, Daniel Comeskey4, Blair Jope‑Street2, Virginia L. Harvey5,6, Manasij Pal Chowdhury5,6, Michael Buckley5,6, Thomas Higham4,7 & Katerina Douka2,7 Samantha Brown1,2, Naihui Wang2, Annette Oertle2, Maxim B. Kozlikin3, Michael V. Shunkov3, Anatoly P. Derevianko3, Daniel Comeskey4, Blair Jope‑Street2, Virginia L. Harvey5,6, Manasij Pal Chowdhury5,6, Michael Buckley5,6, Thomas Higham4,7 & Katerina Douka2,7 Denisova Cave, a Pleistocene site in the Altai Mountains of Russian Siberia, has yielded significant fossil and lithic evidence for the Pleistocene in Northern Asia. Abundant animal and human bones have been discovered at the site, however, these tend to be highly fragmented, necessitating new approaches to identifying important hominin and faunal fossils. Here we report the results for 8253 bone fragments using ZooMS. Through the integration of this new ZooMS-based data with the previously published macroscopically-identified fauna we aim to create a holistic picture of the zooarchaeological record of the site. We identify trends associated with climate variability throughout the Middle and Upper Pleistocene as well as patterns explaining the process of bone fragmentation. Where morphological analysis of bones from the site have identified a high proportion of carnivore bones (30.2%), we find that these account for only 7.6% of the ZooMS assemblage, with large mammals between 3 and 5 more abundant overall. Our analysis suggests a cyclical pattern in fragmentation of bones which sees initial fragmentation by hominins using percussive tools and secondary carnivore action, such as gnawing and digestion, likely furthering the initial human-induced fragmentation. Studying highly fragmented archaeological and paleontological bone assemblages is a particularly challenging task. Robust enough that they can survive a range of depositional environments, bones are still susceptible to numerous taphonomic processes1–4. Research into large assemblages of fragmented bones has been instrumental in exploring depositional histories and accumulation5–8 and the impact of freezing and thawing9, weathering10,11, trampling and gnawing12, and the role of humans modifying and processing bones, either for subsistence rea- sons (e.g. bone marrow exploitation and fuel use)13–16 or for the production of osseous tools17,18. Fragmentation adversely affects taxonomic and anatomical attribution and it is estimated that only a third of bones found in Pleistocene archaeological contexts can be identified using traditional methodologies, i.e. through visual inspection19,20. www.nature.com/scientificreports www.nature.com/scientificreports Zooarchaeology through the lens of collagen fingerprinting at Denisova Cave Without a reliable means of determining taxonomically highly fragmented bones these are often recorded as “unidentified” fauna excluding their integration into detailed zooarchaeological studies and the possibility of identifying new faunal groups and uncommon taxa. p y y g g p Recently the potential of highly fragmented Pleistocene-age bone assemblages has been explored through the use of peptide mass fingerprinting, specifically using ZooMS (Zooarchaeology by Mass Spectrometry)21. ZooMS provides an efficient means of screening and taxonomically identifying bones through the targeted analysis of type I collagen (COL1). The method has been applied to large Pleistocene assemblages22–24, including the Mid- dle and Late Pleistocene site, Denisova Cave, in the Russian Altai25,26. ZooMS analysis of fragmented bones at Denisova Cave has so far resulted in the identification of nine new hominin remains25–27, including two Nean- derthals (Denisova 15 and Denisova 17), three Denisovans (Denisova 19, Denisova 20, and Denisova 21), and 1Institute for Scientific Archaeology, Eberhard Karls University of Tübingen, Tübingen, Germany. 2Max Planck Institute for the Science of Human History, Jena, Germany. 3Institute of Archeology and Ethnography, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia. 4Oxford Radiocarbon Accelerator Unit, RLAHA, University of Oxford, Oxford OX1 3QY, UK. 5Department of Earth and Environmental Sciences, School of Natural Sciences, The University of Manchester, Manchester M13 9PL, UK. 6Manchester Institute of Biotechnology, The University of Manchester, Manchester M1 7DN, UK. 7Department of Evolutionary Anthropology, Faculty of Life Sciences, University of Vienna, Vienna, Austria. *email: brown@shh.mpg.de; douka@shh.mpg.de \| https://doi.org/10.1038/s41598-021-94731-2 Scientific Reports \| (2021) 11:15457 www.nature.com/scientificreports/ Figure 1. Comparison of overall ZooMS results by chamber of Denisova Cave showing samples which could be identified to their most specific ZooMS taxon (shown here as herbivores and predators) in comparison with samples which could only be identified to family/order or those which failed analysis. Figure 1. Comparison of overall ZooMS results by chamber of Denisova Cave showing samples which could be identified to their most specific ZooMS taxon (shown here as herbivores and predators) in comparison with samples which could only be identified to family/order or those which failed analysis. the offspring of a Neanderthal mother and a Denisovan father (Denisova 11)25,28. In the course of identifying these individuals a large assemblage of fauna has been identified which has not been discussed thus far. Here we report the results of 8253 fragmented bones from Denisova Cave that were analysed using ZooMS. Zooarchaeology through the lens of collagen fingerprinting at Denisova Cave These bones were initially studied with the aim of identifying hominin remains and, in light of recent pub- lications detailing the chronology of the site26,29, zooarchaeological findings29–34, and palaeoenvironmental reconstructions35,36 we aim to identify what contribution, if any, these fragmented bones may play in our under- standing of Denisova Cave’s faunal record by reincorporating them into research at the site.h The addition of ZooMS-based determinations into the more traditional zooarchaeological analysis is inher- ently complicated, particularly when analysing morphologically non-diagnostic bones. A first limitation stems from the broad taxonomic groupings created through ZooMS analysis which do not match the often more detailed macroscopic identifications. For large mammals, ZooMS is typically only able to make genus level identifications, with some exceptions in which species can be determined37. Second, because NISP (Number of Individual/Identified Specimens) is the only reliable measurement through which these highly fragmentary bones can be counted, this inevitably leads to an overestimation of the presence/absence of fauna19,20. With these limita- tions in mind, and using different approaches we detail below, we use ZooMS-identified bones to create a holistic view of the fauna assemblage from Denisova Cave and determine broad trends and variation through time. Materials These were sampled with the permission of and in collaboration with the Institute of Archeology and Ethnography of the Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.ii y Bones were specifically chosen as they could not be identified on the basis of morphology and, where possible, fragments > 2 cm in length were preferentially selected, although smaller bones were also part of our analysed assemblage. Assemblages of bones of this size were also assumed to contain morphologically indistinct hominin remains since all previously identified hominins at the site were no bigger than ~ 3 cm in length. Samples were drilled in preparation for analysis and from each bone approximately 20 mg was removed using a diamond covered disc. Care was taken to clean equipment between drilling samples to minimise cross-contamination. Sampling was undertaken at two laboratories, at the School of Archaeology, University of Oxford, U.K. and at the Department of Archaeology, Max Planck Institute for the Science of Human History (MPI-SHH), Jena, Germany. Materials Denisova Cave, situated in the Altai Mountains of Russian Siberia, is unique in its hominin record as the only site where both Neanderthal and Denisovan fossils have been identified (Supplementary Fig. 1)38–41. The cave, situated in the ledge of a southwest-facing rock wall and overlooking the Anui River, has the longest stratigraphic sequence for northern Eurasia, with contexts dating from the Middle Pleistocene. This extensive sequence has made Denisova Cave a type-site for northern Eurasia, allowing for palynological42 and palaeoecological43,44 reconstructions of the region and several zooarchaeological studies29–34,36. Despite particularly favorable condi- Denisova Cave, situated in the Altai Mountains of Russian Siberia, is unique in its hominin record as the only site where both Neanderthal and Denisovan fossils have been identified (Supplementary Fig. 1)38–41. The cave, situated in the ledge of a southwest-facing rock wall and overlooking the Anui River, has the longest stratigraphic sequence for northern Eurasia, with contexts dating from the Middle Pleistocene. This extensive sequence has made Denisova Cave a type-site for northern Eurasia, allowing for palynological42 and palaeoecological43,44 reconstructions of the region and several zooarchaeological studies29–34,36. Despite particularly favorable condi- tions that ensure a high degree of biomolecular preservation at the site, less than 5% of bones excavated can be identified macroscopically29–34,36, making Denisova Cave cave a good candidate for the application of ZooMS. We analysed 8,253 non-diagnostic bone fragments which were excavated from each of the three interconnecting chambers of Denisova Cave. The majority of these bones come from the East Chamber (n = 6288), followed by the Main (n = 1143) and South (n = 822) chambers (Supplementary Information; Supplementary Fig. 1). These were sampled with the permission of and in collaboration with the Institute of Archeology and Ethnography of the Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.ii g g p p y tions that ensure a high degree of biomolecular preservation at the site, less than 5% of bones excavated can be identified macroscopically29–34,36, making Denisova Cave cave a good candidate for the application of ZooMS. We analysed 8,253 non-diagnostic bone fragments which were excavated from each of the three interconnecting chambers of Denisova Cave. The majority of these bones come from the East Chamber (n = 6288), followed by the Main (n = 1143) and South (n = 822) chambers (Supplementary Information; Supplementary Fig. 1). Results ll Overall, of the 8253 analysed bones, 74% were assigned to a specific ZooMS taxon whereas an additional 5% could only be identified to family or order level due to low quality spectra (Supplementary Information). The South Chamber had a high rate of samples that failed to produce enough collagen for taxonomic identification (28%), in comparison with lower rates of failure for the East (16%) and Main (17%) chambers (Fig. 1).ii In total we identified 18 ZooMS taxa amongst the bones we analysed (Table 1). A ZooMS taxon is defined here as the most specific identification ZooMS is able to provide, generally to a genus or family level. For instance, several Canids have been identified within the morphological assemblage at Denisova Cave, including Canis lupus (wolf), Vulpes corsak (corsak fox), Cuon alpinus (dhole), and Alopex lagopus (arctic fox), all of which could Scientific Reports \| (2021) 11:15457 \| https://doi.org/10.1038/s41598-021-94731-2 www.nature.com/scientificreports/ Table 1. The results of ZooMS analysis of 8,253 fragmented bones from Denisova Cave. For the species most likely included in the “ZooMS-taxon” see Table S1. Results ll ZooMS taxon East chamber Main chamber South chamber Total Bison/Yak 1629 337 221 2187 Canidae 140 12 8 160 Capra 209 75 30 314 Cervidae 18 18 Cervidae/Gazella/Saiga 1189 35 32 1256 Crocuta/Panthera 99 21 19 139 Elephantidae 181 40 35 256 Equidae 669 123 58 850 Felidae 7 1 8 Hominin 6 1 7 Leporidae 2 2 Muridae 2 2 Mustelidae 2 2 Ovis 147 108 40 295 Ovis/Capra 302 67 54 423 Rangifer 34 3 1 38 Rhinocerotidae 401 98 48 547 Ursidae 123 20 33 176 Vulpes vulpes 19 19 Bird 9 1 10 Capra/Rangifer 12 2 3 17 Cervidae/Gazella/Saiga/Equidae 15 1 16 Crocuta/Panthera/Mustelidae 11 5 4 20 Equidae/Rhinocerotidae 1 1 Felidae/Crocuta/Panthera 1 1 Felidae/Crocuta/Panthera/Mustelidae 2 2 Felidae/Ursidae 18 4 1 23 Ovis/Capra/Cervidae/Gazella/Saiga 25 25 Ovis/Capra/Rangifer 4 2 6 Ovis/Cervidae/Gazella/Saiga 25 3 28 Unknown 19 3 3 25 Failed 968 183 229 1380 Total 6288 1143 822 8253 h ZooMS taxon East chamber Main chamber South chamber Total Bison/Yak 1629 337 221 2187 Canidae 140 12 8 160 Capra 209 75 30 314 Cervidae 18 18 Cervidae/Gazella/Saiga 1189 35 32 1256 Crocuta/Panthera 99 21 19 139 Elephantidae 181 40 35 256 Equidae 669 123 58 850 Felidae 7 1 8 Hominin 6 1 7 Leporidae 2 2 Muridae 2 2 Mustelidae 2 2 Ovis 147 108 40 295 Ovis/Capra 302 67 54 423 Rangifer 34 3 1 38 Rhinocerotidae 401 98 48 547 Ursidae 123 20 33 176 Vulpes vulpes 19 19 Bird 9 1 10 Capra/Rangifer 12 2 3 17 Cervidae/Gazella/Saiga/Equidae 15 1 16 Crocuta/Panthera/Mustelidae 11 5 4 20 Equidae/Rhinocerotidae 1 1 Felidae/Crocuta/Panthera 1 1 Felidae/Crocuta/Panthera/Mustelidae 2 2 Felidae/Ursidae 18 4 1 23 Ovis/Capra/Cervidae/Gazella/Saiga 25 25 Ovis/Capra/Rangifer 4 2 6 Ovis/Cervidae/Gazella/Saiga 25 3 28 Unknown 19 3 3 25 Failed 968 183 229 1380 Total 6288 1143 822 8253 Table 1. The results of ZooMS analysis of 8,253 fragmented bones from Denisova Cave. For the species most kely included in the “ZooMS-taxon” see Table S1. only be classified as ‘Canidae’ through ZooMS analysis as their peptide mass fingerprints are identical. Vulpes vulpes (red fox) is the only Canid that can be differentiated using ZooMS, as a result of a small difference in their COL1ɑ2 484‒498 peptide45 (Supplementary Table 1). The assemblage is dominated by large vertebrates, with small vertebrates and birds accounting for less than 1% of the identified material examined. Results ll Small mammals from both the morphological assemblage and ZooMS-IDed component, samples which failed ZooMS analysis, and “Unknown” spectra are not included. East chamber. ZooMS analysis was carried out on 6,288 bone fragments from most archaeological layers of the East Chamber (Table 1; Supplementary Table 2). Predators account for 20% of the morphological assemblage of the East Chamber, in comparison with only 7.7% of the ZooMS-IDed assemblage. The overall proportion of predator remains does not vary significantly throughout the layers of the East Chamber in the morphological assemblage. Cave hyaena (Crocuta crocuta spelaea) are the dominant predators between layers 9–13, alongside smaller numbers of Panthera, Canidae, and Felidae29,31. Their behaviours are particularly visible in the archaeo- logical material from layers 9–11 where a large number of bones have clearly passed through the digestive tracts of hyaenas or wolves, leaving acid corrosion marks on bones and dissolving the enamel on teeth29,31. In con- trast, in the ZooMS-IDed component, Crocuta/Panthera are present in relatively low numbers, accounting for approximately 1.9% of all identified bones (Fig. 2). Canidae on the other hand are consistently the dominant predator group in the ZooMS-IDed component for each of the studied layers of the East Chamber. Within the morphological assemblage, Canidae only exceed Crocuta/Panthera in layers 15–1729,31 a period during which forest species like Capreolus pygargus (Siberian roe deer) and Cervus elaphus (red deer) are the major herbivore groups present in the assemblage (see below). In the ZooMS-IDed fauna, Canidae account for 2.7% of all the bones identified within the East Chamber.hi i The two most significant herbivore taxa in the ZooMS-IDed component for the East Chamber are Cervi- dae/Gazella/Saiga and Bison/Yak. The Cervidae/Gazella/Saiga ZooMS-taxon includes C. pygargus, C. elaphus, Megaloceros giganteus (Irish elk), Alces alces (elk), Saiga tatarica borealis (saiga antelope), and Procapra gutturosa (Mongolian gazelle). The only cervid species present at Denisova Cave which can be separated from this group is the Siberian roe deer on the basis of their COL1ɑ2 757–789 (ɑ2 757)45 peptide marker. The ɑ2 757 peptide for Siberian roe deer is present at m/z 3043.4/3059.4 but at m/z 3017.5/3033.5 for Cervidae/Gazella/Saiga. Since this differentiation is based on a single marker it is possible that many of the generically identified Cervidae/Gazella/ Saiga are actually Siberian roe deer which were missing their ɑ2 757 marker in poorer preserved specimens. Results ll This is likely in part a result of our selection of bones which were preferentially 2 cm in length or more. only be classified as ‘Canidae’ through ZooMS analysis as their peptide mass fingerprints are identical. Vulpes vulpes (red fox) is the only Canid that can be differentiated using ZooMS, as a result of a small difference in their COL1ɑ2 484‒498 peptide45 (Supplementary Table 1). The assemblage is dominated by large vertebrates, with small vertebrates and birds accounting for less than 1% of the identified material examined. This is likely in part a result of our selection of bones which were preferentially 2 cm in length or more. Comparing datasets. In order to compare the ZooMS identified fauna with previously-published zoo- archaeological datasets from Denisova Cave we have compiled those datasets, referred to as the ‘morphologi- cal assemblage’, from the literature and converted them into ZooMS-style groupings (following Supplementary Table 1) which can then be directly compared with the new ZooMS results we present here (Fig. 2). In our discussion of percentages, we have excluded samples which failed ZooMS analysis or those which could not be assigned to the most specific ZooMS taxon possible, with the exception of Ovis/Capra and Crocuta/Panthera (see Supplementary Information) in order to make them more compatible with zooarchaeological results. We have also excluded small vertebrates as they were not identified in sufficient numbers for direct comparison with the morphological assemblage. While some results are not included in our comparisons with the zooarchaeo- logical datasets, all results are reported in the Supplementary Information (Supplementary Table 2, 3, 4; External Database 1). We use the term “predator”, rather than “carnivore”, to group the activity of hominins, Canidae, Crocuta/ Panthera, Ursidae and V. vulpes, some of which have varied diets. The term “herbivores” includes Bison/Yak, Capra, Cervidae/Gazella/Saiga, Elephantidae, Equidae, Ovis, and Rhinocerotidae. https://doi.org/10.1038/s41598-021-94731-2 Scientific Reports \| (2021) 11:15457 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Comparison of the overall abundance of fauna between the morphological assemblage and the ZooMS-IDed fauna for the three chambers of Denisova Cave. Only the largest faunal groups have been included. Small mammals from both the morphological assemblage and ZooMS-IDed component, samples which failed ZooMS analysis, and “Unknown” spectra are not included. Figure 2. Comparison of the overall abundance of fauna between the morphological assemblage and the ZooMS-IDed fauna for the three chambers of Denisova Cave. Only the largest faunal groups have been included. Results ll The ɑ2 757 marker is easily lost as a result of collagen degradation, a common problem for instance in the separation of Ovis and Capra which is done on the basis of the same peptide46.h p p p Cervidae/Gazella/Saiga bones account for 54% of the ZooMS-IDed component for layer 15. This number declines significantly in the overlying archaeological layers. In comparison, Bison/Yak, a taxonomic group that contains the remains of steppe bison (Bison priscus) and the less common Baikal yak (Poёphagus mutus), continue Scientific Reports \| (2021) 11:15457 \| https://doi.org/10.1038/s41598-021-94731-2 www.nature.com/scientificreports/ Figure 3. Relative abundance of fauna identified using ZooMS per layer of the East Chamber of Denisova Cave. This information can also be found in Supplementary Table 2. (A) The stratigraphy and corresponding age ranges are highlighted with the most abundant fauna for each layer. The marine-oxygen isotope curve was compiled from benthic δ18O records64; marine-oxygen isotope stages 3, 5, and 7 are highlighted in grey29. (B) Each bar depicts the major taxa identified. Only the largest groups of fauna have been included, excluding small mammals, “Unknown” identifications, and failed samples. Figure 3. Relative abundance of fauna identified using ZooMS per layer of the East Chamber of Denisova Cave. This information can also be found in Supplementary Table 2. (A) The stratigraphy and corresponding age ranges are highlighted with the most abundant fauna for each layer. The marine-oxygen isotope curve was compiled from benthic δ18O records64; marine-oxygen isotope stages 3, 5, and 7 are highlighted in grey29. (B) Each bar depicts the major taxa identified. Only the largest groups of fauna have been included, excluding small mammals, “Unknown” identifications, and failed samples. to increase by percentage throughout the stratigraphic layers. Bison/Yak eventually account for 70% of the ZooMS IDed fauna in layer 9.226 (Fig. 3; Supplementary Table 2). Main chamber. We carried out ZooMS analysis for 1,143 bones from layers 9–12 of the Main Chamber (Table 1; Supplementary Table 3). Predators dominate the morphological assemblage, accounting for more than 30% of all identifiable bone for layers 9–12. In comparison, the dominant predators, Canidae, Crocuta/Panthera, and Ursidae, make up only 5.5% of the ZooMS-IDed component. Our Crocuta/Panthera taxon likely includes C. spelaea, although Panthera spelaea are also present in the earliest Pleistocene layers of the Main Chamber. Crocuta/Panthera dominate the morphological assemblage for layers 9–11, accounting for 9.4% of identifiable bones (Fig. 2)29. Discussion P tt f f Patterns of faunal variability. ZooMS has previously been applied to larger microfauna assemblages and older individual faunal samples47,48, yet our present work is the largest application of ZooMS to an assemblage of fragmented bones with an archaeological association. The oldest archaeological layers we study here (layers 15 and 14) date to between 217 and 163 ka29. For layer 15, from which we analysed nearly 1000 bones, the success rate was 86% (Supplementary Table 2). This is particularly encouraging and paves the way for the future analysis of bone assemblages of similar biomolecular preservation and antiquity.if Significant differences in success rates were observed across layers and the three non-connecting chambers. Bones from the East Chamber were the most successful, however an average of 35% of samples from the youngest Pleistocene contexts (layers 9.2 and 9.3) failed to produce enough collagen for ZooMS analysis. This high failure rate is likely due to the heavy phosphatisation in the soil36,49 which has previously impeded OSL, radiocarbon dating, and genetic analyses for these layers26,29. At present, it seems identifying fossils suitable for biomolecular analysis from Upper Pleistocene contexts at Denisova Cave will continue to be challenging. This is unfortunate given the persistent questions regarding which hominin populations were present in the Altai Mountains dur- ing this period26. g p At Denisova Cave a high degree of bone fragmentation is observed overall. Of the 177,000 bones excavated at the East Chamber more than 95% were less than 2–5 cm in length and could not be identified macroscopically29,31. Using ZooMS however, on average 74% of the fragmented bones we analysed could be assigned to a specific ZooMS taxon and an additional 5% of samples that produced low quality spectra could be identified to family or order levels. This contrast in identification success (< 5% identified morphologically versus ~ 80% based on ZooMS) highlights once again the merits of large-scale application of ZooMS on highly fragmented archaeo- logical assemblages. g g Bones analysed in this study from the Main Chamber were selected from the Upper Palaeolithic layers 9 and 11 (Supplementary Fig. 1), whereas bones from the South Chamber came from both Upper Palaeolithic layer 11 and Middle Palaeolithic layers 12 and 21. For the East Chamber, bones were selected from all major stratigraphic units, from the early Middle Palaeolithic through to the Upper Palaeolithic layers (15–9), and the archaeologi- cally sterile layer 17. Results ll In the ZooMS-IDed component, Crocuta/Panthera account for 1.68% of the fragmented bones for layers 9–11 making them the most commonly identified predators for these contexts. The morphological assemblage identifies Canidae as the major predator group in layer 1229,34, however none were identified in the ZooMS-IDed component for this context.hfii p The major herbivore groups do not differ significantly between the morphological and ZooMS identified fauna. Equidae, Ovis/Capra, and Cervidae/Gazella/Saiga are present in relatively large numbers in each dataset. Bison/Yak remains increase significantly within the ZooMS-IDed fauna, accounting for 35% of the assemblage, more than four times the amount identified within the morphological bones (8.35%). South chamber. We analysed 822 bones from the South Chamber using ZooMS (Table 1; Supplementary Table 4). Crocuta/Panthera, Canidae, and Ursidae are the dominant predator groups in the ZooMS fauna in this Chamber. They account, however, for only 10.5% of this assemblage, three times less than predators in the morphological assemblage (Fig. 2). Ursidae (Ursus (Spelaearctos) savini) account for 9.7% of the ZooMS-IDed fauna for layer 11. Previous zooarchaeological analysis suggested the cave may have been used for hibernation by the small cave bear (U. spelaearctos)32 which could explain the unusually high number of bear bones. Crocuta/ Panthera were the dominant predator group for the ZooMS-IDed fauna in layer 12, accounting for 2.7% of the assemblage which is largely in line with dominant predator groups identified in the morphological assemblage. Bison/Yak (36.6%) and Equidae (10.3%) were the most common herbivores present within the ZooMS-IDed component. https://doi.org/10.1038/s41598-021-94731-2 Scientific Reports \| (2021) 11:15457 \| www.nature.com/scientificreports/ Zooarchaeological analysis of bones from the South Chamber32,33 suggests intense predator presence for layers 9–12, in particular Crocuta/Panthera. Their bones are disproportionately represented in the morphologically identifiable bones for South Chamber (n = 255), second only to Capra sp. remains, which are identified as the likely prey for cave hyena and snow leopard32,33. Fragments of cave hyena coprolites were identified throughout layers 9–12 of the South Chamber and many of the bones of prey taxa show traces of acid corrosion, indicat- ing that they have passed through the digestive tracts of predators32,33. It is noteworthy that only 3.7% of bones recovered from this part of the cave are larger than 5 cm33. We may hypothesize that this large degree of frag- mentation is due to predation. Discussion P tt f f The majority of bones from the ZooMS-IDed component from all three chambers were the remains of prey, suggesting that the formation of the assemblage is not entirely relegated to equifinality in that some processes of the accumulation can be unravelled3,4. In the East Chamber, the earliest evidence for hominins processing carcasses and modifying bone is present in layers 15 and 14 of the East Chamber which coincides with the arrival of the first Denisovans to the site27,35,50. From this period lithic technologies are continuously present throughout the contexts of this chamber and the layers studied for the Main and South chambers35,51. Alongside hominins, previous zooarchaeological studies for Denisova Cave have highlighted the major role carnivores likely played in the formation of the site’s fragmented bone assemblage30,33. Carnivore remains are found in all contexts for the site but their behaviours are particularly visible in Upper Palaeolithic contexts with extensive gnawing marks and digestion of bones likely by hyenas, panthers, leopards, and wolves31,33,35. The overlap and relationship between hominins and predators at Denisova Cave has been investigated through multiple studies. While periods of intensive hominin presence are evident, particularly during the Early Middle and Middle Pal- aeolithic contexts where anthropogenic marks are frequently found on bones29,31, evidence for other predators at the site is continuously documented throughout the Palaeolithic30–33. l f h h f h f h d h h h d l y g Recent analysis of the microstratigraphy of Denisova Cave has furthered this, showing that due to slow sedimentation accumulation rates it is currently not possible to identify when predators like bears, wolves, and hyaenas were occupying the cave as opposed to hominin groups like Denisovans and Neanderthals36. Rather, it seems that many groups were alternating as the site’s main occupants throughout the Pleistocene32,36. Further analyses of the bone assemblage, such as detailed taphonomic studies, bone refitting exercises and extensive dat- ing of specific locations to assess the contemporaneity of various species at the site, will shed further light on this. Early evidence however suggests that at times hominins and other predators may have even benefited from each other’s presence. Results ll In turn, such a high concentration of carnivore processing could explain the low level of protein preservation in this section of the cave; 28% of the fragmented remains analysed using ZooMS failed to produce enough collagen for identification as opposed to 17% and 16% for the East and Main Chambers, respectively (Fig. 1; Table 1; Supplementary Table 4). Faunal patterns and palaeoclimate. Faunal patterns and palaeoclimate. Recent work on building chronologies for the site using radiocar- bon and optical dating26,29 has enabled the three chambers of Denisova Cave to be securely dated and cross- correlated. As a result, stratigraphic contexts from separate sections of the site can now be attributed to specific MIS stages and compared to one another. We identify several trends in the ZooMS-IDed component with ref- erence to depositional age and the environmental conditions prevailing at the time. We note, for instance, that despite the wide variety of taxa the Cervidae/Gazella/Saiga ZooMS identification encompasses, they remain in relatively low abundance throughout the majority of the Pleistocene, except for the earliest layers which cor- respond to MIS 9–7 (337–191 ka). The majority of bones from this phase were excavated in layer 15 of the East Chamber which is attributed to MIS 7 and the Penultimate Interglacial, and coincides with the arrival of the first Denisovans to the site27,35,50. Layers 15 and 14 of the East Chamber have the highest abundance of stone tools of any other phase at Denisova Cave and a large proportion of humanly modified bone27,35. The morphological assemblage from these layers is dominated by the remains of Siberian roe deer (19%) and red deer (10%) that live in forest and grasslands29,31. Cervids were clearly favoured by these early Denisovans, an observation which remains in stark contrast to the faunal record of all later phases documented at the site. Bison/Yak are the most abundant fauna for almost all subsequent (post-MIS 7) layers (Fig. 3; Supplementary Tables 1, 2, and 3). Steppe bison and Baikal yak are known to have favoured more open, steppe and forest-steppe environments31,55. Despite the expansion of forests during interglacial periods however, their remains continue to dominate the ZooMS-IDed component at 39% (MIS 3) and 31% (MIS 5) (Fig. 3; Supplementary Tables 1, 2, and 3). This suggests that rather than the environment being a driving force behind the abundance of fragmented Bos and Bison bones, predators, likely hominins, were preferentially targeting them, mirroring a trend identified in other Pleistocene assemblages analysed with ZooMS22. g y With regards to predators in the ZooMS-IDed component, Canidae are the dominant group for the majority of the Pleistocene layers we studied. From MIS 9–4 their bones account for approximately 5% of the identified remains in our study. Faunal patterns and palaeoclimate. They are found alongside smaller numbers of other predators, including Felidae and V. vulpes. Crocuta/Panthera remains are first identified in the ZooMS-IDed component in layer 15 and they have been macroscopically identified in the morphological assemblage in layers covering the entire Pleistocene. The majority of these bones are probably cave hyaena, however, smaller numbers of snow leopard and Eurasian cave lion are also present throughout the stratigraphy29–34. Crocuta/Panthera become more common than Canidae during MIS 5–3, alongside archaeological evidence of intensive carnivore presence during the same period, par- ticularly in the South Chamber (Fig. 3; Supplementary Tables 1, 2, and 3). Bear remains are identified throughout the Pleistocene layers of Denisova Cave both in the ZooMS-IDed component and the morphological assemblage. The extinct cave bears, U. savini, were mostly vegetarian, and used the cave for hibernation32,33. Animal size and impact on ZooMS‑based fauna patterns. Body mass and size class, assigned based on previously published data56, probably play a disproportionate role in the number of taxa identified using ZooMS. For instance, Elephantidae, Bison/Yak, and Rhinocerotidae remains, all within body class size 6, are 3–5 times more abundant in the ZooMS-IDed component than the morphological dataset (Fig. 2). Bison/Yak remains are the most sensitive to this shift, accounting for only 6.3% of the morphological assemblage29–34 as opposed to 31% of the ZooMS-IDed component. A normalisation of the dataset based on body mass is not easy since it requires a high degree of confidence on other information, such as age and size of the targeted individu- als, and carcass processing practices (e.g. transfer of specific body parts to the cave), which are unknown and/or fluctuate through time in the case of Denisova Cave. l g In the case of mammoths, which were likely present in the Altai region in very small numbers and are not considered to have been a major contributor to hominin diet57, their bones are three times more abundant in the ZooMS identified megafauna (3.84% versus 1.08%). The only exception to this is the ratio of horse bones, for which no significant change in the percentage of their remains exists between the zooarchaeological dataset29–34 and the ZooMS-IDed component. Faunal patterns and palaeoclimate. With these factors in mind, any tentative predictive model for the abundance of large herbivores within a fragmented bone assemblage should consider both body class size and the likelihood that some taxa targeted by hominins were processed both in situ and at the cave site. Discussion P tt f f The close stratigraphic relationship between carnivores and hominins at Denisova Cave and our inability to specifically discern when these groups were dominant at the site may indicate the role scavenging played at Denisova Cave, particularly in regards to cave hyaenas and wolves taking advantage of the intensive hunting practices of Denisovans and Neanderthals36. Additionally, cut marked carnivore bones, particularly of red and polar foxes52, suggests hominins might have been targeting them for their fur. This practice is common in other Pleistocene sites in Russia, such as in the Kostenki region53,54.hf g The most notable difference between the morphological assemblage and the ZooMS-IDed component of Denisova Cave is the abundance of predators in comparison with herbivores. In the morphological assemblage of all three chambers, on average, predator groups account for 30.2% of all identified bones29–34, whereas in the Scientific Reports \| (2021) 11:15457 \| https://doi.org/10.1038/s41598-021-94731-2 www.nature.com/scientificreports/ ZooMS-IDed component predators account for only 7.6%. This discrepancy helps to inform us of the main factor influencing fragmentation rates at the site. Both the ZooMS ID-ed component and the morphological assemblage suggest that carnivores were the driving force behind the high rate of herbivore bone fragmentation. Given that the hominin bones recovered so far are similarly fragmented to herbivore bones (none exceed a few cm in length), we hypothesize that although humans were the main agent responsible for the accumulation and initial dismembering, butchering, and fragmentation of (most) herbivores in the cave, a secondary agent, that is carnivores such as hyenas, caused further processing and reduction in size of both animal and human bone deposited there. This hypothesis is largely in line with archaeological evidence for the site, where bones and teeth are frequently found with gnawing and digestion marks32,33. Methods Analysis was carried out at the ZooMS facility of the Department of Archaeology at the Max Planck Institute for the Science of Human History (MPI-SHH), Jena, Germany and the Manchester Institute of Biotechnology at the University of Manchester, UK. Samples analysed at the MPI-SHH followed published protocols using ammo- nium bicarbonate as the means of collagen extraction after which failed samples were re-attempted following acid soluble protocols23,58–61. Samples analysed at the University of Manchester followed published acid soluble protocols60,61. These protocols have all been optimised for the analysis of COL1 for taxonomic identification using ZooMS despite differences in initial protein extraction. The resulting spectra were screened for diagnostic markers using flexAnalysis 3.4 (Bruker Daltonics) and mMass software62. The spectra were compared against a reference library of known peptide markers21,23,63 which was informed further by previous zooarchaeological analysis carried out at Denisova Cave29–34.hf y Samples were analysed following established protocols. The ammonium bicarbonate buffer protocol23,58,59 involved sample rinsing in ammonium bicarbonate overnight and incubation for 1 h at 65 ℃. The supernatant was treated with 0.4 µg trypsin (Thermo Scientific Pierce ™ Trypsin Protease) and allowed to digest at 37 ℃ for 18 h. The incubated samples were concentrated and desalted using C18 ZipTips (Thermo Scientific Pierce™ C18 Tips) and eluted in a final solution of 50 µl of 50% acetonitrile and 0.1% TFA. 0.5 µl of the resulting solution was mixed with 0.5 µl of α cyano-4-hydroxycinnamic acid solution (10 mg/mL in 50% acetonitrile [ACN] and 0.1% trifluoroacetic acid [TFA]) and allowed to crystallise. The samples were analysed using a Bruker Autoflex Speed LRF MALDI ToF/ToF mass spectrometer. p Samples analysed using acid soluble protocols25,60 were demineralised in 0.6 M hydrochloric acid (HCl) for 18 h. The supernatant was removed into 30 kDa molecular weight cut-off (MWCO) ultrafilters and centrifuged at 3700 rpm for 1 h. The filtrate was then twice washed through with 500 μL of 50 mM ammonium bicarbonate (AmBic) and further centrifuged at 3700 rpm for half an hour after each treatment. The final residue was resus- pended with additional AmBic (200 μL), half of which was removed to create a backup sample set before diges- tion. The remaining 100 μL was then treated with 0.2 μg trypsin (sequencing grade; Promega UK) and incubated at 37 °C for 18 h. Methods The resulting solution was then diluted to 1:10 using TFA and mixed with a matrix solution of 1 μl of α-cyano-4-hydroxycinnamic acid solution (10 mg/mL in 50% ACN/0.1% TFA), allowed to crystallise and analysed using a Bruker Ultraflex II MALDI ToF/ToF mass spectrometer. For samples identified as ovicaprids, the tryptic peptide solution was further purified and fractionated using C18 Ziptips into 10% ACN and 50% ACN fractions (both in 0.1% TFA) and further analysed using MALDI as described above. Conclusionsh The application of ZooMS at Denisova Cave highlights the potential of the method to elucidate early hunting practices and adaptation to new environments. The new data expands our understanding of the site’s faunal record and taphonomy, and the high success rate is illustrative of the potential of the method to other sites and regions with comparable biomolecular preservation and to material of the same or even greater antiquity, extend- ing further back into the Middle Pleistocene.fii g Aside from its effectiveness in screening large numbers of bones for the identification of specific taxa, e.g. hominin fossils, ZooMS is complementary to traditional zooarchaeological practices as it allows a large part https://doi.org/10.1038/s41598-021-94731-2 Scientific Reports \| (2021) 11:15457 \| www.nature.com/scientificreports/ of the morphologically non-identifiable component to be diagnosed taxonomically. This is particularly true at Denisova Cave where traditional zooarchaeological analyses identified less than 5% of the excavated fauna versus ~ 80% identification success using ZooMS in this study.h i g y The new data is also useful for elucidating the taphonomic history of the bones recovered from a site. In our case, this is shown by the differences in prey-predator ratios in the ZooMS-IDed versus the morphologically- identified assemblages. These differences reveal that the main influence in the fragmentation of herbivores/prey bones at Denisova was two-fold and operated at different levels. Hominin processing of carcasses was the main factor for the introduction, deposition and initial fragmentation of many of the herbivore and some of the car- nivore bones at the site. This was followed by secondary and more extreme fragmentation, of both animal and human bones, as a result of predator scavenging and gnawing. Data availability y All ZooMS spectra for identified samples are available on Mendeley Data and are located in two databases. External Database 1: http://dx.doi.org/10.17632/5bwmbhs3fs.1. External Database 2: http://dx.doi.org/10.17632/ bgm2k6gt3j.1. All ZooMS spectra for identified samples are available on Mendeley Data and are located in two databases. External Database 1: http://dx.doi.org/10.17632/5bwmbhs3fs.1. External Database 2: http://dx.doi.org/10.17632/ bgm2k6gt3j.1. Received: 22 January 2021; Accepted: 15 July 2021 Received: 22 January 2021; Accepted: 15 July 2021 Referencesh 1. Brain, C. K. The Hunters Or the Hunted?: An Introduction to African Cave Taphonomy. (University of Chicago Press, 1983). 2. Villa, P. & Mahieu, E. Breakage patterns of human long bones. J. Hum. Evol. 21, 27–48 (1991).i , , g p g J , 3. Rogers, A. R. On equifinality in faunal analysis. Am. Antiq. 65, 709–723 (2000). g p g 3. Rogers, A. R. On equifinality in faunal analysis. Am. 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Author contributions S.B., N.W., A.O., D.C., B.J.S., V.L.H., M.B. performed the laboratory work; S.B., N.W., A.O., B.J.S., V.L.H., M.P.C. analyzed the data; S.B.. created the figures; M.K., M.S., A.D. provided the samples and site-specific expertise; K.D. and T.H. designed the study; S.B. and K.D. wrote the manuscript with the assistance and input of all authors. Acknowledgements g We would like to thank the European Research Council, the Max Planck Society, the Oxford Radiocarbon Accel- erator Unit (ORAU), the University of Manchester and Manchester Institute of Biotechnology, and the Institute of Archeology and Ethnography, Russian Academy of Sciences Siberian Branch for their ongoing support. We would like to thank our volunteers (Miriam Jenkins, Esther Gillespie, Lauren Bell, Marine Caldarola, Raija Heikkila, Laura Doody, Saltanat Amirova, Geoff Church, Lucy Koster, Rachael Holmes, Luke Ghent, Phoebe Ewles-Bergeron, Nicholas Siemens, Marion Sandilands, and Julianna Zavodski) who helped sample the material as well as Sandra Heberstreit, Jana Zech, and Kristine Richter for discussions and help in the laboratory. Funding g Open Access funding enabled and organized by Projekt DEAL. This work has received funding from the ERC under the European Union’s Horizon 2020 Research and Innovation Programme, grant agreement no. 715069 (FINDER) to KD and under the European Union’s Seventh Framework Programme (FP7/2007–2013), grant agreement no. 324139 (PalaeoChron) to TH. This project was supported further through the Royal Society and research fellowship funding to MB (UF120473) and the archaeological field studies were funded by the Russian Foundation for Basic Research (no. 18-09-40100 and 20-29-01011). Many thanks also go to the University of Manchester for the Dean’s Award Scholarship funding to VLH. Competing interests h p g The authors declare no competing interest. www.nature.com/scientificreports/ l l d l d f l d ( ) y 50. Slon, V. et al. Neandertal and Denisovan DNA from Pleistocene sediments. Science 356, 605–608 (2017). k h k f h l l h d h l h l h 51. Derevianko, A. P. & Shunkov, M. V. Formation of the Upper Paleolithic traditions in the Altai. Archaeol. Ethnol. Anthropol. Eurasia 12–40 (2004). 52. Vasiliev, S. A. Faunal exploitation, subsistence practices and Pleistocene extinctions in Paleolithic Siberia. Deinsea 9, 513–556 (2003). ( ) 3. Vereshchagin, N. K. & Kuzmina, I. E. Remains of mammals from the Palaeolithic sites on the Don and upper Desna. Trudy Zool Inst. AN SSSR 72, 77–110 (1977). 54. Borgia, V. Hunting high and low: Gravettian hunting weapons from Southern Italy to the Russian Plain. Open Archaeol. 3, 376–391 (2017). 55. Vasiliev, S. K. Late Pleistocene Bison (Bison p. priscus Bojanis, 1827) from the southeastern part of Western Siberia. Archaeol., Ethnol. Anthropol. Eurasia 34, 34–56 (2008). p 6. Smith, F. A. et al. Body mass of late Quaternary mammals. Ecology 84, 3402 (2003). p 56. Smith, F. A. et al. Body mass of late Quaternary mammals. Ecology 84, 3402 (2003). y y gy 57. Agadjanian, A. K. & Shunkov, M. V. Late Pleistocene mammals of the Northwestern Altai: Report 2. Charysh Basin. Paleontol. J. 52, 1461–1472 (2018). ( ) 8. van Doorn, N. L., Hollund, H. & Collins, M. J. A novel and non-destructive approach for ZooMS analysis: Ammonium bicarbonate buffer extraction. Archaeol. Anthropol. Sci. 3, 281 (2011). https://doi.org/10.1038/s41598-021-94731-2 Scientific Reports \| (2021) 11:15457 \| Additional informationh Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-94731-2. Correspondence and requests for materials should be addressed to S.B. or K.D. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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https://openalex.org/W4200179263	https://bmcoralhealth.biomedcentral.com/track/pdf/10.1186/s12903-021-02019-8	English	null	Radiographic evaluation of a cross-shaped incision technique for thick-gingiva and thin-gingiva patients treated with implant-supported fixed prosthesis	BMC oral health	2,021	cc-by	6,113	© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Luo et al. BMC Oral Health (2021) 21:655 https://doi.org/10.1186/s12903-021-02019-8 Luo et al. BMC Oral Health (2021) 21:655 https://doi.org/10.1186/s12903-021-02019-8 Open Access Abstract Background: To evaluate a cross-shaped incision technique for thick-gingiva and thin-gingiva patients treated with implant-supported fixed prosthesis. Methods: Total 55 patients receiving cross-shaped incision were assigned into thick-gingiva group (29 cases) and thin-gingiva group (26 cases). Follow-up was performed at 3 and 12-month after final restoration. Results: Mesial and distal papilla height was significantly greater in thick-gingiva group than thin-gingiva group at 3 and 12 months, while periodontal depth and crestal marginal bone level around implant had no significant differ- ence between the two groups during follow-up. No case of recession of buccal marginal gingiva was observed in thick-gingiva group. However, the recession of marginal gingiva of buccal aspect of the crown was found in 5 patients (19.2%) with thin-gingiva. Conclusions: The cross-shaped incision may be applied to reconstruct gingival papillae and avoid the gingival reces- sion in patients with thick-gingiva phenotype. Trial registration This study was registered at ClinicalTrials.gov (registration number NCT04706078, date 12 January 2021, Retrospectively registered). Keywords: Cross-shaped incision, Gingiva, Recession of marginal gingiva, Implant, Fixed prosthesis the gingival shrinkage [1]. Food impaction includes verti- cal (the forceful wedging of food into the interproximal space by masticatory pressure) and horizontal (the forc- ing of food interproximally by tongue or cheek pressure) food impaction [2].hi Radiographic evaluation of a cross‑shaped incision technique for thick‑gingiva and thin‑gingiva patients treated with implant‑supported fixed prosthesis Wen Luo1,2, Xinyu Wang1, Yaqian Chen1, Yuping Hong1, Yili Qu1, Yi Man1* and Yingying Wu1* Background Dental implant has been successfully used to restore missing teeth. However, food impaction at the implant site is considered as a common complication in patients with implant prostheses. Food impaction is the phenom- enon in the chewing when the food dregs or fibers are pushed into the clearance by occlusal force or owing to The loss of interproximal contact between fixed implant prostheses and adjacent teeth is the main issue of vertical food impaction, which can be solved through occlusal adjustment, re-making of the prosthesis, inlay- crown or full crown of adjacent teeth [3]. Horizontal food impaction is mostly caused by the absence of interproxi- mal papilla, which leads to the abnormal space under the proximal connection [1, 4, 5]. Sometimes gingival Correspondence: 364662698@qq.com; yywdentist@163.com 1 State Key Laboratory of Oral Diseases and National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, No.14, Sec.3, Renminnan Road, Chengdu 610041, Sichuan, China Inclusion criteria 1. Good general health, no chronic systemic diseases. 2. All subjects included in this study needed to have one missing premolar or molar teeth with adjacent natural teeth. 3. All subjects included in this study had been treated with one bone-level implant insertion in the pre- molar or molar region. The patients had insufficient gingival papilla height (referred to contralateral natu- ral tooth which also had insufficient gingival papilla height) and at least 2 mm of keratinized tissue width around the implant. Various treatment plans and techniques have been proposed to restore the deficient papilla. Hard tissue augmentation during implant placement was suggested as an effective method to obtain desired inter-dental/ inter-implant papillae [7]. Orthodontic procedures are proposed to enhance hard tissue profiles and improve the papillae height [8]. Both surgical and orthodontic management is designed to create the papillae in the pre- surgical or surgical phase. If there is a deficit of papillae during the stage-two surgery, then soft tissue grafting or vascularized interposition periosteal-connective tis- sue (VIP-CT) flap was recommended [9–11]. However, the management would be a great challenge to clini- cians in posterior position. Therefore, a provisional res- toration with proper emergence profile has been widely accepted as the treatment to reconstruct papillae around implant, although the bone may recede more from com- posite resin than a titanium surface [12]. In addition, the pressure against the peri-implant soft tissue may cause discomfort to patients [7]. Therefore, the cross-shaped incision across gingival sulcus could be considered as an effective and simple way for reducing the soft tissue resistance to seat the restoration with implant-supported fixed prosthesis. The cross-shaped incisions went directly to the bone surface. The length of the cross-shaped inci- sions was 1–2 mm in keratinaized gingiva.hf Samples and groupsi Fifty-five subjects were selected from the patients who need treatment with a cross-shaped incision technique of the Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University in China between June 2018 and June 2020. Patients were divided into two groups according to the gingival phenotype determined by a periodontal probe. After the insertion of the probe into the facial aspect of the sulcus through the gingival margin, the simple visual method is based on the transparency of the periodon- tal probe through the gingival margin while probing the buccal sulcus at the midfacial aspect of the tooth. When the outline of the underlying periodontal probe can be seen through the gingival, the gingival phenotype is con- sidered thin. The gingival phenotype is thick in the other case. When the crown shape protrusion is not obvious, the main direction of the probe is parallel to the long axis of the tooth. The peri-implant phenotype was catego- rized as thin-gingiva (26 cases, outline of the probe can be seen through the gingiva) or thick-gingiva (29 cases, outline of the probe cannot be seen through the gingiva) (Fig. 1) [13]. This study aimed to investigate whether different gin- gival phenotypes have the same ability to recover from surgical injury, and we evaluated a cross-shaped inci- sion technique for thick-gingiva and thin-gingiva patients treated with implant-supported fixed prosthesis. Luo et al. BMC Oral Health (2021) 21:655 Luo et al. BMC Oral Health (2021) 21:655 Page 2 of 8 the patients signed informed consent before the implant surgery. incising (such as a cross-shaped incision) is necessary to reduce the soft tissue resistance to seat the restoration. Surgical injury may lead to gingival recession and the absence of interproximal papilla. Gingival phenotype, divided into thin or thick gingiva, affects the dimension of the periodontal tissue. A thick phenotype is prone to pocket formation, while a thin phenotype is prone to gin- gival recession following mechanical or surgical manipu- lation [6]. The regeneration or reconstruction of gingival papillae is a challenge to clinicians. Methods Study designh The present study was performed in accordance with the World Medical Association Declaration of Helsinki and was approved by Ethics Committee of West China Hospi- tal of Stomatology, Sichuan University, China (Approval No. 2009033). The study adheres to CONSORT guide- lines and is registered in the ClinicalTrials.gov (regis- tration number NCT04706078, date 12 January 2021, https://clinicaltrials.gov/ct2/show/NCT04706078). All 1. Active periodontal infections. 1. Active periodontal infections. 2. Heavy smoking (> 10 cigarettes per day). Clinical follow‑up Fig. 1 The phenotype of gingiva determined by a periodontal probe All patients in this study accepted oral hygiene instruc- tion at each visit. All patients were examined at 3-month and 12-month after final restoration (Fig. 2g, h) for the following examinations: 1. Presence/absence of papilla height was assessed visu- ally according to the papilla index proposed by Jemt [14]. 2. Modified Plaque Index (mPI): plaque accumulation around the marginal peri-implant tissue was assessed by the criteria of mPI [15].i 3. Modified Sulcus Bleeding Index (mBI): the bleed- ing tendency of the marginal peri-implant tissue was evaluated using mBI [15]. 4. Probing Depth (PD, mm): PD was assessed at the mid-buccal, mid-oral, mesial and distal aspects of the buccal surfaces of each implant with a standard peri- odontal probe, and final value was determined by the average of four aspects. crown after 3-6 months of healing. Titanium abutments with the lowest gingival height (1–1.5 mm) were selected, and all of the crowns were made of zirconia. Platform switching which referred to the use of a smaller diameter abutment on a larger diameter implant collar was used in all the implants. 5. Gingival margin level (GML): gingival margin level was assessed by calculating the vertical distance between the most apical point of gingival mar- gin at the buccal aspect of the crown and line con- necting the peak of the adjacent mesial and distal natural teeth (PMD) [16]. The length of the natural crown next to the implant supported restoration was recorded to correct any changes in magnification (Fig. 3). 5. Gingival margin level (GML): gingival margin level was assessed by calculating the vertical distance between the most apical point of gingival mar- gin at the buccal aspect of the crown and line con- necting the peak of the adjacent mesial and distal natural teeth (PMD) [16]. The length of the natural crown next to the implant supported restoration was recorded to correct any changes in magnification (Fig. 3). Two weeks later, the try-in was carried out after the fabrication of a definitive abutment and crown. The pro- cedures were as follows: the patients were given 1 ml articaine for local infiltration anesthesia. The healing abutment was disconnected, and the cross-shaped inci- sion was made at the buccal, lingual, mesial and distal aspects across the gingival sulcus using 12# blade. The cross-shaped incisions went directly to the bone surface. Proceduresh The bone-level implants were placed 1 mm below the alveolar bone level according to the manuals. The trial started at 3–6 months after the one-stage surgery. Impression was taken for the fabrication of definitive Luo et al. BMC Oral Health (2021) 21:655 Page 3 of 8 Radiographic follow‑up l d h Periapical radiographs of the implant-supported crown was taken using the parallel photographing technique at each recall examination. To be specific, the landmarks of first bone-implant contact (fBIC) and implant shoulder (IS) were used for measurements. fBIC-IS was defined as the vertical distance the first bone-implant contact to implant shoulder, and the distance was assessed at the mesial and distal aspect of implant, respectively. When the marginal crestal bone was located coronal to the IS, a positive (+) value was given, where a negative (–) value when located apically to the IS, the value was deemed as zero when IS and fBIC coincided. The crestal bone level at the time of impression taking was regarded as baseline (Fig. 4). The known implant length was used for the cali- bration of dimensional distortion in the radiograph (the length of implant was 10 mm in Fig. 4). Clinical follow‑up The length of the cross-shaped incisions was 1–2 mm in keratinaized gingiva (Fig. 2a, b). X-ray was taken to make sure that the abutment and crown were properly seated after try-in of definitive abutments and crowns (Fig. 2c, d). The crown was cleaned after occlusal adjust- ment. Then screw of the abutment was tightened with a torque nearly 35 N cm using the screw driver connected to a torque wrench. Before bonding of the crown, exu- dation in gingival sulcus was stopped by cotton balls for about 1 h. Various methods were used to minimize excess cement extrusion into the peri-implant tissue. Customized abutment replica was made of acrylic resin before the cement. The crown was inserted onto the rep- lica, permitting the extrusion of excess cement (Fig. 2e). The crown was placed onto the abutment (Fig. 2f). Den- tal floss was positioned mesial and distal surfaces of the prosthesis in order to remove the excessive cement after clotting. Antibiotic and anti-inflammatory drugs were not recommended. The patients were requested not to brush the surgical area in 24 h. Chlorhexidine mouth- wash was routinely used to maintain good oral hygiene after surgery and crown placement. Statistical analysis Th l The statistical analysis was performed using Graph- Pad Prism 6.0 program. The data were presented as Luo et al. BMC Oral Health (2021) 21:655 Page 4 of 8 Luo et al. BMC Oral Health (2021) 21:655 Fig. 2 Clinic procedures for patients subjected to cross-shaped incision. a Buccal view of implant sites; b Cross-shaped incision was made across gingival sulcus after the disconnection of healing abutments; c Buccal view of the cross-shaped incision with definitive abutment; d X-ray was taken to make sure the abutment and crown were properly seated; e Customized resin abutment was made; f Buccal view of free gingival around implant after final restoration; g Buccal view of gingival papilla around implant 3 months after final restoration; h Buccal view of gingival papilla around implant 12 months final restoration Page 4 of 8 Fig. 2 Clinic procedures for patients subjected to cross-shaped incision. a Buccal view of implant sites; b Cross-shaped incision was made across gingival sulcus after the disconnection of healing abutments; c Buccal view of the cross-shaped incision with definitive abutment; d X-ray was taken to make sure the abutment and crown were properly seated; e Customized resin abutment was made; f Buccal view of free gingival around implant after final restoration; g Buccal view of gingival papilla around implant 3 months after final restoration; h Buccal view of gingival papilla around implant 12 months final restoration Results MAP: the most apical point of the gingival margin at the buccal aspect of the crown; PMD: the line connecting the peak of the adjacent mesial and distal natural teeth (PMD); GML: the distance from MAP to PMD; Magnification: the length of the natural crown next to the implant supported prosthesis was recorded to correct any changes in magnification △ Significant difference (P < 0.05) between 3 months or 12 months and baseline Fig. 4 Schematic drawing illustrating the landmarks used for periapical radiographs measurement. IS: implant shoulder; fBIC: first bone-implant contact; a the vertical distance the first bone-implant contact to implant shoulder measured from radiograph. b Implant length. x (fBIC-IS): the real vertical distance the first bone-implant contact to implant shoulder and sample characteristics were presented in Table 1. All patients were treated with implants placement in posterior areas. Three months after final restoration, all implants showed stable osseointegration with the absence of pain or inflammation, the absence of peri- implant radiolucency, and the absence of screw or crown loosening. There was no complain in both thick-gingiva group and thin-gingiva group. The Jemt parameters of soft tissue were listed in Table 2. In thick-gingiva group, both mesial and distal papilla filled more than half of the proximal space and in good harmony with the adjacent papillae 3 months after restoration; and the papilla height didn’t change significantly over one year. Although the score of papil- lae height in thin-gingiva group was lower than thick- gingiva group, there was no statistical difference between two groups (P > 0.05). From 3 to 12-month visit, the score of mPI and mBI decreased slightly in both thin-gingiva group and thick-gingiva group, but the change didn’t show significant difference (P > 0.05). At 3-month visit, the PD in thin-gingiva group and thick-gingiva group was 2.16 ± 0.42 mm and 2.18 ± 0.41 mm, respectively, which remained almost the same from 3 to 12-month visit in both groups (P > 0.05). Fig. 4 Schematic drawing illustrating the landmarks used for periapical radiographs measurement. IS: implant shoulder; fBIC: first bone-implant contact; a the vertical distance the first bone-implant contact to implant shoulder measured from radiograph. b Implant length. x (fBIC-IS): the real vertical distance the first bone-implant contact to implant shoulder Fig. 4 Schematic drawing illustrating the landmarks used for periapical radiographs measurement. Results means ± standard deviations (SD), the differences between thin-gingiva group and thick-gingiva group were compared using the paired t test. A difference was considered significant if the P value was < 0.05. During the study period, a total of 55 patients were included. Patients were grouped into 2 groups accord- ing to gingival phenotype. Detailed demographic data Luo et al. BMC Oral Health (2021) 21:655 Page 5 of 8 Fig. 3 Schematic drawing illustrating the landmarks used for the measurement of gingival marginal level. MAP: the most apical point of the gingival margin at the buccal aspect of the crown; PMD: the line connecting the peak of the adjacent mesial and distal natural teeth (PMD); GML: the distance from MAP to PMD; Magnification: the length of the natural crown next to the implant supported prosthesis was recorded to correct any changes in magnification Table 2 Clinical Jemt parameters of soft tissue from 3 to 12 months Data were presented as means ± standard deviations (SD) Significant difference (P < 0.05) between thin-gingiva group and thick-gingiva group; △ Significant difference (P < 0.05) between 3 months or 12 months and baseline Parameters Follow-up (months) Thin-gingiva Thick-gingiva P value Mesial papilla 3 12 2.62 ± 0.62 2.41 ± 0.63 2.77 ± 0.66 2.54 ± 0.58 0.39 0.45 Distal papilla 3 12 2.52 ± 0.64 2.31 ± 0.66 2.69 ± 0.68 2.42 ± 0.58 0.33 0.51 mPI (mm) 3 12 0.89 ± 0.79 1.04 ± 0.84 1.04 ± 0.82 0.88 ± 0.65 0.51 0.47 mBI(mm) 3 12 0.61 ± 0.74 0.75 ± 0.88 0.77 ± 0.95 0.65 ± 0.80 0.49 0.68 PD (mm) 3 12 2.16 ± 0.42 1.94 ± 0.47 2.18 ± 0.41 2.07 ± 0.45 0.81 0.29 GML(mm) Baseline 7.59 ± 1.24 7.17 ± 1.26 – 3 8.23 ± 1.26△ 7.53 ± 1.29 0.008* 12 9.01 ± 1.30△ 8.35 ± 1.48 0.04* Table 2 Clinical Jemt parameters of soft tissue from 3 to 12 months Fig. 3 Schematic drawing illustrating the landmarks used for the measurement of gingival marginal level. Data were presented as means ± standard deviations (SD) Discussionh with thick-gingival phenotype. In thick-gingiva group, the GML remained stable during the whole follow-up period, there has no obvious change about the GML from baseline to 12-month visit (P > 0.05). According to clinical examinations, the recession of marginal gingiva of buccal aspect of the crown was found in 5 patients with thin-gingiva during the follow-up period (Fig. 5).h with thick-gingival phenotype. In thick-gingiva group, the GML remained stable during the whole follow-up period, there has no obvious change about the GML from baseline to 12-month visit (P > 0.05). According to clinical examinations, the recession of marginal gingiva of buccal aspect of the crown was found in 5 patients with thin-gingiva during the follow-up period (Fig. 5).h The purpose of this study was to evaluate clinical and radiographic manifestations of a cross-shaped incision technique for thick-gingiva and thin-gingiva patients treated with implant-supported fixed prosthesis. Our study demonstrated that papillae filled more than half of the proximal space, in good harmony with the adjacent papillae in both thin-gingiva and thick-gingiva groups. In our study, zirconia crowns were fabricated and cemented to abutments with the lowest gingival height. Welander et al. demonstrated an apical shift of the barrier epithe- lium and the marginal bone around AuPt-alloy, where the soft tissue dimensions remained stable around Ti and ZrO2 abutments [17]. Kajiwara et al. demonstrated that greater blood flow was detected around zirconia abut- ment group compared to the titanium abutment or cast- to-abutment in free gingiva [18]. And we proposed that zirconia crowns may promote microcirculatory dynamics in soft tissue and be beneficial for the bone remodeling around implant.l The change of peri-implant hard tissue was shown in Table 3. Implant was inserted into the maxillae or mandible located coronal to the IS, and there has no difference in fBIC-IS between thin-gingiva group and thick-gingiva group. The mean fBIC-IS in thin-gingiva group decreased from 0.40 ± 0.95 at 3-month visit to -0.02 ± 1.01 at 12-month visit; and the value in thick- gingiva group decreased from 0.29 ± 0.59 in 3-month visit to -0.11 ± 0.63 at 12-month visit (P < 0.05). Although both thin-gingiva group and thick-gingiva group showed slight marginal bone resorption, there has no significant difference between thin-gingiva group and thick-gingiva group (P > 0.05). Results IS: implant shoulder; fBIC: first bone-implant contact; a the vertical distance the first bone-implant contact to implant shoulder measured from radiograph. b Implant length. x (fBIC-IS): the real vertical distance the first bone-implant contact to implant shoulder Table 1 Demographic data of the patients Data were presented as means ± standard deviations (SD) Demographics Thin-gingiva Thick-gingiva Patients (n) 29 26 Age range (years) 24–58 21–62 Mean ± SD 40.14 ± 10.75 38.69 ± 11.06 Sex (n: femal/male) 13/16 15/11 Implant location (n: maxilla/madible) 18/11 14/12 Table 1 Demographic data of the patients The GML on the buccal aspect of the crown was assessed by the distance from TBF to PMD. Table 2 showed that at 3-month or 12-month visit, GML in thin-gingiva group increased significantly compared to the baseline (P < 0.05), higher than the GML in patients Luo et al. BMC Oral Health (2021) 21:655 Luo et al. BMC Oral Health (2021) 21:655 Page 6 of 8 Page 6 of 8 Page 6 of 8 △ Significant difference (P < 0.05) between 3 months or 12 months and baseline Discussionh Compared with thin-gingiva group, GML did not change from baseline to the 12-month visit in thick- gingiva group, indicating consistent stability of the gin- gival margin after the cross-shaped incision of gingival sulcus around the crown in patients with thick-gingival phenotype. Author details 1 Based on these results, the zirconia crown is beneficial for the reconstruction of gingival papillae by promoting microcirculatory dynamics in soft tissue around implant. The gingival margin remained stable in patients with thick phenotype gingiva after the cross-shaped incision of gingival sulcus around the crown. The cross-shaped incision has several advantages. It is visible to check the proper placement of crowns and easy to clean excessive cement with the slight lift of incised gingiva. The limi- tation for cross-shaped incision is that the recession of marginal gingiva where the incision is made may hap- pen in thin-gingiva group. The patients included must have at least 2 mm of keratinized tissue width around the 1 State Key Laboratory of Oral Diseases and National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, No.14, Sec.3, Renminnan Road, Chengdu 610041, Sichuan, China. 2 Depart- ment of Stomatology, The First Affiliated Hospital of Hainan Medical University, Haikou 570102, China. Received: 8 July 2021 Accepted: 10 December 2021 Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Declarations Marginal crestal bone in both thin-gingiva and thick- gingiva groups was located coronal to the IS at the begin- ning, then decreased slightly during the following year, in accordance with the results of other studies [21, 22]. The stability of marginal bone may be due to the protection of soft tissue barrier, which serves as a protective seal for the adjacent periodontium [23]. Furthermore, “platform switching” was used in all the implants, which may result in less marginal bone resorption [24, 25]. In turn, the underlying bone provides the support of gingival tissue [6, 26]. Conclusionh The cross-shaped incision may be applied to reconstruct gingival papillae and avoid the gingival recession in patients with thick-gingiva phenotype. For the patients with thin-gingival phenotype, a modified method aimed to reconstruct gingival papillae and avoid the gingival recession need further study. Authors’ contributions C l Q Conceptualization: YM, YQ, YW, WL. Data curation: XW, YC. Investigation: YH. Supervision: YM. All authors read and approved the manuscript. Funding This study was funded by Hainan Natural Science Foundation of China (No. 818MS142) and The National Key Research and Development Program of China (No. 2016YFC1102700). The funder played no role in the design and execution of this study. Consent for publication Not applicable. Consent for publication Not applicable. Discussionh Moreover, securing a rich blood flow in soft tissues around implants is considered to be advantageous for the maintenance of immune function [18], which was reflected by the low score of mPI and mBI in this study. All patients in this study accepted oral hygiene instruc- tion at each visit, so PD in both thick-gingiva and thin- gingiva groups remained healthy. The values of PD even decreased slightly from 3 to 12-month after restora- tion, perhaps due to the emphasized instruction of oral hygiene at each visit. Fig. 5 Recession of marginal gingiva was found in the middle of buccal aspect around crown 3 months after cement The importance of possessing an adequate width and thickness of keratinized mucosa seems to be crucial both for natural teeth and dental implants. A deficiency of (or minimal) keratinized mucosa around implants has been shown to hinder patient oral hygiene, leading to soft tissue inflammation, mucosal recession, and attach- ment loss [19]. Having at least 2 mm of keratinized tissue Fig. 5 Recession of marginal gingiva was found in the middle of buccal aspect around crown 3 months after cement Table 3 Clinical parameters of hard tissue from 3 to 12 months Data were presented as means ± standard deviations (SD) △ Significant difference (P < 0.05) between 3 months or 12 months and baseline Parameters Follow-up (month) Thin-gingiva Thick-gingiva P value Mesial fBIC-IS Baseline 1.01 ± 0.82 0.72 ± 0.69 0.93 3 0.44 ± 1.12△ 0.26 ± 0.76△ 0.67 12 0.03 ± 1.31△ − 0.02 ± 0.74△ 0.91 Distal fBIC-IS Baseline 0.81 ± 0.82 0.71 ± 0.69△ 0.81 3 0.33 ± 0.92△ 0.20 ± 0.74△ 0.58 12 − 0.07 ± 0.98△ − 0.20 ± 0.78△ 0.82 Mean fBIC-IS ((M + D)/2) Baseline 0.91 ± 0.70 0.72 ± 0.62△ 0.93 3 0.40 ± 0.95△ 0.29 ± 0.59△ 0.77 12 − 0.02 ± 1.01△ − 0.11 ± 0.63△ 0.82 Table 3 Clinical parameters of hard tissue from 3 to 12 months Luo et al. BMC Oral Health (2021) 21:655 Page 7 of 8 width had protective effect on peri-implant health, and implants with < 2 mm of keratinized tissue width were more prone to develop peri-implant biologic complica- tions [20]. The patients included in this study had at least 2 mm of keratinized tissue width around the implant. However, the recession of marginal gingiva was detected in patients with thin-gingival phenotype. 1. Du H, Gao M, Qi C, Liu S, Lin Y. Drug-induced gingival hyperplasia and scaffolds: they may be valuable for horizontal food impaction. Med Hypotheses. 2010;74:984–5. Discussionh Gingival phe- notype, thin or thick, may affect the dimension of the periodontal tissue. A thick phenotype is prone to pocket formation, while a thin phenotype is prone to gingival recession following mechanical or surgical manipula- tion [6]. Different from natural teeth, supracrestal fib- ers (gingivo-dental and transseptal fibers) was not in the gingival tissue surrounding the implant abutment. Fur- thermore, the absence of blood vessel branches associ- ated with the periodontal ligament results in restricted blood supply to the peri-implant mucosa. The pressure between the restoration and gingiva typically causes ischemia [7]. Therefore, the peri-implant mucosa can also be defined as “scar-like” tissues. The gingival with thin phenotype around implant is easier to shrink following surgery, which may be the reason of gingival recession in the middle of buccal aspect in patients with cross-shaped incision. Compared with thin-gingiva group, GML did not change from baseline to the 12-month visit in thick- gingiva group, indicating consistent stability of the gin- gival margin after the cross-shaped incision of gingival sulcus around the crown in patients with thick-gingival phenotype. implant. The lack of negative controls was another limita- tion of this study. width had protective effect on peri-implant health, and implants with < 2 mm of keratinized tissue width were more prone to develop peri-implant biologic complica- tions [20]. The patients included in this study had at least 2 mm of keratinized tissue width around the implant. However, the recession of marginal gingiva was detected in patients with thin-gingival phenotype. Gingival phe- notype, thin or thick, may affect the dimension of the periodontal tissue. A thick phenotype is prone to pocket formation, while a thin phenotype is prone to gingival recession following mechanical or surgical manipula- tion [6]. Different from natural teeth, supracrestal fib- ers (gingivo-dental and transseptal fibers) was not in the gingival tissue surrounding the implant abutment. Fur- thermore, the absence of blood vessel branches associ- ated with the periodontal ligament results in restricted blood supply to the peri-implant mucosa. The pressure between the restoration and gingiva typically causes ischemia [7]. Therefore, the peri-implant mucosa can also be defined as “scar-like” tissues. The gingival with thin phenotype around implant is easier to shrink following surgery, which may be the reason of gingival recession in the middle of buccal aspect in patients with cross-shaped incision. Abbreviations fBIC Fi t b i fBIC: First bone-implant contact; GML: Gingival margin level; IS: Implant shoul- der; mBI: Modified Sulcus Bleeding Index; mPI: Modified Plaque Index; PD: Probing Depth; VIP-CT: Vascularized interposition periosteal-connective tissue. Ethics approval and consent to participate The present study was performed in accordance with the World Medical Association Declaration of Helsinki and was approved by Ethics Committee of West China Hospital of Stomatology, Sichuan University, China (Approval No. 2009033). All the patients signed informed consent. Publisher’s Note 6. Müller HP, Heinecke A, Schaller N, Eger T. Masticatory mucosa in subjects with different periodontal phenotypes. J Clin Periodontol. 2000;27:621–6. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. f 7. Yao JW, Wang HL. Assessment of peri-implant soft tissue adaptive pres- sure and time after provisional restorations. Int J Periodontics Restorative Dent. 2019;39:809–15. 7. Yao JW, Wang HL. Assessment of peri-implant soft tissue adaptive pres- sure and time after provisional restorations. Int J Periodontics Restorative Dent. 2019;39:809–15. 8. Salama H, Salama M. 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Welander M, Abrahamsson I, Berglundh T. The mucosal barrier at implant abutments of different materials. Clin Oral Implants Res. 2008;19:635–41. 18. References References 1. Du H, Gao M, Qi C, Liu S, Lin Y. Drug-induced gingival hyperplasia and scaffolds: they may be valuable for horizontal food impaction. Med Hypotheses. 2010;74:984–5. Luo et al. BMC Oral Health (2021) 21:655 Page 8 of 8 2. Bidra AS. Nonsurgical management of inflammatory periimplant disease caused by food impaction: a clinical report. J Prosthet Dent. 2014;111:96–100. 2. Bidra AS. Nonsurgical management of inflammatory periimplant disease caused by food impaction: a clinical report. J Prosthet Dent. 2014;111:96–100. non-matching healing abutments: micro-CT analysis. Clin Oral Implants Res. 2015;26:42–6. 25. Farronato D, Santoro G, Canullo L, Botticelli D, Maiorana C, Lang N. 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Choose BMC and benefit from: Publisher’s Note Kajiwara N, Masaki C, Mukaibo T, Kondo Y, Nakamoto T, Hosokawa R. Soft tissue biological response to zirconia and metal implant abutments compared with natural tooth: microcirculation monitoring as a novel bioindicator. Implant Dent. 2015;24:37–41. 19. Lin GH, Chan HL, Wang HL. The significance of keratinizedmmucosa on implant health: a systematic review. J Periodontol. 2013;84:1755–67. 20. Perussolo J, Souza AB, Matarazzo F, Oliveira RP, Araujo MG. Influence of the keratinized mucosa on the stability of peri-implant tissues and brushing discomfort: a 4-year follow-up study. Clin Oral Implants Res. 2018;29:1177–85. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 21. Cooper LF, Ellner S, Moriarty J, et al. Three-year evaluation of single-tooth implants restored 3 weeks after 1-stage surgery. Int J Oral Maxillofac Implants. 2007;22:791–800. 22. Kan JY, Rungcharassaeng K, Liddelow G, Henry P, Goodacre CJ. Periim- plant tissue response following immediate provisional restoration of scalloped implants in the esthetic zone: a one-year pilot prospective multicenter study. J Prosthet Dent. 2007;97:109–18. 23. Al-Juboori MJ. Interdental implant papillae grow up with temporary abutment displaced at monthly intervals. J Contemp Dent Pract. 2015;16:422–6. 24. Finelle G, Papadimitriou D, Souza A, Katebi N, Gallucci G, Araújo M. Peri-implant soft tissue and marginal bone adaptation on implant with 24. Finelle G, Papadimitriou D, Souza A, Katebi N, Gallucci G, Araújo M. Peri-implant soft tissue and marginal bone adaptation on implant with
https://openalex.org/W4391098140	https://pubs.aip.org/aip/jap/article-pdf/doi/10.1063/5.0188863/19860477/044501_1_5.0188863.pdf	English	null	A new analytical method for modeling a 2D electrostatic potential in MOS devices, applicable to compact modeling	Journal of applied physics	2,024	cc-by	6,003	RESEARCH ARTICLE \| JANUARY 22 2024 A new analytical method for modeling a 2D electrostatic potential in MOS devices, applicable to compact modeling RESEARCH ARTICLE \| JANUARY 22 2024 A new analytical method for modeling a 2D electrostatic potential in MOS devices, applicable to compact modeling J. Appl. Phys. 135, 044501 (2024) https://doi.org/10.1063/5.0188863 J. Appl. Phys. 135, 044501 (2024) https://doi.org/10.1063/5.0188863 Articles You May Be Interested In Articles You May Be Interested In Comparison of junctionless and inversion-mode p-type metal-oxide-semiconductor field-effect transistors in presence of hole-phonon interactions Influence of neighboring coupling on metal-insulator-semiconductor (MIS) deep-depletion tunneling current via Schottky barrier height modulation mechanism J. Appl. Phys. (April 2017) J. Appl. Phys. (April 2017) 24 October 2024 05:17:56 A new analytical method for modeling a 2D electrostatic potential in MOS devices, applicable to compact modeling Cite as: J. Appl. Phys. 135, 044501 (2024); doi: 10.1063/5.0188863 Submitted: 24 November 2023 · Accepted: 22 December 2023 · Published Online: 22 January 2024 Cite as: J. Appl. Phys. 135, 044501 (2024); doi: 10.1063/5.0188863 Submitted: 24 November 2023 · Accepted: 22 December 2023 · Published Online: 22 January 2024 Cite as: J. Appl. Phys. 135, 044501 (2024); doi: 10.1063/5.0188863 Submitted: 24 November 2023 · Accepted: 22 December 2023 · Published Online: 22 January 2024 F. Lime,1,a) B. Iñiguez,1 and A. Kloes2 F. Lime,1,a) B. Iñiguez,1 and A. Kloes2 I. INTRODUCTION very accurate for complex and asymmetrical structures. As a more accurate alternative, one can use Fourier series,7,8 but with the dis- advantage of using complex expressions and infinite series. Another possibility is using the conformal mapping technique. The latter is equivalent to Fourier series, but in some cases, it has the advantage of leading to simpler closed-form expressions.9 In semiconductor technologies, compact models are the neces- sary core component of design tools and circuit simulators. They are needed to reproduce the behavior of the electronic components. These models need to be simple, fast, and efficient in order to allow complex circuit simulations. Over the years, semiconductor devices have been scaled down in order to improve their performance and price per unit. With this size reduction comes new effects to be taken in account, and new architectures have been introduced as well. As a consequence, it becomes more critical to model the device in two or even three dimen- sions, in addition to the common and usual 1D approach. g g For a MOSFET, conformal mapping was first implemented in Ref. 10 The method has also been used to evaluate fringing fields in SOI technologies.11,12 The Poisson equation is usually decomposed into the sum of a 1D and a 2D component. The 1D term is a Poisson equation, while the 2D one reduces to the Laplace equation. This is an approximation to simplify the problem. Then, the Laplace equation is solved over a rectangular domain. This is referred to as the 4-corner problem. This has been done for a Double gate MOSFET in Ref. 13. However, the solution of the 4-corner problem is not analytical and, moreover, is computationally expensive. Because of this, the following approximation is usually made. When the rectangle is very long, one of its edges can be neglected as it tends to infinity. This approximation is called the 2-corner problem.9,14 This method is For field effect transistors, 2D modeling of the surface poten- tial is necessary in order to take into account short channel effects such as Drain Induced Barrier Lowering (DIBL) and channel length modulation, or even narrow and corner effects in long 3D structures.1 The various 2D models for compact modeling include the use of an equivalent characteristic length.2–6 This method has been widely used because of its simplicity, especially when it is not possible to obtain an exact solution. ABSTRACT This paper presents a new conformal mapping method to solve 2D Laplace and Poisson equations in MOS devices. More specifically, it con- sists of an analytical solution of the 2D Laplace equation in a rectangular domain with Dirichlet boundary conditions, with arbitrary values on the boundaries. The advantages of the new method are that all four edges of the rectangle are taken into account and the solution con- sists of closed-form analytical expressions, which make it fast and suitable for compact modeling. The new model was validated against other similar methods. It was found that the new model is much faster, easier to implement, and avoids many numerical issues, especially near the boundaries, at the cost of a very small loss in accuracy. 24 October 2024 05 © 2024 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0188863 17:56 © 2024 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons A https://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0188863 Journal of Applied Physics pubs.aip.org/aip/jap METHOD AFFILIATIONS 1Electronics Engineering Department, Universitat Rovira i Virgili (URV), Tarragona 43007, Spain 1Electronics Engineering Department, Universitat Rovira i Virgili (URV), Tarragona 43007, Spain 2Technische Hochschule Mittelhessen, Competence Center for Nanotechnology and Photonics, Wiesenstrasse 14, Giessen 35390, Germany 2Technische Hochschule Mittelhessen, Competence Center for Nanotechnology and Photonics, Wies Germany a)Author to whom correspondence should be addressed: francois.lime@urv.cat III. THE 2-CORNER PROBLEM The solution can of the Laplace equation be found using a Schwarz–Christoffel conformal mapping. In this case, the method is analytical and consists of the following equation, which allows to map a rectangle of length L and height H of the (x, y)-plane, as shown Fig. 1, to the upper part of the (u, v)-plane,14 with w ¼ w1D þ w2D. With this simplification, we only have to solve Laplace equa- tion (3) in order to obtain the 2D contribution to the potential w2D. In addition, modern devices usually use low semiconductor doping, and as 2D effects are evaluated below the threshold, it can be considered that ρ ≃0, so that Poisson equation (1) reduces to Laplace equation (3). For the sake of simplicity, we consider that this is the case in this paper, and we have w ¼ w2D ¼ V and ρ ¼ 0. In this paper, we propose a new approach to the 4-corner problem, which allowed us to obtain a solution that is analytical and uses closed-form expressions, which are desirable in compact models. First, we will explain how to obtain the solution of the Laplace equation on a rectangular domain with the 2-corner method as it is necessary to understand our new method. In the second part, we will explain how to take into account the fourth boundary condition. In the last part, the accuracy of the new 4-corner model will be discussed. II. LAPLACE AND POISSON EQUATIONS In semiconductor devices, the electrostatic potential w and the charges are governed by the Poisson equation. The 2D Poisson equation can be written as w ¼ u þ iv ¼ cosh π z H , (4) (4) @2w @x 2 þ @2w @y 2 ¼ ρ εsc , (1) with z ¼ x þ iy. (1) This is equivalent to the following transition equations between x, y and u, v: where ρ is the charge density and εsc is the semiconductor permittivity. u x, y ð Þ ¼ cosh π x H cos π y H (5) v x, y ð Þ ¼ sinh π x H sin π y H : (6) 24 October 2024 05 u x, y ð Þ ¼ cosh π x H cos π y H (5) (5) Usually, a long channel transistor is governed by the 1D Poisson equation and 2D effects are to be considered only when the channel length is too short. In these cases, 2D effects can be considered as a kind of perturbation to the 1D solution, and the Poisson equation can then be decomposed into a 1D Poisson equa- tion and a 2D Laplace equation, and v x, y ð Þ ¼ sinh π x H sin π y H : (6) ber 2024 05 (6) The mapping of the rectangle of Fig. 1(a) using (4) is shown Fig. 1(b). The rectangular domain is mapped to the upper half (u, v)-plane, with the right edge of the rectangle on the positive values of the real axis of the complex (u, v)-plane. The top, bottom, 5:17:56 @2w1D @x 2 ¼ ρ εsc , (2) (2) FIG. 1. A rectangular area in the (x, y)-plane of dimensions L ¼ 35; : nm and H ¼ 20; : nm. The space between the lines is 1; : nm (a). (b) is the same rectangular area being mapped into the (u, v)-plane using (4). The circled numbers show the location of the four corners plus the point of coordinates L, H=2 ð Þ. FIG. 1. A rectangular area in the (x, y)-plane of dimensions L ¼ 35; : nm and H ¼ 20; : nm. The space between the lines is 1; : nm (a). I. INTRODUCTION However, this technique is not J. Appl. Phys. 135, 044501 (2024); doi: 10.1063/5.0188863 © Author(s) 2024 J. Appl. Phys. 135, 044501 (2024); doi: 10.1063/5.0188863 135, 044501-1 135, 044501-1 Journal of Applied Physics pubs.aip.org/aip/jap METHOD @2w2D @x 2 þ @2w2D @y 2 ¼ 0, (3) usually preferred over the 4-corner one because its solution con- sists of simple analytical expressions, which are convenient for compact modeling. However, the 2-corner solution becomes inac- curate when the length of the rectangle is less than two times its height. (3) with w ¼ w1D þ w2D. h h l f II. LAPLACE AND POISSON EQUATIONS (b) is the same rectangular area being mapped into the (u, v)-plane using (4). The circled numbers show the location of the four corners plus the point of coordinates L, H=2 ð Þ. Appl. Phys. 135, 044501 (2024); doi: 10.1063/5.0188863 135, 044501-2 Author(s) 2024 J. Appl. Phys. 135, 044501 (2024); doi: 10.1063/5.0188863 © Author(s) 2024 135, 044501-2 Journal of Applied Physics pubs.aip.org/aip/jap METHOD L, H=2 ð Þ. As this point is situated on the boundary of the domain, where the potential is known, the method will not be applied at this particular point and this should not be an issue. and left edges are mapped to the real axis, while the right edge is mapped to a half ellipse around the point (0,0) whose size increases as L increases. This means that when L tends to infinity, or equiva- lently L .. W, the right edge of the rectangle will effectively be mapped to infinity. The result of applying (9) to the domain of Fig. 1 is shown Fig. 2. Basically, the geometry is unaffected except for the right- hand side boundary of the rectangle that is opened up toward infinity. In other words, Eq. (9) maps the upper part of the right- hand side of the rectangle (i.e., the boundary points situated between the circled numbers #1 and #5 in Fig. 1(a)) to the upper side and the lower part (the points between circled numbers #4 and #5) to the bottom edge. The middle point (L, H=2), labeled as circled number #5 in the figure, is mapped to infinity. The vertical left-hand side of the rectangle is mapped between 0 and π but is not completely straight anymore. With that reserve in mind, which will have consequences explained latter, we can say that we just approximately converted the 4-corner problem to a 2-corner one, whose solution can be found with Eqs. (4–8). More specifically, we combine (9) with (4) in order to align the four edges to the real axis of the w-plane. This gives the following map: With this assumption that L .. W, we can then obtain a solu- tion of the Laplace equation, considering V ¼ V0 on a line located on the boundary of the rectangle of the (x, y)-plane, and V ¼ 0 everywhere else on the boundary. It could be, for example, a line between points #2 and #3 in Fig. 1. II. LAPLACE AND POISSON EQUATIONS In the (x, y)-plane, this line is defined between the two points z1 ¼ x1, y1 ð Þ and z2 ¼ x2, y2 ð Þ. In the (u, v)-plane, the line will be located on the real axis between the points u x1, y1 ð Þ and u x2, y2 ð Þ. The solution is then,10,14,15 V x, y ð Þ ¼ V0 π f x, y, x1, y1 ð Þ f x, y, x2, y2 ð Þ ð Þ (7) (7) with f x, y, x0, y0 ð Þ ¼ arctan u x, y ð Þ u x0, y0 ð Þ v x, y ð Þ (8) (8) w ¼ cosh Z1 x, y ð Þ ð Þ (10) w ¼ cosh Z1 x, y ð Þ ð Þ (10) (10) and where u and v are, respectively, given by (5) and (6). This is the solution of the 2-corner problem, that is to say, when the right-hand side boundary condition is not applied. It is strictly valid if the line is located far away from the opposite side of the rectangle so that the potential at the missing boundary is close to 0. In order to take into account the third edge of the rectangle, i.e., the right-hand side boun- dary condition in our example, we need to solve the 4-corner problem. This is required when H ≃L. or, equivalently, w ¼ u þ iv ¼ 1 2 cosh π L H cosh π Lz H þ cosh π Lz H cosh π L H ! , (11) (11) with with u x, y ð Þ ¼ cosh π Lx H cos π y H 2cosh π L H cosh2 π L H sinh2 π Lx H þ cos2 π y H þ 1 ! , (12) v x, y ð Þ ¼ sinh π Lx H sin π y H 2cosh π L H cosh2 π L H sinh2 π Lx H þ cos2 π y H 1 ! : (13) 24 October 2024 05:17:56 IV. A NEW APPROACH FOR THE 4-CORNER PROBLEM When V0 is a function of y, we can use the superposition principle to obtain an approximated solution, as shown in this example for the left-hand side of the rectangular domain, at +5:84 ¼ cosh π ð Þ þ 1=cosh π ð Þ ð Þ=2, obtained from (11) when z ¼ L ¼ H. The boundary is mapped to the real axis as follows. The left-hand side is mapped to the values 1 , u , 1, the top side to 5:84 , u , 1, and the bottom edge to 1 , u , 5:84. As for the upper half of the right side, it is mapped to the interval 1 , u , 5:84, while the lower half corresponds to 5:84 , u , 1. V x, y ð Þ ¼ X N1 n¼1 V0 yn ð Þ þ V0 ynþ1 ð Þ 2π f x, y, 0, ynþ1 ð Þ f x, y, 0, yn ð Þ ð Þ, (17) with yn ¼ y1 þ y2 y1 N n: 24 October 2024 05:17:5 V x, y ð Þ ¼ X N1 n¼1 V0 yn ð Þ þ V0 ynþ1 ð Þ 2π f x, y, 0, ynþ1 ð Þ f x, y, 0, yn ð Þ ð Þ, (17) However, (11) is only an approximation for the 4-corner problem, as the left-hand edge is not exactly mapped to the real axis, but very close to it, as shown by the inset of Fig. 3. As a conse- quence, the value obtained when solving the Laplace equation at the left-hand side will be a little underestimated. The reason for this error is due to the map (9). This can be seen in Fig. 2 as the vertical lines, which are close to the right-hand side are not straight anymore but deformed in the center. This effect becomes weaker the closer we are to the left-hand side of the rectangle but never disappears completely. This means that the approximation is better for higher L=H ratios. However, the accuracy is still acceptable for the limiting case L ¼ H, with a deformation of less than 1% at the left-hand side. From the inset of Fig. IV. A NEW APPROACH FOR THE 4-CORNER PROBLEM Inset: a close-up showing the deviation from the real axis when 1 , u , 1. The maximum deviation is at most 0.0038 at u ¼ 0. The four corners and the point L, H=2 ð Þ are indicated with a circled number. v x, y ð Þ ¼ sinh π Hy L sin π x L 2cosh π H L cosh2 π H L sinh2 π Hy L þ cos2 π x L 1 ! : (15) v x, y ð Þ ¼ sinh π Hy L sin π x L 2cosh π H L cosh2 π H L sinh2 π Hy L þ cos2 π x L 1 ! : (15) The solution of the potential using the Laplace equation can obtained with (7) for a constant value on the boundary between the points z1 and z2, considering z2 to be after z1 in an anticlock- wise way around the boundary. However, when the point L, H=2 ð Þ is between z1 and z2, (7) have to be replaced by V x, y ð Þ ¼ V0 þ V0 π f x, y, x1, y1 ð Þ f x, y, x2, y2 ð Þ ð Þ (16) (16) FIG. 3. The rectangular area of Fig. 1 after being mapped into the w-plane using (11), for the case H ¼ L. The left-hand edge of the rectangle is mapped to the values of the real axis u between 1 and 1. Inset: a close-up showing the deviation from the real axis when 1 , u , 1. The maximum deviation is at most 0.0038 at u ¼ 0. The four corners and the point L, H=2 ð Þ are indicated with a circled number. because the line is going from þ1 to 1 in the (u, v)-plane. This can be useful as the error explained above is mainly located on the left-hand side of the domain, so in some cases, it might be more interesting to apply the boundary condition to the right- hand side. IV. A NEW APPROACH FOR THE 4-CORNER PROBLEM The solution of the 4-corner problem is already known and can be found in some textbooks on complex analysis.15 It is also based on a Schwarz–Christoffel conformal mapping. This method has been implemented for a Double Gate MOSFET in Ref. 13. However, this solution requires finding an inverse function for elliptic integrals. Thus, it is a numerical method and is not analyti- cal. In the following, we propose a new approach that is not based on the Schwarz–Christoffel conformal mapping. This new model does not require the evaluation of elliptic integrals and has the advantage of being analytic. However, we will also show that the new method is only a good approximation to the exact Schwarz– Christoffel approach. (13) The result of the map (11) is shown Fig. 3 for the case H ¼ L. The four corners, labeled with circled numbers 1–4 in Figs. 1 and 3, are mapped to the real axis: two at +1 and the other two FIG. 2. The rectangular area of Fig. 1(a) after being mapped into the (X1, Y1)-plane using (9). The method consists of applying a map, which converts the 4-corner problem into a 2-corner one. Such a map is the following one: Z1 ¼ X1 þ iY1 ¼ ln cosh π L H cosh π Lz H ! : (9) (9) First, it should be shown that the map (9) is conformal, i.e., that Laplace equation is invariant to this mapping. This can be done using the property that any combination of two conformal maps is also conformal. In fact, cosh z ð Þ is conformal as well as ln z ð Þ, except when zj j ¼ 0. This means that (9) is conformal, except at the point #5 in Fig. 1, which is the point of coordinates FIG. 2. The rectangular area of Fig. 1(a) after being mapped into the (X1, Y1)-plane using (9). J. Appl. Phys. 135, 044501 (2024); doi: 10.1063/5.0188863 © Author(s) 2024 J. Appl. Phys. 135, 044501 (2024); doi: 10.1063/5.0188863 135, 044501-3 Journal of Applied Physics METHOD FIG. 3. The rectangular area of Fig. 1 after being mapped into the w-plane using (11), for the case H ¼ L. The left-hand edge of the rectangle is mapped to the values of the real axis u between 1 and 1. IV. A NEW APPROACH FOR THE 4-CORNER PROBLEM 3, it can be seen that the left- hand side of the rectangle is approximately mapped to the real axis between u ¼ 1 and u ¼ 1, with a value of 0:0037 for the maximum deviation from the real axis at u ¼ 0. This represents 0.185% of the length of this side of the rectangle in the (u,v)-plane, which is equal to 2. As this deformation is quite small, we consider the map (11) to be a good approximation for the 4-corner problem, provided that L . H. with The desired number of intermediary values of V0 y ð Þ between y1 and y2 is N 1. Using (17), it is then possible to obtain an approximated ana- lytical and explicit expression for the solution of the Laplace equa- tion, with arbitrary Dirichlet boundary conditions on the rectangular domain. g To illustrate the accuracy of the new method, we show the following example. A domain with H ¼ L ¼ 12 nm is considered with the potential equal to 1 V on the left-hand side of the rectan- gle and 0 everywhere else on the boundary. The results for the 4- and 2-corner cases are plotted against x in Fig. 4, for y ¼ H=2 . It can be observed that, for the 4-corner case, the potential correctly cancels to 0 on the right-hand side, in contrast to the 2-corner one, because the fourth boundary condition is not applied in that case. The 4-corner approach is much more accurate than the 2-corner one, especially when L becomes comparable to H and the 2-corner method fails. This figure also compares the new model with the original Schwarz–Christoffel numerical 4-corner model.13 The agreement with our 4-corner model is excellent. This shows that the approximations of the analytical model have a very low impact on the results, even for the worst case H ¼ L. In addition, our model is much faster and, being analytical, does not suffer from the numerical inaccuracies inherent to the For the cases when L , H, the problem should be rotated 90. This can be done easily in equations (12) and (13) by inverting x and y, L, and H and also changing the sign of u. V. A SIMPLE EXAMPLE FOR A DOUBLE GATE MOSFET As explained below (16), we can assign non zero values to the boundary condition on the right-hand edge of the domain, or to the left-hand one, which is easier to implement. We give the fol- lowing example for the right-hand case. We first calculate the potential for the right-hand side of the channel, i.e., for x . L=2. The contribution from the drain was obtained from (16) as follows. As the boundary condition line is crossing the point L, H=2 ð Þ, we have to use (16) instead of (7), Vdrain x, y ð Þ ¼ VD þ VD π f x, y, L, toxb ð Þ ½ f x, y, L, H toxf : (18) (18) Where toxf and toxb are, respectively, the equivalent thicknesses of the front and back gate dielectrics. In a similar way, contributions from the top and back gate were obtained from (7), FIG. 4. The potential at y ¼ H=2, for the following boundary conditions: V ¼ 1V on the left-hand side and 0 everywhere else, for the case H ¼ L. The 4- and 2-corner cases are compared. Symbols are the 4-corner model without approxi- mation, presented in Ref.13. Vtopgate x, y ð Þ ¼ VGt π f x, y, L, H ð Þ f x, y, 0, H ð Þ ð Þ, (19) Vbackgate x, y ð Þ ¼ VGb π f x, y, 0, 0 ð Þ f x, y, L, 0 ð Þ ð Þ: (20) Vtopgate x, y ð Þ ¼ VGt π f x, y, L, H ð Þ f x, y, 0, H ð Þ ð Þ, (19) Vtopgate x, y ð Þ ¼ VGt π f x, y, L, H ð Þ f x, y, 0, H ð Þ ð Þ, (19) (19) 4-corner Schwarz–Christoffel method. These inaccuracies of the Schwarz–Christoffel model come from the use of complex inte- grals and interpolations, generally over quantities, which tend to infinity. Vbackgate x, y ð Þ ¼ VGb π f x, y, 0, 0 ð Þ f x, y, L, 0 ð Þ ð Þ: (20) (20) FIG. 5. (a) The calculated 2D potential from our model, for a Double Gate MOSFET with tsc ¼ 12 nm, tox ¼ 1:6 nm, and L ¼ 25 nm. (b) Corresponding equipotentials. IV. A NEW APPROACH FOR THE 4-CORNER PROBLEM This gives u x, y ð Þ ¼ cosh π Hy L cos π x L 2cosh π H L cosh2 π H L sinh2 π Hy L þ cos2 π x L þ 1 ! , (14) (14) 135, 044501-4 135, 044501-4 J. Appl. Phys. 135, 044501 (2024); doi: 10.1063/5.0188863 © Author(s) 2024 J. Appl. Phys. 135, 044501 (2024); doi: 10.1063/5.0188863 Journal of Applied Physics METHOD pubs.aip.org/aip/jap FIG. 4. The potential at y ¼ H=2, for the following boundary conditions: V ¼ 1V on the left-hand side and 0 everywhere else, for the case H ¼ L. The 4- and 2-corner cases are compared. Symbols are the 4-corner model without approxi- mation, presented in Ref.13. VI. CONCLUSIONS In this work, a new technique to model a 2D potential in MOS devices has been presented. It is based on a conformal mapping method to solve the Laplace equation on a rectangular domain. The new model takes into account all four edges of the rectangle, and only needs the values on the boundary to be defined. The solution presents the advantage of being constituted of closed- form analytical expressions. These are similar in complexity to the solution of the 2-corner problem. The accuracy was found to be excellent and the new technique was found to be a very good approximation of the 4-corner method. Compared to the 4-corner method, the new model is much faster, easier to implement, and avoid many numerical issues, especially near the boundaries. The trade-off is a small, negligible loss in accuracy. Thanks to these characteristics, the new model is suitable for compact modeling and should be especially useful when modeling very short-channel FETs. Summing all the contributions gives the contribution for the right-hand side of the channel VR x, y, VD ð Þ ¼ Vdrain x, y, VD ð Þ þ Voxt x, y, VD ð Þ þ Voxb x, y, VD ð Þ: (21) (21) The contribution from the left side was obtained “mirroring” VR with respect to the middle of the channel, so that the total potential, including the contributions from the top and back gates, is Vtot x, y ð Þ ¼ VR x, y, VD ð Þ þ VR L x, y, VS ð Þ þ Vtopgate x, y ð Þ þ Vbackgate x, y ð Þ: (22) (22) To validate our model, we compared it with a similar method published in Ref. 13, which used a Schwarz–Christoffel conformal mapping, that is to say, the original 4-corner method, without approximation, which is not analytical. We implemented their method and reproduced the example given in their paper, which consists of DGMOS with 25 nm for the gate length, a silicon thick- ness of 12 nm, a gate oxide thickness of 1.6 nm, and an aluminum gate. The results are shown in Fig. 5. We used equivalent gate dielectric thicknesses teq ox ¼ toxεsc=εox in order to avoid the disconti- nuity in the two relative permittivity values, which are εsc ¼ 11:8 for the semiconductor and εox ¼ 7 for the dielectrics. VI. CONCLUSIONS As an improvement over,13 a linear boundary condition in the gate dielec- tric was also considered with 10 intermediary values, i.e., N = 10 in (17). Figure 5(a) shows the 2D potential obtained from our model, i.e., Equation (22), and (b) shows the corresponding equipotentials. In this former figure, the source is located on the left, the drain on the right, the top gate on the top and the back gate on the bottom of the rectangle. The linear potential in the dielectrics, along with the ten intermediary steps can also be seen in Fig. 5(a). In Fig. 5(c), our model is compared with both the Schwartz–Cristoffel 4-corner method of Ref. 13 and with Technology Computer-Aided Design (TCAD) simulations done with Silvaco Atlas. Figure 5(c) shows two cuts of the 2D potential of (a): a vertical one at x ¼ L=2 and a horizontal one at y ¼ H=2. The comparison between the two 4-corner approaches shows very similar results, which validates our method. The two characteristics are nearly identical, but our analyt- ical model should give a better representation of the 2D potential in the gate dielectrics, especially near the boundary conditions and the corners, because in those areas it is difficult to evaluate the numerical approach with a high enough precision. The small dis- crepancy with TCAD could be due to the source/drain junction built-in potential, that is to say, the boundary value at the source and drain end of the channel, as we could achieve a much better fit with the lower value of this built-in potential. This may be explained as in reality, this potential is not applied exactly at the V. A SIMPLE EXAMPLE FOR A DOUBLE GATE MOSFET (c) Horizontal and vertical cuts of the potential shown in (a), at the center of the channel. Lines are our model, dotted lines are from TCAD Silvaco, and symbols are the 4-corner Schwarz–Christoffel model from Kolberg et al.13 24 October 2024 05:17:56 24 October 2024 05:17:56 J. Appl. Phys. 135, 044501 (2024); doi: 10.1063/5.0188863 135, 044501-5 © Author(s) 2024 Journal of Applied Physics pubs.aip.org/aip/jap METHOD boundary of the channel but more inside the source and drain regions.16 The contribution from the top and back gate dielectrics Voxt and Voxb were obtained from (17). Considering, as an approximation, a linear potential drop in the dielectrics, the following Dirichlet boundary condition were considered in the front and back gate insulators: V0xt y ð Þ ¼ VGt þ VS VGt ð Þ H y ð Þ=toxf and V0xb y ð Þ ¼ VGb þ VS VGb ð Þy=toxb. In this case, we also used equivalent oxide thicknesses in order to avoid discontinuity in the permittivity. The authors have no conflicts to disclose. The authors have no conflicts to disclose. DATA AVAILABILITY Data sharing is not applicable to this article as no new data were created or analyzed in this study. Author Contributions F. Lime: Conceptualization (lead); Investigation (lead); Methodology (lead); Validation (lead); Writing – original draft (lead). B. Iñiguez: Investigation (equal); Supervision (equal); Writing – review & editing (equal). A. Kloes: Investigation (equal); Supervision (equal); Writing – review & editing (equal). 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https://openalex.org/W2121802483	http://www.scielo.cl/pdf/ejb/v17n2/a08.pdf	English	null	Assessment of somaclonal variation in somatic embryo-derived plants of yacon [Smallanthus sonchifolius (Poepp. and Endl.) H. Robinson] using inter simple sequence repeat analysis and flow cytometry	Electronic Journal of Biotechnology	2,014	cc-by	5,190	a r t i c l e i n f o Article history: Received 24 September 2013 Accepted 31 December 2013 Available online 28 January 2014 Keywords: DNA polymorphism Molecular markers Ploidy level Proembryogenic callus Article history: Received 24 September 2013 Accepted 31 December 2013 Available online 28 January 2014 Keywords: DNA polymorphism Molecular markers Ploidy level Proembryogenic callus Background: Yacon (Smallanthus sonchifolius) is a root crop native to the Andean region. Low sexual reproductive capacity is a major constraint facing the genetic breeding of this crop. Biotechnological techniques offer alternative ways to widen genetic variability. We investigated somaclonal variation in regenerants of yacon derived from in vitro somatic embryogenesis using simple sequence repeat (ISSR) analysis and ﬂow cytometry. Background: Yacon (Smallanthus sonchifolius) is a root crop native to the Andean region. Low sexual reproductive capacity is a major constraint facing the genetic breeding of this crop. Biotechnological techniques offer alternative ways to widen genetic variability. We investigated somaclonal variation in regenerants of yacon derived from in vitro somatic embryogenesis using simple sequence repeat (ISSR) analysis and ﬂow cytometry. Results: Twenty tested ISSR primers provided a total of 7848 bands in 60 in vitro regenerants and control plant. The number of bands for each primer varied from 3 to 10, and an average of 6.95 bands was obtained per ISSR primer. Eight primers were polymorphic and generated 10 polymorphic bands with 7.19% mean polymorphism. ISSR analysis revealed genetic variability in 6 plants under study. These regenerants had Jaccard's distances 0.104, 0.020, 0.040, 0.106, 0.163 and 0.040. Flow cytometric analysis did not reveal changes of relative nuclear DNA content in regenerants suggesting that the plants obtained via somatic embryogenesis had maintained stable octoploid levels. Results: Twenty tested ISSR primers provided a total of 7848 bands in 60 in vitro regenerants and control plant. The number of bands for each primer varied from 3 to 10, and an average of 6.95 bands was obtained per ISSR primer. Eight primers were polymorphic and generated 10 polymorphic bands with 7.19% mean polymorphism. ISSR analysis revealed genetic variability in 6 plants under study. These regenerants had Jaccard's distances 0.104, 0.020, 0.040, 0.106, 0.163 and 0.040. Flow cytometric analysis did not reveal changes of relative nuclear DNA content in regenerants suggesting that the plants obtained via somatic embryogenesis had maintained stable octoploid levels. Iva Viehmannova a,⁎, Zuzana Bortlova a, Jan Vitamvas b, Petra Hlasna Cepkova a, Katerina Eliasova c, Eva Svobodova a Martina Travnickova a a Department of Crop Sciences and Agroforestry in Tropics, Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague, Kamycka 129, Prague 165 21, Czech Republic b Department of Dendrology and Forest Tree Breeding, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences Prague, Kamycka 129, Prague 165 21, Czech Republic c Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojova 263, Prague 252 43, Czech Republic a r t i c l e i n f o Conclusions: Our ﬁndings show that indirect somatic embryogenesis could be used in yacon improvement to widen genetic variability, especially when low sexual reproductive capacity hinders classical ways of breeding. © 2014 Pontiﬁcia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. Electronic Journal of Biotechnology 17 (2014) 102–106 Electronic Journal of Biotechnology 17 (2014) 102–106 Contents lists available at ScienceDirect Contents lists available at ScienceDirect 3458/$ – see front matter © 2014 Pontiﬁcia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. dx.doi.org/10.1016/j.ejbt.2013.12.011 Assessment of somaclonal variation in somatic embryo-derived plants of yacon [Smallanthus sonchifolius (Poepp. and Endl.) H. Robinson] using inter simple sequence repeat analysis and ﬂow cytometry Assessment of somaclonal variation in somatic embryo-derived plants of yacon [Smallanthus sonchifolius (Poepp. and Endl.) H. Robinson] using inter simple sequence repeat analysis and ﬂow cytometry va a,⁎, Zuzana Bortlova a, Jan Vitamvas b, Petra Hlasna Cepkova a, Katerina Eliasova c, a Martina Travnickova a Iva Viehmannova a,⁎, Zuzana Bortlova a, Jan Vitamvas b, Petra Hlasna Cepkova a, Katerina Eliasova c, Eva Svobodova a, Martina Travnickova a 2.3. Histological analysis of embryogenic structures The use of ﬂow cytometry for detection of DNA content is also a very convenient method to reveal somaclonal variation in regenerated plants, as in vitro cultivation is also being associated with ploidy level changes [13,19]. Especially in vitro long-term cultivation, plant regeneration via indirect morphogenesis and preexistence of ploidy variation in explant of polysomatic species may result in ploidy instability of in vitro regenerated plants [20,21]. Flow cytometry has several advantages compared to other methods; unlike chromosome countings, it is a rapid method for ploidy testing since it allows the examination of large numbers of cells as well as different types of tissues and cell layers [22]. In the present study, we examined somaclonal variation in plants regenerated via somatic embryogenesis, where somatic embryos were initiated using different growth regulators at various levels. Two methods were employed, ISSR markers and ﬂow cytometry. To our knowledge this is the ﬁrst report in S. sonchifolius on the assessment of somaclonal variation in regenerated plantlets from indirect somatic embryogenesis. The use of ﬂow cytometry for detection of DNA content is also a very convenient method to reveal somaclonal variation in regenerated plants, as in vitro cultivation is also being associated with ploidy level changes [13,19]. Especially in vitro long-term cultivation, plant regeneration via indirect morphogenesis and preexistence of ploidy variation in explant of polysomatic species may result in ploidy instability of in vitro regenerated plants [20,21]. Flow cytometry has several advantages compared to other methods; unlike chromosome countings, it is a rapid method for ploidy testing since it allows the examination of large numbers of cells as well as different types of tissues and cell layers [22]. To comﬁrm regeneration of plants via somatic embryos, proembryogenic callus and somatic embryos at the cotyledonary stage were ﬁxed by immersion in 50% FAA (formaldehyde:acetic acid:ethanol:water, 1:1:9:9) for 48–96 h. Dehydration was carried out according to standard procedures with tissues being passed through ethanol–butanol dehydration series, followed by embedding in parafﬁn wax at 58–60°C [24,25]. The parafﬁn blocks were cut into ribbons of thin sections (13 μm) with a rotary microtome (Thermo Shandon Finesse, UK). The microtome sections were stained with 0.1% alcian blue in 3% acetic acid and 0.1% nuclear fast red in 5% Al2(SO4)3 [26] and photographed under a Nikon Eclipse 80i (Nikon, Japan) microscope equipped with a camera Nikon Digital Sight DS-5Mc (Nikon, Japan). 2.3. Histological analysis of embryogenic structures In the present study, we examined somaclonal variation in plants regenerated via somatic embryogenesis, where somatic embryos were initiated using different growth regulators at various levels. Two methods were employed, ISSR markers and ﬂow cytometry. To our knowledge this is the ﬁrst report in S. sonchifolius on the assessment of somaclonal variation in regenerated plantlets from indirect somatic embryogenesis. In total, 60 randomly selected regenerated plants were subjected to molecular and ploidy analysis. Evaluation was performed immediately after plant regeneration, and after the third subculture. 1. Introduction explained because of high levels of vegetative propagation and long term selection for desired agronomic traits in yacon [7]. Moreover, low germination of pollen, few achenes with seeds and low seed viability cause sexual reproduction of yacon to be very rare [8]. Yacon [Smallanthus sonchifolius (Poepp. and Endl.) H. Robinson] from the Asteraceae family is a perennial herb native to the Andean region and is known to have been cultivated since pre-Inca times. Yacon storage roots are a particularly abundant source of fructooligosaccharides, consisting of a series of inulin type β (2 →1) fructans, which have a positive effect on human health [1,2]. The aerial part of yacon shows strong antimicrobial activity [3], and the whole plant contains phenolic compounds with antioxidative activity [4]. Low sexual reproductive capacity is also a major constraint facing genetic breeding of this crop [8]. Biotechnological techniques, such as induction and selection of somaclonal variation, in vitro chromosome doubling and genetic transformation offer alternative ways to widen genetic variability of the crop [9]. Numerous studies have been reported on in vitro propagation of S. sonchifolius via shoot tips, axillary buds or somatic embryos [10,11,12]. Nevertheless, in these studies, genetic ﬁdelity of regenerated plants had not been assessed, though especially the presence of a disorganized growth phase in tissue culture is considered as one of the factors that may cause somaclonal variation [13]. Although occurrence of uncontrolled variation during the culture process is mostly an unexpected and undesired phenomenon [14], it can also be an important tool for plant breeding via generation of new varieties with useful agronomic traits [13]. In different landraces, there appears to be signiﬁcant variation in morphological traits [5], as well as in antioxidative activity and fructooligosaccharide content [6]. However, genetic divergence among landraces, as revealed by molecular markers, is very low. This can be Somaclonal variation resulting from point mutations or the activation of mobile elements can be detected by DNA-based marker systems, such as randomly ampliﬁed polymorphic DNA (RAPD), ampliﬁed fragment length polymorphism (AFLP), simple sequence repeat (SSR) and inter I. Viehmannova et al. / Electronic Journal of Biotechnology 17 (2014) 102–106 103 simple sequence repeat (ISSR) [13,15,16]. The low reproducibility of RAPD, high cost of AFLP and the need to know the ﬂanking sequences to develop species speciﬁc primers for SSR polymorphism are the major limitations of the ﬁrst three methods. 2.1. Plant material Experiments were carried out on an octoploid clone of S. sonchifolius (2n = 8x = 58), originated in Ecuador. In vitro plants were cultured on MS medium [23] containing 1 mg l-1 thiamine, 100 mg l-1 myo-inositol, 30 g l-1 sucrose and 8 g l-1 agar, at pH 5.7. Cultures were maintained for 30 d at 25/23°C under a 16/8 h light/dark regime with 36 μmol m-2 s-1 cool white ﬂuorescent light. Young petioles were excised from the plants. The petioles were cut into ca. 5 mm long segments and placed with the abaxial surface toward the medium, ﬁve pieces to each ﬂask. These segments were used as an initial culture for somatic embryogenesis experiments. 2. Materials and methods Flow cytometry measurement was performed using a Partec II ﬂow cytometer (Partec GmbH, Monster, Germany) equipped with an HBO mercury arc lamp. Sample preparation followed the two-step methodology according to Dolezel et al. [27]. Approximately 1 cm2 of leaf tissue from both the sample and an appropriate amount of internal reference standard (Bellis perennis L., 2C = 3.38 pg; Schonswetteer et al. [28]) were chopped with razor blade in 0.5 ml of ice-cold Otto I buffer (0.1 M citric acid, 0.5% Tween 20). The suspension was ﬁltered through a 42-μm nylon mesh and incubated for 10 min at room temperature. The staining solution consisted of 1 ml of Otto II buffer (0.4 M Na2HPO4.12H2O) supplemented by AT-selective ﬂuorescent dye 4′,6-diamino-2-phenylindol and 2-mercaptoethanol in ﬁnal concentrations of 4 μg ml- 1 and 2 μl ml-1, respectively. After a 5 min incubation at room temperature, the samples were analysed with a ﬂow cytometer. The stained nuclei were analysed at a concentration of 5000 per sample. Obtained histograms were evaluated using the FloMax software (ver. 2.4d; Partec GmbH, Münster, Germany). For each analysed sample, DNA-ratios were counted by dividing the mean of the dominant (G0/G1) peak of the yacon sample by the mean of the G0/G1 peak of the internal standard. Coefﬁcient of variation of the G0/G1 peaks was also recorded. 2.2. Regeneration of plants via somatic embryogenesis Petiole segments were cultivated in Erlenmeyer ﬂasks (100 ml) with 25 ml of MS medium containing 30 g l-1 sucrose, 1 mg l-1 thiamine, 100 mg l-1 myo-inositol, 8 g l-1 agar (pH 5.7) and different plant growth regulators (PGRs) at various concentrations. The treatments consisted of combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) at a concentration of 1 mg l-1 and either zeatin or N6-benzyladenine (BA) at concentrations 0.01, 0.05, or 0.1 mg l-1. Of these media, two were adopted from the study of Correa et al. [12] in this experiment, namely 2,4-D (1 mg l-1) in combination with BA (0.01 mg l-1, or 0.1 mg l-1). The media were denoted as A, B, C, D, E, F in order of appearance. Medium without PGRs was used as control. Cultures were maintained at 25/23°C under a 16/8 h light/dark regime with 36 μmol m-2 s-1 cool white ﬂuorescent light. After 8 weeks of culture, proembryogenic calli were periodically subcultured onto the same medium every 20 d. A total of 20 explants were used per treatment in two replications. 1. Introduction ISSR marker is a technique that overcomes most of these limitations. Moreover, it is a very simple, fast, cost-effective, highly polymorphic and reliable method [17]. It requires only a small quantity of a DNA sample and it does not need any prior sequence information to design the primer [18], thus it is suitable for the assessment of the genetic variation among in vitro regenerants. In vitro regenerated plants were isolated and transferred individually to MS medium, and maintained together with control plants in a sustainable manner described above, with regular subcultures of 30–40 d. All in vitro experiments were arranged as a completely randomized design. Statistical analysis of data obtained from ﬂow cytometric analysis, was performed by analysis of variance (ANOVA) and the signiﬁcantly different means were identiﬁed by using the Tukey's HSD test (p = 0.05) [StatSoft STATISTICA 9.0]. 2.3. Histological analysis of embryogenic structures 3.1. ISSR and ﬂow cytometric analysis All 20 tested ISSR primers provided clear and scorable bands with satisfactory intensity. A total of 7848 bands were generated from the control plant and 60 in vitro regenerants. The size of the ampliﬁcation fragments ranged from 180 to 2500 bp. The number of bands for each primer varied from 3 to 10, and an average of 6.95 bands was obtained per ISSR primer. Eight tested primers generated polymorphic bands, most of which provided one polymorphic band from their DNA proﬁles. Only ‘UBC823’ and ‘UBC841’ generated two polymorphic bands. Fig. 2 shows polymorphic ampliﬁcation patterns obtained with ISSR primer ‘UBC834’. The total polymorphism scored is given in Table 1. In four initiation media tested (i.e. A, B, E and F) at least one variable regenerant in a different primer fragment proﬁle was revealed. Regenerant F5 was detected as the most variable, showing polymorphism in primers `UBC823', `UBC834', `UBC836', `UBC840', `UBC841', `UBC844', `UBC845' and `UBC856'. In most primers, polymorphic bands were absent in the DNA pattern of variable regenerants when compared to that one of the control plant, with the exception of primers ‘UBC836’ and ‘UBC841’ where extra bands were scored. The PCR reaction for each ISSR primer was performed in two repetitions, only clear and completely reproducible bands were included in data evaluation. The bands were scored as presence (1) and absence (0) for each regenerant and control plant and were transformed into a binary character matrix. Genetic dissimilarity was calculated with Jaccard's distances using Darwin 5.0 software [29]. Table 1 Table 1 Primers used in ISSR polymorphism analysis, number and size of ampliﬁed fragments. Primers code (UBC) Sequence 5′–3′ Annealing temperature (°C) Total number of bands ampliﬁed Number of scorable bands per primer No. and frequency of polymorphic bands per primer Polymorphic regenerants Range of ampliﬁcation (pb) UBC807 (AG)8T 46.5 420 7 0 350–1000 UBC809 (AG)8G 48.0 420 7 0 450–1250 UBC810 (GA)8T 50.0 540 9 0 300–1350 UBC823 (TC)8C 47.5 232 4 2 (50%) A6, E1, E9, F5, F9 700–1300 UBC824 (TC)8G 48.0 600 10 0 300–1100 UBC828 (TG)8A 50.0 420 7 0 550–2500 UBC829 (TG)8C 52.5 480 8 0 500–1300 UBC834 (AG)8YT 52.0 239 4 1 (25%) F5 300–1200 UBC835 (AG)8YC 50.0 420 7 0 350–2500 UBC836 (AG)8YA 48.0 423 9 1 (11%) B8, E1, F5 500–2000 UBC840 (GA)8YT 46.5 478 8 1 (11%) E9, F5 180–1000 UBC841 (GA)8YC 52.0 423 9 2 (22%) A6, F5 200–1300 UBC844 (CT)8RC 47.5 297 5 1 (20%) A6, E9, F5 600–1250 UBC845 (CT)8RG 47.5 179 3 1 (33%) F5 800–1300 UBC847 (CA)8RC 52.5 360 6 0 550–2000 UBC851 (GT)8CT G 52.5 540 9 0 650–1900 UBC854 (TC)8RG 50.0 180 3 0 500–1300 UBC855 (AC)8YT 53.0 360 10 0 700–1350 UBC856 (AC)8YA 54.0 597 10 1 (10%) A6, E9, F5 500–1300 UBC873 (GACA)4 46.5 240 4 0 1000–1300 Total 7848 139 10 (7.19%) Table 1 Primers used in ISSR polymorphism analysis, number and size of ampliﬁed fragments. via somatic embryogenesis (Fig. 1b, c) was achieved from each of the tested initiation media and in vitro plantlets did not show any evident morphological abnormalities. Ten randomly selected plants from the six treatments were subjected to ISSR and ﬂow cytometric analysis to reveal potential somaclonal variation. reaction for individual primers. The cycling conditions were as follows: initial denaturation step for 5 min at 94°C, followed by 40 cycles of 1 min at 94°C (denaturing), 1 min at speciﬁc annealing temperature (Table 1), 2 min at 72°C (extension), and 1 cycle for 10 min ﬁnal extension step at 72°C. Electrophoretic separation was performed with 5.5 μl of ampliﬁed products on 2% agarose gel in 1× TBE buffer. Gels were run for about 3–3.5 h at 55 V. DNA ampliﬁcation products were stained with SYBR® Safe DNA Gel Stain (Invitrogen, USA). 2.5. DNA extraction and ISSR analysis Total DNA was extracted from leaf material obtained from 60 regenerated plantlets and initial plant material was used as control. From the fresh leaves were taken the samples of about 100 mg weight. For the extraction of genomic DNA the Invisorb® Spin Plant Mini Kit (Stratec Molecular, Germany) was used. Twenty ISSR primers (The University of British Columbia Biotechnology Laboratory, Canada) were tested. Polymerase chain reaction (PCR) ampliﬁcations were performed in a 20 μl reaction volume containing 1 μl of template DNA at concentration 50 ng μl-1, 0.5 μl of primer at concentration 0.5 μM, 10 μl PPP Master Mix (Top-Bio, Czech Republic), 0.2 μl BSA (Fermentas, Germany), 8.3 μl PCR H2O (Top-Bio, Czech Republic). Ampliﬁcations were performed in a T100™Cycler (Bio-Rad, USA). The PCR was carried out with modiﬁcations of the annealing temperature to optimize the The proembryogenic calli were transferred into Erlenmeyer ﬂasks (100 ml) with 25 ml of embryo induction medium. Full strength MS medium without PGRs, containing 30 g l-1 sucrose, 1 mg l-1 thiamine and 100 mg l-1 myo-inositol and 8 g l-1 agar (pH 5.7) was used. The cul- tures were kept at 25/23°C under a 16/8 h light/dark regime. A total of ten calli were used per treatment in three replications. I. Viehmannova et al. / Electronic Journal of Biotechnology 17 (2014) 102–106 104 3. Results and discussion In our experiment, callus from petiole segments started to appear by the 3rd week on all initiation media tested. These calli were yellowish-green, friable and fast growing. Histological examination conﬁrmed numerous meristematic tissues consisting of small and dense cells in the callus, suggesting proembryogenic structure formation (Fig. 1a). The induction frequency of proembryogenic callus in the treatments varied from 70 to 100%. The maturation of somatic embryos and development into plantlets was achieved after transfer of proembryogenic calli to induction media without auxin as was reported in the previous study by Correa et al. [12]. Histological analysis conﬁrmed the bipolar character of developed structures and showed no vascular connection with the callus. Successful regeneration of plants The ISSR data was used to calculate the Jaccard's distances. It revealed rather lower genetic variability in tested samples, i.e. 0.104, 0.020, 0.040, 0.106, 0.163 and 0.040 for the regenerants A6, B8, E1, E9, Fig. 1. Somatic embryogenesis in yacon. (a) Proembryonic callus with meristematic centres (bar, 0,5 mm). (b) Early cotyledonary embryo (bar, 0.8 mm). (c) Germinated well developed embryo (bar, 1.5 mm). (Ct: Cotyledon; Hy: Hypocotyl; ME: Meristematic Centres; Ra: Radicle; SA: Shoot Apex). Fig. 1. Somatic embryogenesis in yacon. (a) Proembryonic callus with meristematic centres (bar, 0,5 mm). (b) Early cotyledonary embryo (bar, 0.8 mm). (c) Germinated well developed embryo (bar, 1.5 mm). (Ct: Cotyledon; Hy: Hypocotyl; ME: Meristematic Centres; Ra: Radicle; SA: Shoot Apex). 105 I. Viehmannova et al. / Electronic Journal of Biotechnology 17 (2014) 102–106 Fig. 2. ISSR proﬁle of control plant and in vitro regenerants (F1–F10) of S. sonchifolius using primer ‘UBC834’ (L: ladder; CP: control plant). Linear histograms of relative nuclear DNA content showed in all cases two peaks, the ﬁrst corresponding to somatic nuclei arrested in the G0/G1 phase of the cell cycle, and belonging to the internal standard Bellis perennis, and the second representing nuclei of the yacon sample in the G0/G1 phase (Fig. 3a, b). The DNA-ratios of regenerated plantlets, obtained from different initiation media, varied from 1.708 to 1.722, and they were not signiﬁcantly different to that of the control plant (1.709), suggesting that the plants obtained via somatic embryogenesis had maintained stable octoploid level. Coefﬁcients of variation did not exceed 2.48 in any of the measurements. Absence of alterations in ploidy level indicates that there is no correlation between molecular and genomic changes. 3. Results and discussion Somaclonal variation in terms of genome changes is usually associated with aneuploidy or polyploidy [35]. In many diploid and tetraploid species, increase of ploidy level in regenerants was reported, especially when plants were recovered from long-term callus or suspension cultures [20,36]. Octoploid yacon, however, seems to be stable and not susceptible to ploidy level alteration, as demonstrated also in a previous study where extremely low frequency of polyploids was obtained after in vitro induction of polyploidization by means of colchicin and oryzalin [37]. Moreover, histograms in the present study showed only two peaks, indicating no polysomaty. Thus, pre-existence of ploidy variability in initial explants might be excluded. Fig. 2. ISSR proﬁle of control plant and in vitro regenerants (F1–F10) of S. sonchifolius using primer ‘UBC834’ (L: ladder; CP: control plant). F5 and F9, respectively. The other regenerants had a distance value of 0 compared to the control plant, indicating no genetic variation. To our best knowledge, no study on somaclonal variation in plants of yacon regenerated via somatic embryogenesis has been made before. ISSR markers proved to be a powerful technique to reveal somaclonal variation. Based on our results, it can be concluded that somatic embryogenesis probably may not be favourable for large-scale propagation and conservation purposes in yacon, as the risk of genetic instability cannot be excluded. Nevertheless, it seems to be an effective technique to widen genetic variability and it can be used in yacon improvement, especially when low sexual reproductive capacity hinders classical ways of breeding. Besides, somatic embryos may serve as a valuable source for studies of biochemical, physiological and morphological processes during embryo developmental stages, as the lack of zygotic embryos in yacon does not allow us to carry out this type of research. ISSR analysis was found to be a reliable method, enabling rapid evaluation of somaclonal variability by fast scanning of the whole genome as reported by Rathore et al. [30]. In our case, the method revealed genetic variability in 6 among 60 plants under study. Eight primers were polymorphic and generated 10 polymorphic bands with 7.19% mean polymorphism. Similarly, many recent studies have used ISSR analysis to determine somaclonal variation of in vitro regenerants, e.g. in Nothapodytes foetida 7.53% polymorphism [31], in Aloe vera 15.1% polymorphism [30], and in Cymbopogon pendulus 9.4% polymorphism [32] was detected. Since ISSR analysis is not effective in detection of epigenetic changes, Linacero et al. 3. Results and discussion [33] applied a modiﬁed method of ISSR using methylation sensitive restriction enzymes to digest DNA, followed by PCR ampliﬁcation, to detect both epigenetic and genetic changes in somatic-embryo derived plants of Secale cereale. In yacon, however, detailed research on epigenetic changes still need to be carried out. References The use of aluminium lake of nuclear fast red in plant material successively with alcian blue. Biol Plant 1973;15:294–7. http://dx.doi.org/10.1007/BF02922713. [27] Dolezel J, Greilhuber J, Suda J. Flow cytometry with plant cells. Weinheim: Wiley-VCH; 2007. [8] Mansilla R, López C, Flores M, Espejo R. Reproductive biology study in ﬁve accessions of Smallanthus sonchifolius (Poepp. & Endl.) Robinson. Ecol Appl 2010;9:167–75. [28] Schonswetter P, Suda J, Popp M, Weiss-Schneeweiss H, Brochmann C. 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Afr Crop Sci Conf Proc 2003;6:363–6. p p g [7] Milella L, Martelli G, Salava J, Fernández E, Ovesna J, Greco I. Total phenolic content, RAPDs, AFLPs and morphological traits for the analysis of variability in Smallanthus sonchifolius. Genet Resour Crop Evol 2011;58:545–51. http://dx.doi.org/10.1007/s10722-010-9597-x. [26] Benes K, Kaminek M. Acknowledgements PCR-based markers can be used for genetic discrimination purposes to locate and isolate mutations linked to these markers [19], however, some important variations like genomic mutations may not be detected [34]. For this reason, ploidy level detection is a useful complementary approach [19]. This research was ﬁnancially supported by an Internal Grant Agency of the Czech University of Life Science Prague CIGA (Project No. 20115004) and an Internal Grant Agency of Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague IGA (Project No. 20135119). Fig. 3. Representative ﬂow cytometric histograms documenting the relative DNA content of in vitro plants. (a) Control plant; (b) Randomly selected in vitro regenerant. The peak indicated as “” corresponds to the internal reference standard (Bellis perennis). Fig. 3. Representative ﬂow cytometric histograms documenting the relative DNA content of in vitro plants. (a) Control plant; (b) Randomly selected in vitro regenerant. The peak indicated as “” corresponds to the internal reference standard (Bellis perennis). I. Viehmannova et al. / Electronic Journal of Biotechnology 17 (2014) 102–106 106 [19] Jin SX, Mushke R, Zhu HG, Tu L, Lin ZX, Zhang YX, et al. Detection of somaclonal variation of cotton (Gossypium hirsutum) using cytogenetics, ﬂow cytometry and molecular markers. Plant Cell Rep 2008;27:1303–16. http://dx.doi.org/10.1007/s00299-008-0557-2. References References In: Vasil IK, Thorpe TA, editors. Plant cell and tissue culture. 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https://openalex.org/W2887920589	https://www.aclweb.org/anthology/D18-1103.pdf	English	null	Rapid Adaptation of Neural Machine Translation to New Languages	null	2,018	cc-by	4,034	1Code to reproduce experiments at https://github. com/neubig/rapid-adaptation Graham Neubig, Junjie Hu Language Technologies Institute, Carnegie Mellon University {gneubig,junjieh}@cs.cmu.edu and Blunsom (2013); Bahdanau et al. (2015)), re- quire large amounts of training data, and creating high-quality systems in low-resource languages (LRLs) is a difﬁcult challenge where research ef- forts have just begun (Gu et al., 2018). Another hurdle, which to our knowledge has not been cov- ered in previous research, is the time it takes to create such a system. In a crisis situation, time is of the essence, and systems that require days or weeks of training will not be desirable or even fea- sible. Abstract This paper examines the problem of adapt- ing neural machine translation systems to new, low-resourced languages (LRLs) as effectively and rapidly as possible. We propose meth- ods based on starting with massively multi- lingual “seed models”, which can be trained ahead-of-time, and then continuing training on data related to the LRL. We contrast a num- ber of strategies, leading to a novel, simple, yet effective method of “similar-language reg- ularization”, where we jointly train on both a LRL of interest and a similar high-resourced language to prevent over-ﬁtting to small LRL data. Experiments demonstrate that massively multilingual models, even without any explicit adaptation, are surprisingly effective, achiev- ing BLEU scores of up to 15.5 with no data from the LRL, and that the proposed similar- language regularization method improves over other adaptation methods by 1.7 BLEU points average over 4 LRL settings.1 In this paper we focus on the question: how can we create MT systems for new language pairs as accurately as possible, and as quickly as possible? To examine this question we propose NMT meth- ods at the intersection of cross-lingual transfer learning (Zoph et al., 2016) and multilingual train- ing (Johnson et al., 2016), two paradigms that, to our knowledge, have not been used together in pre- vious work. Our methods, laid out in §2 follow the process of training a seed model on a large num- ber of languages, then ﬁne-tuning the model to im- prove its performance on the language of interest. We propose a novel method of similar-language regularization (SLR) where training data from a second similar languages is used to help prevent over-ﬁtting to the small LRL dataset. Proceedings of the 2018 Conference on Empirical Methods in Natural Language Processing, pages 875–880 Brussels, Belgium, October 31 - November 4, 2018. c⃝2018 Association for Computational Linguistics Proceedings of the 2018 Conference on Empirical Methods in Natural Language Processing, pages 875–880 Brussels, Belgium, October 31 - November 4, 2018. c⃝2018 Association for Computational Linguistics 875 3“Related” could mean different things: typologically re- lated or having high lexical overlap. In our experiments our LRLs are all selected to have an helper that is highly similar in both aspects, but choosing an appropriate helper when this is not the case is an interesting problem for future work. 2 Training Paradigms In this paper, we consider the setting where we have a source LRL of interest, and we want to translate into English.2 All of our adaptation methods are based on ﬁrst training on larger data including other languages, then ﬁne-tuning the model to be speciﬁcally tailored to the LRL. We ﬁrst discuss a few multilingual training paradigms from previous literature (§2.1), then discuss our proposed adaptation methods (§2.2). In the following, we will consider adaptation methods that focus on tailoring a more general model (i.e. bi-source or universal) to a more spe- ciﬁc model (i.e. single-source or bi-source). 1 Introduction When disaster strikes, news and social media are invaluable sources of information, allowing hu- manitarian organizations to rapidly mitigate crisis situations and save lives (Vieweg et al., 2010; Neu- big et al., 2011; Starbird et al., 2012). However, language barriers looms large over these efforts, especially when disasters occur in parts of the world that use less common languages. In these cases, machine translation (MT) technology can be a valuable tool, with one widely-heralded suc- cess story being the deployment of Haitian Creole- to-English translation systems during the earth- quakes in Haiti (Lewis, 2010; Munro, 2010). In the experiments in §3, we attempt to answer two questions: (1) Which method of creating mul- tilingual systems and adapting them to an LRL is the most effective way to increase accuracy? (2) How can we create the strongest system possible with a bare minimum of training time? The re- sults are sometimes surprising – we ﬁrst ﬁnd that a single monolithic model trained on 57 languages can achieve BLEU scores as high as 15.5 with no training data in the new source language whatso- ever. In addition, the proposed method starting with a universal model then ﬁne-tuning with the SLR proves most effective, achieving gains of 1.7 However, data-driven MT systems, particularly neural machine translation (NMT; Kalchbrenner 875 BLEU points averaged over several language pairs compared to previous methods adapting to only the LRL. model that has wide coverage of vocabulary and syntax of a large number of languages, but also has the drawback in that a single model must be able to express information about all the languages in the training set within its limited parameter bud- get. Thus, it is reasonable to expect that this model may achieve worse accuracy than a model created speciﬁcally to handle a particular source language. In the following, we will consider adaptation methods that focus on tailoring a more general model (i.e. bi-source or universal) to a more spe- ciﬁc model (i.e. single-source or bi-source). model that has wide coverage of vocabulary and syntax of a large number of languages, but also has the drawback in that a single model must be able to express information about all the languages in the training set within its limited parameter bud- get. Thus, it is reasonable to expect that this model may achieve worse accuracy than a model created speciﬁcally to handle a particular source language. 2.2 Adaptation to New Languages As noted in the introduction, there are two major requirements: the accuracy of the system is im- portant and the training time required from when we learn of a need for translation to when we can ﬁrst start producing adequate results. Throughout the discussion, we will compare various adapta- tion paradigms with respect to these two aspects. 4In contrast to Gu et al. (2018), who train on 10 languages. Malaviya et al. (2017); Tiedemann (2018) train NMT on over 1,000 languages, but only as a feature extractor for down- stream tasks; MT accuracy itself is not evaluated. 2.1 Multilingual Modeling Methods We use three varieties of multilingual training: Single-source modeling (“Sing.”) is the ﬁrst method, using only parallel data between the LRL of interest and English. This method is straightfor- ward and the resulting model will be most highly tailored to the ﬁnal test language pair, but the method also has the obvious disadvantage that training data is very sparse. 2Translation into LRLs, is a challenging and interesting problem in it’s own right, but beyond the scope of the paper. 2.2.1 Adaptation by Fine-tuning In our experiments (§3.1) this en- tails training systems on 58 source languages, to our knowledge the largest reported in NMT exper- iments.4 This paradigm allows us to train a single 876 LRL train dev test HRL train aze 5.94k 671 903 tur 182k bel 4.51k 248 664 rus 208k glg 10.0k 682 1,007 por 185k slk 61.5k 2,271 2,445 ces 103k Table 1: Data sizes in sentences for LRL/HRL pairs word embeddings, 512-dimensional hidden states, and a standard LSTM-based decoder. Following standard practice (Sennrich et al., 2016; Denkowski and Neubig, 2017), we break low-frequency words into subwords using the sentencepiece toolkit.6 There are two alter- natives for creating subword units: jointly learning subwords over all source language, or separately learning subwords for each source language, then taking the union of all the subword vocabularies as the vocabulary for the multilingual model. Previ- ous work on multilingual training has preferred the former (Nguyen and Chiang, 2017), but in this pa- per we use the latter for two reasons: (1) because data in the LRL will not affect the subword units from the other languages, in the cold-start sce- nario we can postpone creation of subword units for the LRL until directly before we start train- ing on the LRL itself, and (2) we need not be concerned with the LRL being “overwhelmed” by the higher-resourced languages when calculating statistics used in the creation of subword units, be- cause all languages get an equal share.7 In the ex- periments, we use a subword vocabulary of 8,000 for each language. on all languages but the LRL (All−). 6https://github.com/google/ sentencepiece, using the unigram training setting. 7 Preliminary experiments found both comparable: with scores of 20.1 and 19.4 for separate and joint respectively. 8http://statmt.org/moses 9https://github.com/artetxem/undreamt 2.2.1 Adaptation by Fine-tuning Our ﬁrst adaptation method, inspired by Zoph et al. (2016) is based on ﬁne-tuning to the source language of interest. Within our experiments, we will test this setting, but also make two distinctions between the types of adaptation: Bi-source modeling (“Bi”) trains an MT system with two source languages: one LRL that we would like to translate from, and a second highly related high-resource language (HRL): the helper source language.3 This method is inspired by Johnson et al. (2016), who examine multilingual translation models to/from English and two highly related languages such as Spanish/Portuguese or Japanese/Korean. The advantage of this method is that it allows the LRL to learn from a highly simi- lar helper, potentially increasing accuracy. Seed Model Variety: Zoph et al. (2016) per- formed experiments taking a bilingual system trained on a different language (e.g. French) and adapting it to a new LRL (e.g. Uzbek). We can also take universal model and adapt it to the new language, a setting that we examine (to our knowl- edge, for the ﬁrst time) in this work. Warm vs. Cold Start: Another contrast is whether we have training data for the LRL of inter- est while training the original system, or whether we only receive training data after the original model has already been trained. We call the former warm start, and the latter cold start. Intuitively, we expect warm-start training to perform better, as having access to the LRL of interest during the training of the original model will ensure that it can handle the LRL to some extent. However, the cold-start scenario is also of interest: we may want to spend large amounts of time training a strong model, then quickly adapt to a new language that we have never seen before in our training data as data becomes available. For the cold-start models, we start with a model that is only trained on the HRL similar to the LRL (Bi−), or a model trained All-source modeling (“All”) trains not only on a couple source languages, but instead creates a uni- versal model on all of the languages that we have at our disposal. 3.1 Experimental Setup We perform experiments on the 58-language-to- English TED corpus (Qi et al., 2018), which is ideal for our purposes because it has a wide variety of languages over several language families, some high-resourced and some low-resourced. Like Qi et al. (2018), we experiment with Azerbaijani (aze), Belarusian (bel), and Galician (glg) to En- glish, and also additionally add Slovak (slk), a slightly higher resourced language, for contrast. These languages are all paired with a similar HRL: Turkish (tur), Russian (rus), Portuguese (por), and Czech (ces) respectively. Data sizes are shown in Table 1. 2.2.2 Similar-Language Regularization One problem with adapting to a small amount of data in the target language is that it will be very easy for the model to over-ﬁt to the small train- ing set. To alleviate this problem, we propose a method of similar language regularization: while training to adapt to the language of interest, we also add some data from another similar HRL that has sufﬁcient resources to help prevent over- ﬁtting. We do this in two ways: Corpus Concatenation: Simply concatenate the data from the two corpora, so that we have a small amount of data in the LRL, and a large amount of data in the similar HRL. Balanced Sampling: Every time we select a mini- batch to do training, we either sample it from the LRL, or from the HRL according to a ﬁxed ra- tio. We try different sampling strategies, includ- ing sampling with a 1-to-1 ratio, 1-to-2 ratio, and 1-to-4 ratio for the LRL and HRL respectively. We also compare with two additional baselines: phrase-based MT implemented in Moses,8 and unsupervised NMT implemented in undreamt.9 Moses is trained on the bilingual data only (train- ing multilingually reduced average accuracy), and undreamt is trained on all monolingual data available for the LRL and English. 5Found in examples/stanford-iwslt/ 3.2 Experimental Results All-→Bi All-→Bi 1-1 Hours Training BLEU 0 1 2 3 4 5 6 7 8 9 10 0 0.03 0.06 0.09 0.12 0.15 0.18 0.21 bel-rus Sing. Bi All-→Sing. All-→Bi All-→Bi 1-1 Hours Training BLEU Table 2: BLEU for single-source (Sing.), bi-source (Bi), and all-source universal (All) models, with adapted counterparts. 1-1, 1-2, 1-4 indicate balanced sampling from §2.2. Bold indicates highest score. Table 2: BLEU for single-source (Sing.), bi-source (Bi), and all-source universal (All) models, with adapted counterparts. 1-1, 1-2, 1-4 indicate balanced sampling from §2.2. Bold indicates highest score. Figure 1: Example of adaptation on the aze-eng and bel-eng development sets language regularization (“→Bi”), we can see that this helps signiﬁcantly over adapting directly to the LRL, particularly in the cold-start case where we can observe gains of up to 1.7 BLEU points. Finally, in our data setting, corpus concatenation outperforms balanced sampling in both the cold- start and warm-start scenarios. language regularization (“→Bi”), we can see that this helps signiﬁcantly over adapting directly to the LRL, particularly in the cold-start case where we can observe gains of up to 1.7 BLEU points. Finally, in our data setting, corpus concatenation outperforms balanced sampling in both the cold- start and warm-start scenarios. capacity, training on a highly resourced language is effective. Comparing with the phrase-based baseline, as noted by Koehn and Knowles (2017) NMT tends to underperform on low-resource set- tings when trained only on the data available for these languages. However, multilingual training of any variety quickly remedies this issue; all out- perform phrase-based handily. How Can We Adapt Most Efﬁciently? Finally, we revisit adapting to new languages efﬁciently, with Figure 1 showing BLEU vs. hours training for the aze/tur and bel/rus source language pairs (others were similar). We can see that in all cases the cold-start models (All−→) either outperform or are comparable in ﬁnal accuracy to the from- scratch single-source and bi-source models. In ad- dition, all of the adapted models converge faster than the bi-source from-scratch trained models, in- dicating that adapting from seed models is a good strategy for rapid construction of MT systems in new languages. Comparing the cold-start adap- tation strategies, we can see that in general, the higher the density of target language training data, the faster the training converges to a solution, but the worse the ﬁnal solution is. 3.2 Experimental Results This suggests that there is a speed/accuracy tradeoff in the amount of similar language regularization we apply dur- ing ﬁne-tuning. More interestingly, examining the cold-start re- sults, we can see that even systems with no data in the target language are able to achieve non- trivial accuracies, up to 15.5 BLEU on glg-eng. Interestingly, in the cold-start scenario, the All− model bests the Bi−model, indicating that mas- sively multilingual training is more useful in this setting. In contrast, the unsupervised NMT model struggles, achieving a BLEU score of around 0 for all language pairs – this is because unsupervised NMT requires high-quality monolingual embed- dings from the same distribution, which can be trained easily in English, but are not available in the low-resource languages we are considering. Does Adaptation Help? Regarding adaptation, we can ﬁrst observe that regardless of the origi- nal model and method for adaptation, adaptation is helpful, particularly (and unsurprisingly) in the cold-start case. When adapting directly to only the target language (“→Sing.”), adapting from the massively multilingual model performs better, in- dicating that information about all input languages is better than just a single language. Next, compar- ing with our proposed method of adding similar 3.2 Experimental Results Table 2 shows our main translation results, with warm-start scenarios in the upper half and cold- start scenarios in the lower half. Does Multilingual Training Help? To answer this question, we can compare the warm-start Sing., Bi, and All settings, and ﬁnd that the an- swer is a resounding yes, gains of 7-13 BLEU points are obtained by going from single-source to bi-source or all-source training, corroborating previous work (Gu et al., 2018). Bi-source mod- els tend to perform slightly better than all-source models, indicating that given identical parameter Models are implemented using xnmt (Neu- big et al., 2018), commit 8173b1f, and start with the recipe for training on IWSLT TED5. The model consists of an attentional neural machine translation model (Bahdanau et al., 2015), using bi-directional LSTM encoders, 128-dimensional 877 0 1 2 3 4 5 6 7 8 9 10 0 0.03 0.06 0.09 0.12 0.15 0.18 0.21 bel-rus Sing. Bi All-→Sing. All-→Bi All-→Bi 1-1 Hours Training BLEU 0 1 2 3 4 5 6 7 8 9 0 0.03 0.06 0.09 0.12 0.15 aze-tur Sing. Bi All-→Sing. All-→Bi All-→Bi 1-1 Hours Training BLEU Figure 1: Example of adaptation on the aze-eng and bel-eng development sets Strategy aze/tur bel/rus glg/por slk/ces Avg. Phrase-based 5.9 10.5 22.3 23.0 15.4 Unsupervised NMT 0.0 0.3 0.4 0.0 0.2 Warm Start Sing. 2.7 2.8 16.2 24.0 11.4 Bi 10.9 15.8 27.3 26.5 20.1 All 9.7 16.7 26.5 25.0 19.5 Bi →Sing. 11.4 16.3 27.5 27.1 20.6 All→Sing. 10.1 17.5 28.2 27.4 20.8 All→Bi 11.7 18.3 28.8 28.2 21.8 All→Bi 1-1 10.2 18.3 28.8 28.3 21.4 All→Bi 1-2 11.0 17.5 29.1 28.2 21.4 All→Bi 1-4 11.1 17.9 28.5 27.9 21.3 Cold Start Bi− 3.8 2.5 8.6 5.4 5.1 All− 3.7 3.5 15.5 7.3 7.5 Bi−→Sing. 8.7 11.8 25.4 26.8 18.2 All−→Sing. 8.8 15.3 26.5 27.6 19.5 All−→Bi 10.7 17.4 28.4 28.0 21.2 All−→Bi 1-1 10.5 16.0 28.0 28.2 20.7 All−→Bi 1-2 10.7 17.1 28.3 27.9 21.0 All−→Bi 1-4 11.0 17.4 28.4 27.6 21.1 Table 2: BLEU for single-source (Sing.), bi-source (Bi), and all-source universal (All) models, with adapted counterparts. 1-1, 1-2, 1-4 indicate balanced sampling from §2.2. Bold indicates highest score. 0 1 2 3 4 5 6 7 8 9 0 0.03 0.06 0.09 0.12 0.15 aze-tur Sing. Bi All-→Sing. 5 Conclusion This paper examined methods to rapidly adapt MT systems to new languages by ﬁne-tuning. In both warm-start and cold-start scenarios, the best re- sults were obtained by adapting a pre-trained uni- versal model to the low-resource language while regularizing with similar languages. Melvin Johnson et al. 2016. Google’s multilingual neu- ral machine translation system: Enabling zero-shot translation. TACL. Nal Kalchbrenner and Phil Blunsom. 2013. Recurrent continuous translation models. In Proc. EMNLP, pages 1700–1709. 4 Related Work While adapting MT systems to new languages is a long-standing challenge (Schultz and Black, 2006; Jabaian et al., 2013), multilingual NMT is highly promising in its ability to abstract across 878 language boundaries (Firat et al., 2016; Ha et al., 2016; Johnson et al., 2016). Results on multi- lingual training for low-resource translation (Gu et al., 2018; Qi et al., 2018) further demonstrates this potential, although these works do not con- sider adaptation to new languages, the main focus of our work. Notably, we did not examine par- tial freezing of parameters, another method proven useful for cross-lingual adaptation (Zoph et al., 2016); this is orthogonal to our multi-lingual train- ing approach but the two methods could poten- tially be combined. Finally, unsupervised NMT approaches (Artetxe et al., 2017; Lample et al., 2018, 2017) require no parallel data, but rest on strong assumptions about high-quality comparable monolingual data. As we show, when this assump- tion breaks down these methods fail to function, while our cold-start methods achieve non-trivial accuracies even with no monolingual data. Dzmitry Bahdanau, Kyunghyun Cho, and Yoshua Ben- gio. 2015. Neural machine translation by jointly learning to align and translate. In Proc. ICLR. Michael Denkowski and Graham Neubig. 2017. Stronger baselines for trustable results in neural ma- chine translation. In Proc. WNMT. Orhan Firat, Kyunghyun Cho, and Yoshua Bengio. 2016. Multi-way, multilingual neural machine translation with a shared attention mechanism. In Proc. NAACL, pages 866–875. Jiatao Gu, Hany Hassan, Jacob Devlin, and Victor OK Li. 2018. Universal neural machine translation for extremely low resource languages. Proc. NAACL. Thanh-Le Ha, Jan Niehues, and Alexander Waibel. 2016. Toward multilingual neural machine trans- lation with universal encoder and decoder. arXiv preprint arXiv:1611.04798. Bassam Jabaian, Laurent Besacier, and Fabrice Lefevre. 2013. Comparison and combination of lightly supervised approaches for language porta- bility of a spoken language understanding system. IEEE Transactions on Audio, Speech, and Language Processing, 21(3):636–648. Acknowledgements Philipp Koehn and Rebecca Knowles. 2017. Six chal- lenges for neural machine translation. Proc. WNMT. The authors thank Jaime Carbonell, Xinyi Wang, Rebecca Knowles, Arya McCarthy, and anony- mous reviewers for their constructive comments on this paper. Guillaume Lample, Ludovic Denoyer, and Marc’Aurelio Ranzato. 2017. Unsupervised machine translation using monolingual corpora only. This work is sponsored by Defense Advanced Research Projects Agency Information Innovation Ofﬁce (I2O). Program: Low Resource Languages for Emergent Incidents (LORELEI). Issued by DARPA/I2O under Contract No. HR0011-15- C0114. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the ofﬁcial poli- cies, either expressed or implied, of the U.S. Gov- ernment. The U.S. Government is authorized to reproduce and distribute reprints for Government purposes notwithstanding any copyright notation here on. Guillaume Lample, Myle Ott, Alexis Conneau, Lu- dovic Denoyer, and Marc’Aurelio Ranzato. 2018. Phrase-based & neural unsupervised machine trans- lation. arXiv preprint arXiv:1804.07755. William D. Lewis. 2010. Haitian Creole: how to build and ship an MT engine from scratch in 4 days, 17 hours, & 30 minutes. In Proc. EAMT. Chaitanya Malaviya, Graham Neubig, and Patrick Lit- tell. 2017. Learning language representations for ty- pology prediction. In Proc. EMNLP. Robert Munro. 2010. Crowdsourced translation for emergency response in Haiti: the global collabora- tion of local knowledge. In Proc. AMTA Workshop on Collaborative Crowdsourcing for Translation. References Graham Neubig, Yuichiroh Matsubayashi, Masato Hagiwara, and Koji Murakami. 2011. Safety infor- mation mining - what can NLP do in a disaster -. In Proc. IJCNLP, pages 965–973. Mikel Artetxe, Gorka Labaka, Eneko Agirre, and Kyunghyun Cho. 2017. Unsupervised neural ma- chine translation. arXiv preprint arXiv:1710.11041. Mikel Artetxe, Gorka Labaka, Eneko Agirre, and Kyunghyun Cho. 2017. Unsupervised neural ma- chine translation. arXiv preprint arXiv:1710.11041. 879 Graham Neubig, Matthias Sperber, Xinyi Wang, Matthieu Felix, Austin Matthews, Sarguna Pad- manabhan, Ye Qi, Devendra Singh Sachan, Philip Arthur, Pierre Godard, John Hewitt, Rachid Riad, and Liming Wang. 2018. XNMT: The extensible neural machine translation toolkit. In Proc. AMTA, Boston. Toan Q. Nguyen and David Chiang. 2017. Transfer learning across low-resource, related languages for neural machine translation. pages 296–301. Ye Qi, Devendra Sachan, Matthieu Felix, Sarguna Pad- manabhan, and Graham Neubig. 2018. When and why are pre-trained word embeddings useful for neural machine translation? In Proc. NAACL, New Orleans, USA. Tanja Schultz and Alan W Black. 2006. Challenges with rapid adaptation of speech translation systems to new language pairs. In Proc. ICASSP, volume 5. IEEE. Rico Sennrich, Barry Haddow, and Alexandra Birch. 2016. Neural machine translation of rare words with subword units. In Proc. ACL, pages 1715–1725. Kate Starbird, Grace Muzny, and Leysia Palen. 2012. Learning from the crowd: Collaborative ﬁltering techniques for identifying on-the-ground Twitterers during mass disruptions. In Proc. ISCRAM. J¨org Tiedemann. 2018. Emerging language spaces learned from massively multilingual corpora. arXiv preprint arXiv:1802.00273. Sarah Vieweg, Amanda L Hughes, Kate Starbird, and Leysia Palen. 2010. Microblogging during two nat- ural hazards events: what Twitter may contribute to situational awareness. In Proc. CHI, pages 1079– 1088. Barret Zoph, Deniz Yuret, Jonathan May, and Kevin Knight. 2016. Transfer learning for low-resource neural machine translation. In Proc. EMNLP, pages 1568–1575. 880

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